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Sensing sequence-specific DNA micro-environment with nucleotide-independent nitroxides

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Sensing sequence-specific DNA micro-environment with nucleotide-independent nitroxides

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Content SENSING SEQUENCE-SPECIFIC DNA MICRO-ENVIRONMENT WITH NUCLEOTIDE-INDEPENDENT NITROXIDES by Anna M. Popova A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (CHEMISTRY) May 2011 Copyright 2011 Anna M. Popova ii Dedication To L.M., our High School Mom. iii Acknowledgements During these years at USC I‟ve met lots of talented and enthusiastic people who contributed to my development, who were helpful and inspiring, who became very special and dear to me. It is an honor for me to thank you all. Peter, from the beginning till very end you were encouraging and extremely supportive. Working in your laboratory I„ve open to myself an exciting and puzzling world of biomolecules and learned how beautifully physico-chemical tools can be applied to unravel their dynamics and functionality. I strongly believe that unique experience I obtained working with you will contribute to my future endeavors in Life Sciences. Qi, Gian, Yun, Maria, Glenna, Dewi, Bo, Xiao, Phuong, Yuan, Peter – being with all you guys was a great pleasure. Without warm and friendly environment you created, without help and advice you were always open for, my work would be impossible. I will miss our long group meetings and scientific disputes, the time when new ideas were hatching. I am especially grateful to Gian - the blithe spirit of our lab. Dear friend, your openness and optimism have warmed and cheered me up during the rainy days of my PhDs. My interactions with Profs. Hanna Reisler, Ralf Langen and Steve Bradforth have helped me to progress from a naïve student to a confident young scientist. Joining the lab, passing the Quals or sending job applications - your strong support, invaluable advice and scientific knowledge have guided me through, providing experience and judgment. iv I want to sincerely thank Prof. Prakash for being an outstanding teacher and Prof. Warshel for his enthusiasm and inspiration to always look beyond the class material. The graduate courses you developed have influenced my research interests most strongly. My life at USC Chemistry Department was like Los Angeles weather, always sunny and warm, in many ways due to a friendly atmosphere created by people such as Michele Dea, Heather Connor, and Marie de la Torre. Ross Lewis and Allan Kershaw were my 9- 1-1 service, providing an instant relief in technical and instrumental blockages. Dr. Michael Quinlan (as students like to conclude their last lab report) was an awesome and simply the best Chem105 person, and I am waiting forward to see his “storybook”, beyond just the lab manual, being published. Lastly, I owe my deepest gratitude to Chemistry Department, WiSE program and USC College for their financial support, making student living a lot simpler. Joining USC was not just a turning point in my life but a wonderful adventure I will never forget. Here, I‟ve found how inspiring and ultimately rewarding the roads of science and learning can be. I‟ve met Vadim, my dear man, who continues to surprise me how beautiful the World is. I‟ve lived among diverse people and nature – an opportunity which would be hard to realize without USC support. Finally, I would like to thank a beloved city of Saint-Petersburg (Russia) for being my sweet home, and my family and friends for their love, protection and spiritual support. From the bottom of my heart I thank my grandparents Nina and Leonid for their tremendous devotions, belief and care for my future. v Table of Contents Dedication ii Acknowledgements iii List of Tables x List of Figures xi Abstract xiv Chapter 1 Sensing nucleic acid dynamics with SDSL: an overview 1. Chapter 1 Introduction        2. Site-directed spin labeling technique for studies of macromolecule dynamics 2.1. Nitroxide spin labeling       .. 2.2. Basic principles of EPR       …. 2.3. Effect of molecular motions      .... 2.4. Analysis of nitroxide spectra in the slow-motional regime          …. 3. Probing nucleic acid dynamics using cw-EPR 3.1. Overview of the previous studies    ….. 3.2. Probing structural and dynamic features in DNA with nucleotide-independent nitroxides     … 4. Chapter 1 References        … 1 5 6 9 11 14 17 21 vi Chapter 2 Development of nucleotide-independent nitroxide probes for nucleic acids studies 1. Chapter 2 Introduction 1.1. Nitroxide probes and attachment rigidity   ... 1.2. R5-series of nitroxides      ….. 2. Chapter 2 Materials and Methods 2.1. Materials          …. 2.2. Nitroxide spin-labeling      …... 2.3. Characterization of the nitroxide-labeled DNA  .. 2.4. cw-EPR experiments ...     ….. 3. Chapter 2 Results 3.1. Nitroxide probes utilizing pyrroline substitution . 3.2. Nitroxide probes with a pi-bond containing linker ... 3.3. A benzo-fused nitroxide probe      . 3.4. Nitroxide probes with a shorter linker   ... 3.5. A bifunctional nitroxide probe      . 4. Chapter 2 Discussion      … 5. Chapter 2 Conclusions       .. 6. Chapter 2 References        … 25 27 28 29 29 30 33 35 39 41 41 45 46 48 Chapter 3 Site-specific DNA structural and dynamic features revealed by nucleotide-independent nitroxide probes 1. Chapter 3 Introduction  .      … 2. Chapter 3 Materials and Methods 2.1. Materials          ….. 2.2. DNA labeling and purification      …. 2.3. DNA thermal denaturation       ….. 50 55 55 56 vii 2.4. EPR measurements       .... 2.5. Modeling of sterically accessible nitroxide rotamer space          ... 3. Chapter 3 Results 3.1. Model DNA system and nitroxide labeling  … 3.2. DNA structure perturbation     . 3.3. Site-dependent variations in R5 spectra    . 3.4. Site-dependent variations of R5a spectra  ... 3.5. DNA base mutations affect R5a spectra  …. 3.6. Effect of a cosolvent: sucrose vs. Ficoll 70  …. 4. Chapter 3 Discussion 4.1. Perturbations due to R5 and R5a nitroxide probes  ... 4.2. Comparison between R5 and R5a    4.3. Coupling between nitroxide dynamics and DNA: modulation of nitroxide internal motions  .. 4.4. Coupling between nitroxide dynamics and DNA: correlation to DNA local motions    . 5. Chapter 3 Conclusions      ….. 6. Chapter 3 References        … 56 58 58 60 62 67 71 72 74 76 77 82 84 86 Chapter 4 A nucleotide-independent nitroxide probe reports on site-specific stereomeric environment in DNA 1. Chapter 4 Introduction       . 2. Chapter 4 Materials and Methods 2.1. DNA oligonucleotides and nomenclature  .. 2.2. Preparation of R p -R5a and S p -R5a oligonucleotides … 2.3. DNA thermal denaturation        . 2.4. EPR sample preparation and measurements  ... 91 95 96 97 97 viii 2.5. EPR spectra fitting      . 3. Chapter 4 Results 3.1. Preparation and characterization of diastereopure R5a labeled DNA          … 3.2. R5a reports different R p and S p spectra at a given DNA site           .. 3.3. Site-specific variations in R p -R5a and S p -R5a spectra .. 3.4. Temperature dependence of R p -R5a and S p -R5a spectra.. 3.5. Linear combination of R p -R5a and S p -R5a spectra reproduces the mixed-diastereomer spectrum  . 4. Chapter 4 Discussion 4.1. Modulations of R p -R5a and S p -R5a by DNA local structure          ... 4.2. Modulation of R p -R5a and S p -R5a by DNA local motions            … 4.3. Mixed vs. pure diastereomers: advantages and limitations in probing DNA environment   ………….. 5. Chapter 4 Conclusions       .. 6. Chapter 4 References          98 99 101 107 108 111 114 117 119 121 122 Chapter 5 Nitroxide sensing DNA micro-environment: effect of a single nucleotide substitution 1. Chapter 5 Introduction       . 2. Chapter 5 Materials and Methods 2.1. Materials and abbreviations     …. 2.2. Nitroxide labeling and purification    ... 2.3. EPR sample preparation and measurements  ... 2.4. EPR spectra fitting      … 3. Chapter 5 Results          . 125 128 129 129 130 131 ix 4. Chapter 5 Discussion      .... 5. Chapter 5 Conclusions       .. 6. Chapter 5 References        … 136 139 140 Chapter 6 Studying biologically relevant DNA with a nucleotide- independent nitroxide probe 1. Chapter 6 Introduction       . 2. Chapter 6 Materials and Methods     ... 3. Chapter 6 Results and Discussion 3.1. Probing DNA local environment at bcl-2 major break point region         ….. 3.2. Probing DNA local environment in GGGCCC and GCGCGC sequences        . 3.3. Probing DNA local environment at the restriction site of HhaI          .. 4. Chapter 6 Conclusions       .. 5. Chapter 6 References        … 142 144 145 152 156 161 162 Chapter 7 Future work 165 Bibliography 167 x List of Tables 1.1 Experimental methods applied to study structural dynamics in nucleic acids       ……………………………………. 2.1 Results summary for the R5-series  …………………… 2.2 A set of estimated sulfur-sulfur distances in CS67 DNA duplex and HO1942 probe  …………………………………….. 3.1 Thermodynamic parameters of duplex formation for R5a labeled CS duplexes   …………………………………………… 3.2 Sterically allowed conformational space of R5a as estimated by NASNOX   ………………………………………… 4.1 Free energy parameters of duplex formation for R5a labeled CS duplexes      …   ………… 4.2 Parameters for fitting experimental R5a spectra, acquired at 5C        ………………………. 4.3 Parameters for fitting CS7 and CS9 diastereomer spectra at -5, 15  and 25C        …………………………… 5.1 Parameters obtained from simulations of R5a spectra measured at different temperatures  ………………………………. 3 31 44 61 79 101 104 111 136 xi List of Figures 1.1 Timescale of dynamic processes for nucleic acids ……………… 1.2 Nitroxide radicals and a nitroxide EPR spectrum ………………. 1.3 Orientation sensitivity of an EPR spectrum  ……………. 1.4 Effects of molecular disorder and rotational motions on a nitroxide spectrum     ……………………….. 1.5 Schematic of a motional model used in MOMD spectral simulations           …… 1.6 Nitroxide labeling schemes used for probing rotational dynamics in DNA and RNA molecules    ………………………… 2.1 R5 nitroxide and CS DNA      …………….. 2.2 EPR spectra obtained with HO3927 and HO1820 attached to position CS7 of the DNA duplex      ………….. 2.3 Comparison between HO3317, HO368 and HO1820 spectra measured at room temperature   .    …….. 2.4 EPR spectra obtained with HO3178 attached to positions CS2 and CS7 of the DNA duplex         …………… 2.5 CS2 and CS7 HO1642 spectra acquired at different temperatures ….. 2.6 Comparison of the 5 C CS7 spectra obtained using different nitroxides            .. 2.7 HO3992 spectra, with the label at positions 2, 7, and 12 of the CS DNA              2 6 9 10 13 15 27 34 35 36 37 38 40 xii 2.8 Characterization of CS67_HO1942   ……………... 2.9 CS67_HO1942 spectra obtained with DNA in a single- or double- stranded form            3.1 The phosphorothioate labeling scheme  ……………... 3.2 MALDI-TOF spectrum of an R5a labeled DNA strand.   … 3.3 UV melting data for a wild type CS DNA and CS14_R5a         ………………. 3.4 EPR spectra of R5 labeled CS DNA duplexes     . 3.5 Comparisons of R5 spectra       ……………... 3.6 Reproducibility of CS 9_R5 and CS2_R5a spectra . …………….. 3.7 Estimated R5 rotational correlation time ( )  ……………… 3.8 EPR spectra of R5a labeled CS DNA duplexes ………………… 3.9 EPR spectra of R5a at positions 2 and 24 in the mutant and wild type CS duplexes     .     ….. 3.10 R5a spectra obtained in different cosolvents  ……………… 3.11 Proposed R5a/DNA interactions at the 5‟ terminal site CS2           …………….. 4.1 Nitroxide labeled DNA system    ………………. 4.2 Separation of R p and S p diastereomers  ……………… 4.3 R p -R5a and S p -R5a spectra measured at 5ºC  …………… 4.4 Motional parameters obtained from fitting 5ºC spectra ……………. 4.5 Temperature dependence of R p -R5a and S p -R5a spectra …………… 42 43 52 59 62 63 64 65 66 68 71 73 80 92 100 103 106 109 xiii 4.6 R p and S p spectra obtained at different temperatures overlaid to their best fits     ………………………………... 4.7 Comparisons between measured spectra of R5a attached to mixed- diastereomers and those computed by weighted average of the individual R p -R5a and S p -R5a spectra          . 4.8 Results of the mixed-diastereomer spectral simulations …………… 4.9 Spatial orientations of R p and S p nitroxides at CS2 ……………….. 110 112 113 116 5.1 Nitroxide labeled CS oligonucleotides and diastereomer separation         … …………. 5.2 Comparisons between cw-EPR spectra obtained for R5 labeled wild type and mutant DNA duplexes  . …………………………. 5.3 Comparisons between cw-EPR spectra obtained for R5a labeled wild type and mutant DNA duplexes   . …………… …….. 5.4 Simulations results for R5a spectra measured at 25 C, using wild type and mutant DNA     …………… ………… 5.5 Simulations results for R p -R5a spectra experimentally obtained at 5 C and 15C      . …………… ………… 6.1 A scheme of t(14;18) chromosomal translocation ………………… 6.2 Bisulfite reactivity pattern at the bcl-2 major break point region (Mbr)         ………………… 6.3 cw-EPR spectra of a single R5a attached to different sites of 44-nt bcl-2 DNA      …………… …………… 6.4 Pairwise comparisons between R5 and R5a bcl-2 spectra, obtained at room temperature   …………… ………………... 6.5 Results of bisulfite probing for DNA duplexes containing GGGCCC and GCGCGC sequences  …………… …………… ….. 127 132 133 134 135 146 147 149 150 153 xiv 6.6 G/C DNA constructs and their characterization  . ………….. 6.7 Pairwise comparisons between R5a spectra of G 3 C 3 and (GC) 3 ….. 6.8 Results of R5a scanning at HhaI restriction site . ………….. 6.9 Effect of cytosine methylation on R5a spectra   ……………. 154 155 159 160 xv Abstract In site-directed spin labeling, a covalently attached nitroxide probe containing a chemically stable unpaired electron is utilized to obtain information on the local environment of the parent macromolecule. Studies presented in this dissertation examine feasibility of probing local DNA structural and dynamic features using a class of nitroxides that are linked to chemically substituted phosphorothioate positions at the DNA backbone (R5-series). Two members of this family, designated as R5 and R5a, were attached to multiple sites of a dodecameric DNA duplex without severely perturbing the native B-form conformation. Measured X-band electron paramagnetic resonance (EPR) spectra, which report on nitroxide rotational motions, were found to vary depending on the location of the label and the identity of a phosphorothioate diastereomer (R p or S p ). Spectral simulations and molecular modeling have been used to define basic principles for correlating observed variation in EPR spectra with site-specific structural and dynamic features in DNA. Overall, these studies advance our understanding of coupling between DNA and R5/R5a, which may ultimately enable the use of nucleotide- independent probes to obtain quantitative description of sequence-specific properties in large biologically relevant DNA molecules. 1 Chapter 1 Sensing nucleic acid dynamics with SDSL: an overview 1. Chapter 1 Introduction Nucleic acid molecules (DNA and RNA) are among the major players in biology. They are involved in storage and transmission of genetic information, and carry out essential structural, regulatory and catalytic functions in all living organisms. Understanding of nucleic acids structure and conformational transitions is fundamentally important for elucidating mechanisms of their diverse functions. Over the years, experimental and theoretical studies have provided information about structure of RNA and DNA molecules at different hierarchical level and resolution. An abundance of structural data has brought an increasing attention to nucleic acid dynamic behavior, which is yet poorly understood. The limiting factor is a complexity of nucleic acids dynamic processes, spanning timescales ranging over 12-15 orders of magnitude, and involving motions from localized bond vibrations to collective fluctuations of the entire domains (Figure 1.1). Local and global motions form a basis for RNA and DNA conformational transitions, taking place during macromolecule folding, binding, and catalysis. Thus, development of experimental methodologies capable to study nucleic acid structural dynamics is an 2 important step toward understanding these and other cellular functions. Over the past two decades a number of biophysical and biochemical methods have emerged, providing an unprecedented view on complexity and variety of molecular motions (1-18). Table 1.1 outlines experimental techniques adapted to study structural dynamics of RNA and DNA with references to the most recent review articles. Figure 1.1: Timescale of dynamic processes for nucleic acids. While a number of methodologies is available, each of them has defined capabilities and limitations. For example, NMR-based approaches enable studying molecular motions over a broad range of timescales in a residue independent, non-invasive manner with an atomic-level resolution. However, resonance assignment remains a big challenge, limiting their application to relatively small systems. Compared to NMR methods, which are especially useful for monitoring local fluctuations, recent fluorescence based FRET experiments have been effective in capturing inter-domain motions in systems as big as a ribosome (19). Unfortunately, structural dynamics faster than milliseconds is beyond the current technical capabilities. In addition, methods like hydroxyl radical footprinting and SAXS are limited to study large conformational changes, for example, accompanying RNA folding transitions. Overall, describing local and global motions in RNA and DNA 3 molecules over 12-15 orders of a second, and identifying their role in biological functions is a significant challenge. To overcome current limitations, to increase temporal and structural frames of motional sensitivity - development of new physical and chemical approaches, and instrumentation is needed. Table 1.1: Experimental methods applied to study structural dynamics in nucleic acids. 4 Table 1.1 (cont.): Experimental methods applied to study structural dynamics in nucleic acids. This dissertation is primarily focused on the development of a biophysical method, called site-directed spin labeling (SDSL) to monitor local dynamics of DNA molecules in the nanosecond regime. SDSL takes advantage of a chemically stable nitroxide radical 5 covalently attached to the specific site of a macromolecule, whose signal is monitored using Electron Paramagnetic Resonance (EPR) spectroscopy. Continuous wave (cw) EPR spectra are sensitive to reorientational motions of the nitroxide, modulated by structural and dynamic properties of a parent macromolecule at the labeling site. Work reported in this dissertation explores a group of phosphorothioate reactive nitroxides (R5-series) and their applicability in probing local DNA structure and dynamics using cw-EPR measurements. In the following text, the physical basis for cw-EPR spectra and their motional sensitivity are first described, followed by a brief overview of the literature and the accomplished work. 2. Site-directed spin labeling technique for studies of macromolecule dynamics 2.1. Nitroxide spin labeling Most of the biological molecules lack a naturally occurring paramagnetic center and application of EPR spectroscopy methods to these systems relies on a spin label functionalization. Nitroxide radical derivatives (Figure 1.2A) containing an unpaired electron in the p  orbital of the N-O bond have been used as EPR reporters. Compared to other types of free radicals, nitroxides are kinetically and thermodynamically stable molecules (20), giving a persistent EPR signal under physiological conditions. Different chemical methods for covalent attachment of a nitroxide label have been explored (21- 23), increasing capabilities of SDSL in structural, dynamic and functional studies of biological molecules. 6 Figure 1.2: Nitroxide radicals and a nitroxide EPR spectrum. (A) Chemical structure of nitroxide probes commonly used in macromolecule studies. (B) A diagram of allowed transitions between energy levels corresponding to different spin states of the unpaired electron (m s = 1/2) and 14 N (m I = 1; 0) at the resonance. The resulting first-derivative spectrum of a nitroxide is shown below. Picture was adopted from (24). 2.2. Basic principles of EPR EPR signal of an unpaired electron (m s = 1/2) originates from transition between two energy states, corresponding to orientations of the electron magnetic moment parallel and anti-parallel to the external magnetic field (Figure 1.2B). A magnetic field oscillating at the microwave frequency “flips” an electron from the low to high energy level, when the resonance conditions are satisfied: (1) 7 where g denotes a spectroscopic g-factor,  is a Bohr magneton, and H is an applied magnetic field. g  H term defines the Zeeman interactions of the electron spin with a static magnetic field. In the nitroxide, an unpaired electron is localized on p  orbital of the nitrogen 14 N with a nuclear spin I=1. Interactions of an electron spin with the magnetic field produced by 14 N results in splitting of an electron signal into 2(I)+1=3 spectral lines of equal intensity (Figure 1.2B). To account for an electron-nuclear coupling or so-called hyperfine interactions, equation 1 should be modified: (2) where m I represents a nuclear spin number and A is a hyperfine splitting factor. Although EPR measures the net absorption energy at the resonance, an EPR signal is recorded as a first derivative of the absorption signal (Figure 1.2B). Due to limited capabilities of the available microwave frequency sources, EPR signal is obtained by sweeping the magnetic field using a set frequency value (Figure 1.2B). Accordingly, g- factor in equation 2 defines the center-field position of a nitroxide spectrum and A-factor determines amount of peak splitting. Magnetic moment of an electron is about 2000 larger than magnetic moment of a proton, giving rise to one of the major advantages of SDSL technique – its high sensitivity. Compared to NMR, EPR measurements require small amount of material on the order of 0.1-1 nmole and are not limited by the size of a macromolecule, as usually no intrinsic EPR signal is present. 8 A unique feature of an EPR spectrum is its orientational sensitivity. Figure 1.3 demonstrates that central position and slitting of the spectral lines (specified by g- and A- factors) depend on orientation of the nitroxide molecule in the magnetic field. Asymmetry of the nitroxide p  orbital, where the unpaired electron is localized, results in orientational dependence (i.e., anisotropy) of the Zeeman and hyperfine interactions, which are best described by the g and A tensors: and (3) Typical values of (xx,yy,zz) components for a nitroxide label in aqueous environment are (2.0085, 2.0065, 2.0027) for g-tensor and (7,7,35) for A-tensor (25). In the absence of molecular motions, effective g and A values in equation 2 will depend on orientation of the nitroxide molecular frame (Figure 1.3) with respect to the magnetic field, specified by ( , ) polar angles:        2 2 2 2 2 cos sin sin cos sin ) , ( zz yy xx g g g g    (4)        2 2 2 2 2 2 2 2 2 cos sin sin cos sin ) , ( zz yy xx A A A A    (5) Anisotropy of magnetic interactions has been used to study molecular ordering in lipid membranes (26). Figure 1.4A is an example of how angular disorder of a nitroxide modulates the resulting EPR spectrum, causing broadening of the resonance lines. 9 2.3. Effect of molecular motions Sensitivity of an EPR spectrum (i.e., EPR lineshape) to rotational motions is an important characteristic most frequently explored in SDSL of macromolecules. In solution, nitroxide coupling with the lattice is weak and spin-spin relaxation pathways characterized by the spin- spin relaxation time (T 2  15-30 ns) have a dominant effect on conventional cw-EPR measurements (25). Molecular motions with rotational correlation time (  r ) in the ps-ns range modulate T 2 relaxation and have an observable effect on a cw-EPR spectrum. Motional sensitivity arises from magnetic anisotropy of the Zeeman and hyperfine interactions. According to the magnetic anisotropy principle, two spins (or conformations) A and B with different orientations in the external magnetic field, have two distinct resonance signals characterized by different g values 1 . A slow exchange between rotational states A and B results in broadening of the signals due to dynamic fluctuations of the local magnetic fields. As the rate of exchange 1 Hyperfine interactions are not considered for simplicity. Figure 1.3: Orientation sensitivity of an EPR spectrum. Each spectrum shown was simulated with one of the nitroxide principle axis (x, y, z) oriented parallel to the direction of the magnetic field (H). Picture was adopted from (24). 10 increases, leading to averaging of the magnetic anisotropy, EPR signals of A and B will coalesce into a single line. The line width of the resulting spectrum is a product of A and B relaxation times, the rate of rotational exchange and separation of the resonance positions. Reorientational motions of a nitroxide with respect to the magnetic field, modulate an EPR spectrum in a similar manner: large  r leads to broadening of the spectral lines, decrease in their amplitude, as well as appearance of the characteristic spectral features (1.4B). Figure 1.4: Effects of molecular disorder and rotational motions on a nitroxide spectrum. (A) The spectra shown were obtained by simulations using different amount of angular disorder in the rigid-limit motional regime. (B) EPR spectra were simulated varying rotational correlation time (  r ) of a nitroxide moving isotropically in solution. 11 In addition to dynamics in the nanosecond timescale, information about slower motions of the nitroxide, with  r on the order of the spin-lattice relaxation time T 1  1-15 s, can be obtained from saturation transfer or pulse EPR measurements, sensitive to T 1 relaxation pathways (25). 2.4. Analysis of nitroxide spectra in the slow-motional regime Motions of a nitroxide, covalently attached to a macromolecule, such as DNA or RNA, are modulated by structural and dynamic features of the parent macromolecule at the labeling site (here referred as local environment). Therefore analysis of a nitroxide spectrum for rotational dynamics is often used to obtain information about local macromolecule environment. At the conventional X-band frequency (~9.34 GHz), nitroxide rotational motions in the 1-30 ns range (traditionally referred as “slow” motions) lead to partial averaging of the elements of A- and g-tensors. Thus, the most significant spectral changes are observed in this motional time window (Figure 1.4B). Obtaining detailed quantitative description of nitroxide dynamics from a measured spectrum is not a trivial task. Partial averaging of magnetic anisotropy by the “slow” motions produce irregular lineshapes, rendering semi-empirical lineshape analysis, relied on measurements of the amplitude and width of spectral lines, particularly complex. In cases, when relatively large changes in a nitroxide spectrum are observed, parameters such as central line width ( H pp ) and outer splitting (2A zz ) (Figure 1.4B) can be useful in assessment of relative nitroxide mobility (i.e., a combined effect of the rate and amplitude of motions). However, to 12 provide quantitative description of nitroxide dynamics and to overcome limitations of the semi-empirical spectral analysis, approaches based on computer simulations of an EPR spectrum have been developed and employed. The most popular spectral simulation approach, developed by Freed and coworkers (27), relies on a Stochastic Liouville equation (SLE) to find a solution for a time- dependent Hamiltonian. SLE treats magnetic interactions quantum mechanically, while rotational motions are described using classical diffusion models. A family of computer programs has been developed that solve SLE and calculate an EPR spectrum at each step of an iterative spectral fitting. Depending on a nitroxide motional model (e.g., involvement of the local and global modes) a number of input parameters are usually selected and subsequently varied to minimize differences between an experimental and simulated spectra. Microscopic Order Macroscopic Disorder (MOMD) is one of the currently available models (28), and it has been used in analysis of the nitroxide spectra reported in this dissertation (Chapters 4 and 5). In MOMD, rotational diffusion of a macromolecule is slow on an EPR timescale and macromolecules are thought to be randomly oriented in the applied magnetic field (i.e. disordered). At the same time, rotational diffusion of a nitroxide is locally constrained (i.e., ordered) by the macromolecule. MOMD spectral simulations provide information about both the rate and amplitude of the nitroxide local motions (Figure 1.5). Motional amplitude is an available for a nitroxide angular space defined by the strength and asymmetry of an ordering potential. In spite of availability and growing usage of EPR fitting programs, uniqueness of a spectral fit and consequently 13 reliability of the obtained motional parameters is a frequently discussed issue. To address this problem, one has to involve adequate spectral, geometrical or motional constrains obtained from experiments or molecular modeling. Figure 1.5: Schematic of a motional model used in MOMD spectral simulations. Brownian diffusion rate constants (R ‖ , R  ) and coefficient of an ordering potential (c 20 ) are the spectral fitting parameters. Global tumbling is considered slow (i.e., in a rigid-limit regime) and not included in simulations. Picture was adopted from (29). Analysis of EPR spectra for motional dynamics of a nitroxide can be extended to data obtained using multiple EPR frequencies. Compared to cw-EPR spectra acquired at the X-band (9 GHz), high-frequency measurements (140, 250 GHz) are sensitive to faster motions, reaching a picosecond time window. Multi-frequency studies enable expanding a time-frame of EPR motional sensitivity, and provide additional spectral constrains facilitating analysis of complex dynamics of the nitroxide labeled biomolecules (30). 14 While multi-frequency spectral analysis is beyond the scope of this dissertation, it is an important direction for future work. 3. Probing nucleic acid dynamics using cw-EPR 3.1. Overview of the previous studies Analysis of a cw-EPR spectrum (see section 2.4) provides information on overall rotational motions of a nitroxide in a nanosecond regime. These motions include: 1) global macromolecule tumbling; 2) torsional oscillations around bonds, connecting the nitroxide moiety to the macromolecule (i.e., internal motions); and 3) macromolecule fluctuations at the labeling site (i.e., macromolecule motions). Sensitivity of a nitroxide spectrum to all these motional modes has been previously explored. For example, molecular interactions have been monitored using a decrease in the rate of global tumbling and thus in observed nitroxide mobility (31). Besides that, nitroxide internal motions, modulated by the macromolecule structure at the labeling site, are effectively reporting on secondary, tertiary structure and conformational changes of a parent molecule (24, 32, 33). However, determining contribution of macromolecule motions to the overall nitroxide mobility is a complex multifaceted task. In SDSL of nucleic acids, a group of cw-EPR studies have been primarily devoted to probing dynamic behavior in RNA and DNA molecules. Below, some of the major works are briefly described. Robinson and co-workers first introduced a nucleotide derivative, containing a nitroxide moiety rigidly fused to the base (Q, Figure 1.6A). Rigid coupling of the probe to a DNA molecule enabled to eliminate contribution of internal motions to an EPR 15 spectrum. The authors used Q to monitor collective bending and twisting modes in DNA molecules of varying length and sequence (34-36). Analysis of cw-EPR spectra was based on a dynamic model accounting for nitroxide motions in the fast-motional limit (  r  10 -11 -10 -9 s), tracing fast collective deformations (i.e., bending and twisting modes) and local base fluctuations. The results were fit to a modified weakly bending rod theory, relating amplitude of the bending and twisting deformations, obtained from spectral simulations, to the corresponding sequence-dependent force constants. Figure 1.6: Nitroxide labeling schemes used for probing rotational dynamics in DNA and RNA molecules. (A) Rigidly coupled nitroxide probes Q (34) and Ҫ(37); (B) Nitroxides with flexible attachment to the base: DUTA, DUAT (38), 4-thioU-Ra (39); sugar: 2-amino-TEMPO (40); and phosphate: R5/R5a (31, 41). 16 Although rigid attachment is a highly preferable characteristic, some data suggest that nitroxides, rigidly coupled to a DNA base, may alter its dynamic behavior (37). While probes like Q and Ҫ (Figure 1.6A) are better suited for probing collective and global motions, nitroxides connected to the base, sugar and phosphate moieties via several bonds of varying rigidity were introduced to assess preferentially local motions in nucleic acid molecules. Bobst and co-workers investigated DNA molecules by attaching different nitroxides to a pyrimidine base via linkers comprised of 2-12 chemical bonds (Figure 1.6B, DUTA and DUAT) (38). Obtained cw-EPR spectra were simulated using sets of two rotational correlation times  ‖ and   , for rotations of a nitroxide parallel and perpendicular to the principal diffusion axis.   was found to be independent from the tether length thus attributed to rotational dynamics of the labeled pyrimidine (29). Lastly, several reports, suggestive of varying local mobility in RNA molecules have to be mentioned. Sigurdsson (40) and Qin (39) used 2’-sugar and thiouracil modifying nitroxides (Figure 1.6B, 2’- amino-TEMPO and 4-thioU-Ra) respectively, to monitor mobility of the different uracil nucleotides at the bulge/loop and helical sites of two RNA molecules. In all the studies described, effect of macromolecule motions on an EPR spectrum is analyzed using either qualitative correlations between observed nitroxide mobility and features of the macromolecule environment or model-specific spectral simulations. These reports demonstrate the major challenges for cw-EPR, as a tool to study macromolecule motions. First is an analysis of EPR spectra for the nitroxide rotational dynamics (section 2.4). While quantitative analysis can be achieved, it always relies on a specific model, 17 chosen to describe macromolecule and nitroxide motions. How effectively the model reflects molecular behavior, often remains unclear. Second, for the probes with a flexible attachment, contributions of the internal and macromolecule motions have to be deconvoluted. Finally, rotational fluctuations of a macromolecule, reported by cw-EPR, should be ascribed to specific local and collective dynamical modes and quantified. Although no common rules exist to detangle and characterize contributions of nitroxide and macromolecule motions to an observed spectrum, a number of experimental and theoretical strategies can be helpful in data analysis. First, as demonstrated in protein and nucleic acid studies, sensitivity of a cw-EPR spectrum to particular mode/s of motions can be modulated using chemical modifications of a nitroxide and a macromolecule. Second, factors differentially affecting molecular motions, such as temperature, pressure, solvent viscosity, and macromolecule size can be explored. cw- EPR data obtained at multiple frequencies provide additional constrains to a particular motional model used in spectral simulation analysis. Lastly, presence of high resolution structural data, enabling molecular modeling studies of different complexity is often advantageous. Many of these strategies have been explored in SDSL studies reported in this dissertation. 3.2. Probing structural and dynamic features in DNA with nucleotide-independent nitroxides Qin et al. has previously developed a phosphorothioate labeling scheme for attaching a nitroxide (designated as R5) to a specific modified phosphate position of the nucleic 18 acid backbone (Figure 1.6B) (31, 41). R5 is called a nucleotide-independent nitroxide, as it can be covalently linked to any nucleotide within an arbitrary DNA or RNA molecule. The phosphorothioate scheme potentially enables simple and efficient scanning of a given nucleic acid sequence with a nitroxide, by systematically attaching the probe and interrogating local environment at every single nucleotide. The major focus of this dissertation was to explore applicability of the phosphorothioate labeling scheme to monitor sequence-dependent motions in DNA by cw-EPR. Using a model B-form DNA duplex and other biologically relevant sequences, it was demonstrated that probes in the R5-series are effectively reporting on variations in DNA local environment in a site- and stereo-specific manner. In addition, basic principles for correlating observed variation in cw-EPR spectra with structural and dynamic features in DNA have been investigated in many details. Below, the key results of the accomplished work are briefly described with references to the respective dissertation chapters. First, R5-series of phosphorothioate reactive probes with different chemical structure of the nitroxide moiety or/and the length and rigidity of a DNA/nitroxide linker have been investigated (Chapter 2). As a result of this work, several probes were selected as potential reporters of local and global motions in DNA. R5a (Figure 1.6B) is one of the R5-derivatives, which has little effect on DNA thermodynamic stability and global conformation, and was used in subsequent cw-EPR studies. R5 and R5a are linked to the nucleic acid backbone via three single bonds (Figure 1.6B), rotations around which may reduce sensitivity of a probe to DNA motions. In R5a, 19 a bromine substituent at the nitroxide moiety reduces rotational flexibility to a substantial degree, but does not eliminate it completely. Thus, sensitivity of R5 and R5a to DNA local environment has been investigated by attaching the probes to multiple sites of a model B-form DNA duplex (Chapter 3). The resulting EPR spectra were shown to vary in a position and sequence dependent manner, demonstrating coupling of the nitroxides to site-specific structural and dynamic features in DNA. The phosphorothioate (ps) group, introduced at a target site during the solid-phase oligonucleotide synthesis, adopts one of two diastereomeric configurations (R p and S p ), however the effect of ps stereochemistry on nitroxide behavior was unclear. To address this issue, R p and S p have been separated, and the resulting spectra were shown to vary according to diastereomer identity and location of the labeling site in a model DNA duplex (Chapter 4). Using molecular modeling and spectral simulations, it was suggested that while R p -R5a nitroxide effectively reports on structural features at the major groove, S p -R5a is primarily sensitive to DNA backbone motions. In addition, the data obtained with mixed-diastereomers are an average sum of individual R p -R5a and S p -R5a spectra. These findings importantly define advantages and limitations of a SDSL methodology using R5-series of nitroxides. A better mechanistic understanding of dynamic coupling between a nitroxide probe and a macromolecule improves our capability to extract information from cw-EPR data. Toward this goal, one may explore site-specific macromolecule mutations, modulating structural and dynamic environment at the labeling site, and monitor their effect on a nitroxide spectrum. As a first step, a stereo-specific effect of single thymine to uracil base 20 substitution on rotational dynamics of R5 and R5a nitroxides was investigated (Chapter 5). This study demonstrates an increased sensitivity of R p -R5a nitroxide to subtle changes in DNA local structure and provides additional information about R5a/DNA sequence- specific interactions. In Chapter 6, R5a nitroxide scanning was coupled with cw-EPR measurements to probe local structural and dynamic properties in DNA sequences involved in protein- DNA recognition and human cancer (Chapter 6). Lastly, Chapter 7 outlines directions for the future work. 21 4. 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(2001) Electron Paramagnetic Resonance (EPR) and Spin-Labeling, In Encyclopedia of Life Sciences, pp 1-5, John Wiley & Sons, London. 23 25. Fajer, P. G. (2000) Electron spin resonance spectroscopy labeling in peptide and protein analysis, In Encyclopedia of Analytical Chemistry (Meyers, R. A., Ed.), pp 5725-5761, John Wiley & Sons, Chichester. 26. Smirnova, T. I., and Smirnov, A. I. (2007) High-field ESR spectroscopy in membrane and protein biophysics, In Biological Magnetic Resonance (Hemminga, M. A., and Berliner, L. J., Eds.), pp 165-251, Springer, New York. 27. Freed, J. H. (1976) Theory of Slow Tumbling ESR Spectra for Nitroxides, In Spin Labeling Theory and Applications (Berliner, L. J., Ed.), pp 52-132, Academic Press, New York. 28. Meirovitch, E., Nayeem, A., and Freed, J. (1984) Analysis of protein lipid interactions based on model simulations of Electron-Spin Resonance spectra, J Phys Chem 88 , 3454-3465. 29. Liang, Z., Freed, J., Keyes, R., and Bobst, A. (2000) An electron spin resonance study of DNA dynamics using the slowly relaxing local structure model, J Phys Chem B 104, 5372-5381. 30. Earle, K. A., and Smirnov, A. I. (2004) High Field ESR: Applications to protein structure and dynamics, In Biological Magnetic Resonance (Berliner, L. J., and Grinberg, O. G., Eds.), pp 469-517, Plenum Press, New York. 31. Qin, P. Z., Butcher, S. E., Feigon, J., and Hubbell, W. L. (2001) Quantitative analysis of the isolated GAAA tetraloop/receptor interaction in solution: a site- directed spin labeling study, Biochemistry 40, 6929-6936. 32. Hubbell, W. L., Cafiso, D. S., and Altenbach, C. (2000) Identifying conformational changes with site-directed spin labeling, Nat Struct Biol 7, 735- 739. 33. Budamagunta, M., Hess, J., Fitzgerald, P., and Voss, J. (2007) Describing the structure and assembly of protein filaments by EPR spectroscopy of spin-labeled side chains, Cell Biochem Biophys 48, 45-53. 34. Okonogi, T. M., Reese, A. W., Alley, S. C., Hopkins, P. B., and Robinson, B. H. (1999) Flexibility of duplex DNA on the submicrosecond timescale, Biophys J 77, 3256-3276. 35. Okonogi, T. M., Alley, S. C., Reese, A. W., Hopkins, P. B., and Robinson, B. H. (2000) Sequence-dependent dynamics in duplex DNA, Biophys J 78, 2560-2571. 24 36. Okonogi, T. M., Alley, S. C., Harwood, E. A., Hopkins, P. B., and Robinson, B. H. (2002) Phosphate backbone neutralization increases duplex DNA flexibility: a model for protein binding, Proc Natl Acad Sci 99, 4156-4160. 37. Cekan, P., Smith, A. L., Barhate, N., Robinson, B. H., and Sigurdsson, S. T. (2008) Rigid spin-labeled nucleoside C: a nonperturbing EPR probe of nucleic acid conformation, Nucleic Acids Res 36, 5946-5954. 38. Bobst, A. M., and Keyes, R. S. (1998) Spin-labeled nucleic acids, In Biological Magnetic Resonance (Berliner, L. J., Ed.), pp 238-338, Plenum Press, New York. 39. Qin, P. Z., Hideg, K., Feigon, J., and Hubbell, W. L. (2003) Monitoring RNA base structure and dynamics using site-directed spin labeling, Biochemistry 42, 6772-6783. 40. Edwards, T. E., Okonogi, T. M., Robinson, B. H., and Sigurdsson, S. T. (2001) Site-specific incorporation of nitroxide spin-labels into internal sites of the TAR RNA; structure-dependent dynamics of RNA by EPR spectroscopy, J Am Chem Soc 123, 1527-1528. 41. Qin, P. Z., Haworth, I. S., Cai, Q., Kusnetzow, A. K., Grant, G. P., Price, E. A., Sowa, G. Z., Popova, A., Herreros, B., and He, H. (2007) Measuring nanometer distances in nucleic acids using a sequence-independent nitroxide probe, Nat Protoc 2, 2354-2365. 25 Chapter 2 Development of nucleotide-independent nitroxide probes for nucleic acids studies 1. Chapter 2 Introduction 1.1. Nitroxide probes and attachment rigidity Site-Directed Spin Labeling (SDSL) methodology is based on chemical modification of a molecule of interest with a spin probe. Chemically stable nitroxide derivatives, containing an unpaired electron at the p  orbital of the N-O bond, have been synthesized and subsequently used for functionalization of proteins, nucleic acids and smaller molecules (i.e., lipids, steroids, fatty acids) (1-3). A great number of spin-labeling strategies developed over the years stems from a variety of systems under investigation, their chemical composition and biological functions. One important property of a nitroxide probe is its attachment rigidity. Rotational motions of a nitroxide probe independent of the target macromolecule will average its magnetic anisotropy (see Chapter 1), thus lowering orientational and motional sensitivity of EPR measurements. However, one can take advantage of a “natively” flexible probe by monitoring changes in nitroxide mobility upon confinement of its local environment, resulted from binding, conformational changes, macromolecule folding etc. A large amount of available to date SDSL studies suggests that a single labeling scheme may not 26 be used to address all problems, and that nitroxide rigidity should be “tuned” accordingly. In the following paragraphs some applications of “rigid” and “flexible” nitroxide probes developed for protein and nucleic acid studies are briefly described. For example, nucleosides containing a nitroxide moiety rigidly fused to the base (Q and Ç, see Figure 1.6 of Chapter 1) were particularly useful in probing collective and global motions in DNA and RNA systems ((4, 5), Qin and Nguyen unpublished). Superior for probing motions of large subunits and domains, Q and Ç, are likely to have little sensitivity to changes in the local environment. In addition, preparation of Q and Ç labeled oligonucleotides relies on complicated synthetic schemes and is currently limited to DNA. Due to less stringent modification chemistry, non-rigid nitroxide probes (see Figure 1.6 of Chapter 1) were directly used in RNA and DNA studies to monitor site- specific structural and dynamic properties (here referred as local environment) (2). Similarly, in SDSL of proteins, the most commonly utilized cysteine reactive MTSL probe (6) is sensitive to protein secondary and tertiary structure near the labeled site. However, flexible attachment of the probe makes the data only indirectly related to peptide backbone dynamics (7). To overcome this limitation a non-natural amino acid (TOAC) containing a six-member nitroxide ring rigidly coupled to the α-carbon was introduced (8). A significant drawback of this method is its dependence on the solid- phase peptide synthesis or the unnatural amino acid mutagenesis. 27 1.2. R5-series of nitroxides In this study a series of R5-nitroxides and their applicability to monitor structural and dynamic properties in DNA is explored. R5-series takes its origin from an R5 label (Figure 2.1A). R5 utilizes a single phosphorothioate (ps) modification introduced at a target site of the DNA or RNA backbone. R5 has several unique features compared to other probes used in SDSL of nucleic acids (2). First, R5 is a nucleotide-independent probe, as it can be attached to any nucleotide within an arbitrary polyoligonucleotide sequence. Second, R5 uses a simple chemistry, resulting in 90-100% yield of the labeled oligonucleotide over a 12 hour period (Figure 2.1A) (9). Third, R5 is the only known probe for functionalization of the nucleic acid phosphodiester backbone (2). Lastly, several lines of evidence suggest that R5 minimally affects native structure of DNA and RNA molecules (10-12). These features overall account for the use of R5 nitroxide in many nucleic acid studies (10, 11, 13, 14). Figure 2.1: R5 nitroxide and CS DNA. (A) R5 labeling scheme. Rotations around t1, t2 and t3 bonds define internal motions of the nitroxide. (B) Sequence and secondary structure of CS DNA. 28 However, relatively high flexibility of the R5 linker becomes an important issue for some SDSL applications. R5 is attached to DNA/RNA via three single bonds (Figure 2.1A), rotations about which in the nanosecond regime reduce sensitivity of cw-EPR observables to the macromolecule motions. The major goal of this work was to isolate minimally perturbing R5 derivative/s for which advantages of the ps labeling scheme would be coupled with reduced mobility of the linker. In this study, a series of R5 derivatives, utilizing chemical modification of the pyrroline ring or/and DNA-nitroxide linker (Table 2.1), was investigated. All the nitroxides were attached to the same interior and terminal sites of a B-form DNA duplex (CS, Figure 2.1B), and their mobility was monitored using X-band cw-EPR at several temperatures. Spectral analysis indicates that in most cases, chemical modification strategies used were effective in reducing intrinsic flexibility of the original R5. Among R5-derivatives studied, R5a appeared to be the best nitroxide for probing local motions in DNA. Functional properties of other nitroxides indicate their potential usefulness in other applications, such as monitoring global/segmental dynamics and local conformational changes in biologically active DNA and RNA systems. 2. Chapter 2 Materials and Methods 2.1. Materials Spin-labeling reagents used in this work were supplied by Kálmán Hideg (University of Pécs, Hungary). CS DNA oligonucleotides (Figure 2.1B), including those containing site-specific ps modification/s were obtained by the solid-phase chemical synthesis 29 (Integrated DNA Technology, Coralville, IA). In this work, spin-labeled DNA or its spectrum are designated by the nucleotide number at which the nitroxide is introduced. 2.2. Nitroxide spin-labeling For all the nitroxide reagents (see Table 2.1) except HO1819 and HO2303, a labeling reaction was carried out following R5a labeling procedure described in the previous work (14). After incubation for 24 hours, excess of the nitroxide precursor and unreacted DNA were removed with anion-exchange HPLC (9). Purified and desalted samples have been lyophilized and stored at -20C. HO1819 and HO2303 were incubated with ps-modified oligonucleotides at varying conditions, such as temperature, solvent and concentration of a catalyst (pyridine or triethylamine) (Table 2.1). 2.3. Characterization of the nitroxide-labeled DNA For mass spectrometry analysis, aqueous DNA (10-20 M) was mixed with a matrix in 1:2 ratio (v/v). The matrix solution contained 35 mg/ml of 3-hydroxypicolinic acid, 7 mg/ml of di-ammonium hydrogen citrate and 15/85% acetonitrile/water. Purified 12-mer ([M+H]+ ion, 3612.416 Da) and 14-mer ([M+H]+ ion, 4279.915 Da) deoxyoligonucleotides were added to the sample as internal standards. After continuous mixing, samples were manually deposited in 0.8-1 L size drops onto a stainless steel sample plate (Applied Biosystems, Foster City, CA) and air-dried. MALDI-TOF measurements were carried using Voyager-DE STR system (Applied Biosystems). Linear 30 mode acquisition was used to monitor positive ions in the mass range of 1500–7000 Da. Mass calibration was performed with a software provided by the vendor, using average mass values of the standards. Thermal-denaturation of CS67_HO1942 DNA duplex was carried out following a published procedure (14). 2.4. cw-EPR experiments Details of EPR measurements and DNA sample preparation were previously described (14). Briefly, nitroxide labeled oligonucleotides (~0.3 nmol) were incubated with a 10% excess of the complimentary DNA strand in 100 mM NaCl, 50 mM Tris-HCl (pH=7.5) overnight at room temperature. Unless indicated, sucrose was added to a final amount of 34% (w/w) to reduce DNA global tumbling. cw-EPR spectra were obtained on a Bruker EMX spectrometer (Bruker BioSpin, Inc., Billerica, MA) at room temperature or using a liquid nitrogen temperature controller to maintain the temperature at 5, 15 or 25C. All EPR spectra were baseline corrected and normalized to the same number of spins. 31 Table 2.1: Results summary for the R5-series. 32 Table 2.1 (cont.): Results summary for the R5-series. 33 3. Chapter 2 Results Table 2.1 provides a summary of this study. Every nitroxide labeling reagent listed in Table 2.1 was incubated with a ps-modified CS oligonucleotide (Figure 2.1B) (15) and formation of labeled DNA was monitored using anion-exchange HPLC. After chromatography purification (see Methods), mass spectrometry was used to confirm formation of the expected product. All the data reported here were obtained without separation of ps-diastereomers. The following text is structured to describe and compare results obtained for each probe in the R5-series. For HO368, being a reactive precursor for R5, and HO1820, a precursor for R5a, both names are used interchangeably. Other spin labels are named according to their precursors (Table 2.1). 3.1. Nitroxide probes utilizing pyrroline substitution To reduce independent nitroxide flexibility, HO1820 and HO3927 probes utilize a chemically substituted 4-position of the pyrroline ring. This strategy has been originally implemented to derivatize MTSL nitroxide used in protein studies (16). Figures 2.2 and 2.3 provide comparisons between HO3927, HO1820 (R5a precursor) and consequently R5 (HO368 is a R5 precursor) spectra, obtained at room temperature. Observed broadening and amplitude reduction of HO1820 and HO3927 spectral lines indicate that Br- and CH 3 - substituents to a significant extend lower flexibility of the original R5 probe. This result well agrees with molecular modeling data, indicating that 4-Br and 4- CH 3 sterically hinder rotations around the t2 and t3 bonds (Figure 2.1A). 34 Figure 2.2: EPR spectra obtained with HO3927 and HO1820 attached to position CS7 of the DNA duplex. Measurements were done at room temperature. Due to complications related to purity of the original HO3927 reagent (Table 2.1), information on HO3927 is currently limited to one spectrum (i.e. CS7_ HO3927 at room temperature, Figure 2.2). This precludes thorough comparison between HO3927 and HO1820. However, considering that differences between the Van der Waal’s radii of Br (~185 pm) and CH 3 (~200 pm) groups are small, they should provide similar sterical barriers for rotational oscillations around the t3 and t2 bonds. Indeed, within accuracy of our data, HO1820 and HO3927 spectra at position CS7 are identical (Figure 2.2). 35 Figure 2.3: Comparison between HO3317, HO368 and HO1820 spectra measured at room temperature. HO3317 features intermediate nitroxide mobility between that of HO368 and HO1820. 3.2. Nitroxide probes with a pi-bond containing linker Varying the length and rigidity of the nitroxide linker is an alternative approach in controlling independent fluctuation of the probe. For example, Bobst group introduced a series of nitroxide modified pyrimidines, where a nitroxide moiety was connected to the base by tethers comprised of 2 to 13 chemical bonds of varying rigidity (17). In this work, linkers containing a double (HO1642), a triple (HO3317) bonds and a benzene ring (HO3178) have been utilized (Table 2.1). Here, an increase in the length of the linker is 36 compensated by larger rotational rigidity of a pi-bond containing segment. For HO3178 and HO1642, EPR spectra were obtained at two sites of the CS duplex and several temperatures (Figures 2.4 and 2.5). Presence of unseparated products in HO3317 spin- labeling reagent or/and alternative labeling pathways resulted in two nitroxide labeled species of different mass (Table 2.1, entry 6). Accordingly, HO3317 spectra were acquired at room temperature only (Figures 2.3). Figure 2.4: EPR spectra obtained with HO3178 attached to positions CS2 and CS7 of the DNA duplex. 37 Figure 2.5: CS2 and CS7 HO1642 spectra, acquired at different temperatures. 38 Figure 2.6: Comparison of the 5 C CS7 spectra obtained using different nitroxides. From top to bottom spectra are placed in the order of decreasing nitroxide mobility. 39 At two DNA site, HO1642, HO3178 and HO3317 nitroxides report higher nitroxide flexibility than R5a (i.e., HO1820) (Figures 2.3 and 2.6). Compared to R5 (i.e., HO368), HO3178 shows similar amplitude and width of spectral lines, HO3317 has an intermediate line broadening and HO1642 reports a significant amount of nitroxide immobilization, overall indicating equal or lower nitroxide mobility (Figures 2.3 and 2.6). These results may suggest that in HO3317 and HO3178, uniaxial rotations around the segments containing a triple bond or a benzene ring (shown in red in Figures 2.3 and 2.4), are fast and relatively unrestricted. In fact, we expected HO3317 linker to be more flexible than a bulkier HO3178, yet the data show the opposite behavior. Most likely, problems associated with HO3317 labeling (see above) may account for this discrepancy. 3.3. A benzo-fused nitroxide probe HO3992 is a probe containing a benzo-fused pyrroline ring of R5 nitroxide. When compared at 5 C, HO3992 features the broadest spectra among all the previously described probes, including HO1820 and HO3927 (Figures 2.7 and 2.6). Although, similar to R5 and R5a, HO3992 linker consists of three single bonds, rotations around them are significantly restricted. Temperature dependence of HO3992 spectra (Figure 2.7) suggests that nitroxide immobilization was likely achieved by non-specific interactions with DNA. Namely, higher temperature data reveal presence of at least two, mobile and immobile (i.e., DNA interacting) nitroxide populations, not distinguishable in 5C spectra. Notably, that degree of nitroxide immobilization is clearly site-specific (Figure 7). 40 Figure 2.7: HO3992 spectra, with the label at positions 2, 7 and 12 of the CS DNA. Arrows indicate spectral features, characteristic for multiple component spectra. 41 Overall, dynamic behavior of HO3992 is analogous to the one observed in protein studies, where large aromatic substituents at the nitroxide pyrroline ring induced a pronounced drop in mobility of the MTSL probe (16). The suggested property of nitroxides containing bulky groups to establish local interactions with a macromolecule may be useful in probing macromolecule long range motions and conformational changes. 3.4. Nitroxide probes with a shorter linker HO2303 and HO1819 are the nitroxide probes, which unlike R5-derivatives described so far, are designed for a labeling mechanism distinct from S N alkylation. Here, nucleophilic attack directly on to the 4-sp2 carbon of the pyrroline is expected, with 4-Br serving as a leaving group. This reaction should result in a nitroxide moiety attached to the DNA or RNA phosphorus atom using two instead of three single bonds. We speculate that shortening of a nitroxide linker will help reduce its flexibility. Although moderate reactivity was previously demonstrated using thiol reagents (18), all attempts to attach HO2303 and HO1819 to the DNA backbone were unsuccessful (Table 2.1, entries 9 and 10). This may be explained by weaker nucleophilicity of a phosphorothioate compared to a thiol. 3.5. A bifunctional nitroxide probe R5 probe and simplicity of its phosphorothioate-based labeling chemistry have inspired a class of bifunctional probes, such as HO1942. HO1942 requires two ps- 42 modified sites, which increases nitroxide attachment rigidly to the nucleic acid backbone (Figure 2.8A). This feature makes bifunctional probes very useful in monitoring collective motions and global tumbling in nucleic acid systems. Figure 2.8: Characterization of CS67_HO1942. (A) A schematic of HO1942-labeled DNA construct. (B) Analysis of UV thermal melting curves. 43 Figure 2.9: CS67_HO1942 spectra obtained with DNA in a single- (ss) or double- (ds) stranded form. Measurements were done at room temperature in (A) water or (B) 34% (w/w) sucrose solution. In (A) and (B) the top and bottom rows indicate original spectra and spectra corrected for the residual amount of a free-nitroxide. In this study, HO1942 was successfully linked to the CS strand, containing two phosphorothioates at adjacent positions 6 and 7 (CS67, Figure 2.8A). Labeling yield was at least 80% after 22 hour incubation at room temperature. Apparently, single DNA 44 strand is sufficiently flexible to make labeling reaction proceed as fast and efficient as using an R5a precursor (i.e., HO1820). However, formation of a rigid double helical structure substantially compromises thermodynamic stability of CS67_HO1942 DNA, with an observed drop in melting temperature being ~17  (Figure 2.8B). Apparently, HO1942 nitroxide may not fit well geometrical criteria of the DNA double helix. Data in Table 2.2 suggest that HO1942 linkers are quite short to accommodate the probe in all four possible diastereomeric configurations without perturbing DNA structure. To obtain an undistorted EPR signal, CS67_HO1942 duplex was prepared using a 2 fold excess of the unlabeled complementary strand. 3 and 5 fold excesses produced no observable changes in the CS67_HO1942 spectrum. The resulting data (Figure 2.9) indicate that bifunctional nitroxide is substantially immobilized relative to the DNA duplex. In 34% sucrose, HO1942 shows a spectrum with broadened lines and clearly defined features at the low- and high-field regions, characteristic for a low mobility nitroxide. Such lineshapes were not observed for any of the monofunctional R5-derivatives studied. These results overall suggest that bifunctional probes, analogous to HO1942 may provide a chemically simpler alternative to rigidly coupled probes such as Q and Ç. The next step in this direction is to design probes with longer linkers which will allow system S-S distance, Å HO1942 ~6.5 CS67 R p R p 7.2 CS67 S p S p 8.0 CS67 R p S p 9.5 CS67 S p R p 7.0 Table 2.2: A set of estimated sulfur-sulfur distances in CS67 DNA duplex and HO1942 probe. For HO1942, the nitroxide was modeled and distances approximated using WebLab ViewerPro 3.7. For DNA, NMR structure was used (15) with sites 6 and 7 being ps-modified. 45 nitroxide accommodation into DNA and RNA molecules without losing in attachment rigidity. Developing reaction strategies with high R p /S p stereoselectivity of nitroxide labeling will further advance the use of bifunctional probes and EPR data interpretation. 4. Chapter 2 Discussion Using a B-form DNA molecule, work reported here demonstrates how means of chemical modification can be applied to tune rotational dynamics of the R5 nitroxide. Our results indicate that substituents at position 4 of the pyrolline ring, as large as Br- and CH 3 - help to restrict rotational motions of the nitroxide linker. Also, pi-bond systems, for example those containing a triple bond or a benzene ring are not always effective in reducing nitroxide flexibility. Presence of bulky fragments in a nitroxide structure may favor site-specific interactions with DNA and modulate rotational dynamics of a nitroxide in a temperature-dependent manner. Lastly, we demonstrated that R5 bifunctionalization is effective in achieving tight dynamic coupling to a DNA molecule. Overall, dynamic behavior of any R5-derivative is complex as it depends on chemical structure of the nitroxide ring, the linker, as well as the specifics of the macromolecule environment. Clearly, more detailed studies are needed to better understand each nitroxide probe described here and explain observed site- and temperature-dependent variations in their spectra. This is a possible direction for future studies. Among R5-derivatives studied, R5a emerges as the best nitroxide for probing local motions in DNA. First, R5a labeling reaction is > 90% efficient over a 24 hour period. Second, 4-Br substituent of R5a substantially hinders torsional oscillations around the 46 linking bonds, giving rise to broadened spectra, compared to R5 and other monofunctional probes investigated. Unlike HO3992, R5a has minimal propensity for DNA-nitroxide interaction, with more details provided in Chapters 4 and 5. Further experimental (Chapter 3) and computational studies suggest that R5a has little effect on stability of a DNA duplex and its native conformation. All these properties made R5a an important player in the research of our laboratory. This study provides a necessary foundation for the future development and application of R5-derived nitroxides. Up to date, R5a is one of the best studied probes in the R5-series (14, 19). Motional behavior of R5a and its sensitivity to local macromolecule environment have been monitored in DNA duplexes of varying length and sequence in a site- and stereo-specific manner (Chapters 3-6). Although, other probes in the R5-series were not investigated in such details as R5 and R5a, their functional properties may be useful in future applications. For example, one can monitor presence of spectral components resulted from site-dependent HO3992/DNA contacts to study conformational changes accompanying folding and binding processes. In addition, HO1942 and other bifunctional derivatives open a new way to probe collective and global motions in RNA and DNA molecules avoiding complex synthetic procedures. 5. Chapter 2 Conclusions Chemical modification strategies were effectively used to reduce intrinsic flexibility of R5 probe. Among R5-derivatives investigates, several nitroxides with useful structural and dynamic properties were determined. HO1820 emerged as the best candidate for 47 probing local motions in DNA. HO3992 propensity for DNA contacts may be useful in detecting conformational changes. Rigidly coupled to the DNA backbone, HO1942 provides a new way to monitor collective and global motions. This study may help to expand applicability of R5-nitroxides in structural and dynamic studies of nucleic acids. 48 6. Chapter 2 References 1. Marsh, D. (1981) Electron spin resonance: spin labels, Mol Biol Biochem Biophys 31, 51-142. 2. Sowa, G. Z., and Qin, P. Z. (2008) Site-directed spin labeling studies on nucleic acid structure and dynamics, Prog Nucleic Acid Res Mol Biol 82, 147-197. 3. Hideg, K., and Hankovszky, O.H. (1989) Chemistry of spin-labeled amino acids and peptides. Some new mono- and bifunctionalized nitroxide free radicals, In Biological Magnetic Resonance (Berliner, L. J., and Reuben, J., Ed.), pp 427-488, Plenum Press, New York. 4. Okonogi, T. M., Alley, S. C., Harwood, E. A., Hopkins, P. B., and Robinson, B. H. (2002) Phosphate backbone neutralization increases duplex DNA flexibility: a model for protein binding, Proc Natl Acad Sci 99, 4156-4160. 5. Okonogi, T. M., Reese, A. W., Alley, S. C., Hopkins, P. B., and Robinson, B. H. (1999) Flexibility of duplex DNA on the submicrosecond timescale, Biophys J 77, 3256-3276. 6. Berliner, L. J., Grunwald, J., Hankovszky, H. O., and Hideg, K. (1982) A novel reversible thiol-specific spin label: papain active site labeling and inhibition, Anal Biochem 119, 450-455. 7. Columbus, L., and Hubbell, W. L. (2002) A new spin on protein dynamics, Trends Biochem Sci 27, 288-295. 8. Toniolo, C., Valente, E., Formaggio, F., Crisma, M., Pilloni, G., Corvaja, C., Toffoletti, A., Martinez, G. V., Hanson, M. P., Millhauser, G. L., and et al. (1995) Synthesis and conformational studies of peptides containing TOAC, a spin- labelled C alpha, alpha-disubstituted glycine, J Pept Sci 1, 45-57. 9. Qin, P. Z., Haworth, I. S., Cai, Q., Kusnetzow, A. K., Grant, G. P., Price, E. A., Sowa, G. Z., Popova, A., Herreros, B., and He, H. (2007) Measuring nanometer distances in nucleic acids using a sequence-independent nitroxide probe, Nat Protoc 2, 2354-2365. 10. Cai, Q., Kusnetzow, A. K., Hubbell, W. L., Haworth, I. S., Gacho, G. P., Van Eps, N., Hideg, K., Chambers, E. J., and Qin, P. Z. (2006) Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence- independent nitroxide probe, Nucleic Acids Res 34, 4722-4730. 49 11. Cai, Q., Kusnetzow, A. K., Hideg, K., Price, E. A., Haworth, I. S., and Qin, P. Z. (2007) Nanometer distance measurements in RNA using site-directed spin labeling, Biophys J 93, 2110-2117. 12. Price, E. A., Sutch, B. T., Cai, Q., Qin, P. Z., and Haworth, I. S. (2007) Computation of nitroxide-nitroxide distances in spin-labeled DNA duplexes, Biopolymers 87, 40-50. 13. Grant, G. P., Boyd, N., Herschlag, D., and Qin, P. Z. (2009) Motions of the substrate recognition duplex in a group I intron assessed by site-directed spin labeling, J Am Chem Soc 131, 3136-3137. 14. Popova, A. M., Kalai, T., Hideg, K., and Qin, P. Z. (2009) Site-specific DNA structural and dynamic features revealed by nucleotide-independent nitroxide probes, Biochemistry 48, 8540-8550. 15. Leporc, S., Mauffret, O., Tevanian, G., Lescot, E., Monnot, M., and Fermandjian, S. (1999) An NMR and molecular modelling analysis of d(CTACTGCTTTAG)·d(CTAAAGCAGTAG) reveals that the particular behaviour of TpA steps is related to edge-to-edge contacts of their base-pairs in the major groove, Nucleic Acids Res 27, 4759-4767. 16. Columbus, L., Kalai, T., Jeko, J., Hideg, K., and Hubbell, W. L. (2001) Molecular motion of spin labeled side chains in alpha-helices: analysis by variation of side chain structure, Biochemistry 40, 3828-3846. 17. Bobst, A. M. and Keyes, R.S. (1998) Spin-labeled nucleic acids, In Biological Magnetic Resonance (Berliner, L. J., Ed.), pp 238-338, Plenum Press, New York. 18. Kalai, T., Balog, M., Jeko, J., and Hideg, K. (1998) 3-substituted 4-bromo- 2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxyl radicals as versatile synthons for synthesis of new paramagnetic heterocycles, Synthesis-Stuttgart 10, 1476- 1482. 19. Popova, A. M., and Qin, P. Z. (2010) A nucleotide-independent nitroxide probe reports on site-specific stereomeric environment in DNA, Biophys J 99, 2180- 2189. 50 Chapter 3 Site-specific DNA structural and dynamic features revealed by nucleotide-independent nitroxide probes 1. Chapter 3 Introduction DNA is a universal carrier of genetic information and is a key to gene maintenance and expression in most life forms. The linear sequence of a DNA molecule, constituted with four basic building blocks (four nucleotides), defines a host of physical attributes, such as shape (three-dimensional structure), dynamics, and electrostatics. These properties govern DNA interactions with proteins, other nucleic acid molecules, and small molecule ligands, thus dictating biological functions. Many studies have been devoted to understand DNA structure, and a large amount of information is available. Various spectroscopic measurements, including solution (1, 2) and solid-state NMR (3, 4), EPR (5, 6), and fluorescence (7-9) have revealed complex DNA dynamics that involve correlated and uncorrelated processes occurring on timescales ranging from picosecond to millisecond. It has been suggested that site-specific DNA dynamics are linked to functions. For example, sequence-dependent mobility variations of cytosine nucleotides have been linked to site-specific DNA methylation, which is a mechanism of gene regulation (3, 4, 10). Furthermore, altered flexibility at DNA lesion sites has been 51 proposed to play a key role in DNA recognition by repair proteins (11-13). However, current knowledge on sequence dependent DNA dynamics is primitive, and methods for probing site-specific dynamic features in large DNA and DNA/protein complexes are lacking. In SDSL, a chemically inert nitroxide radical is covalently linked to a specific site of a macromolecule, and EPR spectroscopy is used to obtain information on structural and dynamic features of the labeling site. This technique requires only a small amount of a labeled sample (~ 500 picomoles), and is capable of studying high molecular weight complexes under physiological conditions. SDSL has matured as a tool for studying protein structure and dynamics (14). In nucleic acid studies, SDSL has been applied to obtain distance constraints, to probe local and global dynamics, and to monitor conformational changes (15). We have reported a phosphorothioate scheme where a nitroxide is attached to a phosphorothioate group that is chemically substituted at a specific location of a nucleic acid (Figure 3.1A) (16, 17). The phosphorothioate scheme allows efficient nitroxide attachment at an arbitrary nucleotide within a desired sequence. It opens up the possibility of “nitroxide scanning”, where a nitroxide is systematically moved along a stretch of primary sequence in order to obtain information at the level of an individual nucleotide. Figure 3.1A shows two nitroxide probes, R5 and R5a, which are attached using the phosphorothioate scheme and differ only in the 4-substituent of the pyrroline ring. R5 has been used to monitor changes in global molecular tumbling associated with RNA/RNA interactions (16). More recently, R5 and R5a have been employed to study 52 nanosecond dynamics of an RNA element within a large folded ribozyme (18). In addition, distance measurements using a pair of R5’s have been reported (19, 20). Figure 3.1: (A) The phosphorothioate labeling scheme. During the solid phase chemical synthesis, a phosphorothioate is introduced at a specific nucleotide. The modified oligonucleotide is further reacted with 2,2,5,5-tetramethyl-1-oxylpyrroline derivatives (HO368 or HO1820), resulting in covalently linked R5 or R5a, respectively. Three torsion angles of the DNA-nitroxide linker are t1(O5’-P-S-CS), t2(P-S-CS-C3), t3(S-CS-C3-C2). (B) Sequence and secondary structure of the CS DNA duplex. Phosphorothioate groups labeled with nitroxides in this study are shown in green. (C) NMR structure of the CS DNA (21). Work reported here explores feasibility of probing local DNA environment, defined 53 as structural and dynamic features at the labeling site, by monitoring rotational diffusive motions of R5 or R5a. Nitroxide rotational motions (equivalent to nitroxide dynamics) are reported by cw-EPR spectroscopy, in which the spectral lineshape is dictated by rotational averaging of anisotropic Zeeman and hyperfine tensors of a nitroxide. At the commonly used X-band, the cw-EPR spectrum lineshape changes drastically as the nitroxide rotational correlation time vary between 0.1 ns and 50 ns. If nitroxide dynamics in this regime are affected by the parent molecule, the observed EPR spectrum will vary according to structural and dynamic features at the labeling site. In such cases, the EPR spectrum serves as a reporter on the local environment. EPR lineshape analysis is one of the most commonly used source of information in protein SDSL studies (22). It has also been successfully applied to extract information from nitroxide probes attached to base and sugar positions of nucleic acids (15). Particularly, a number of research groups, including Bobst (23), Robinson (5, 24, 25), and more recently Sigurdsson (6), have studied DNA duplexes using nitroxides attached to specific base positions, and their work has revealed complex DNA dynamic behaviors that include both collective and local modes of motion. Coupling between an EPR lineshape and the local macromolecule environment depends on the chemical scheme of nitroxide labeling. While R5 and R5a have been employed in a number of nucleic acid SDSL studies, there were no prior report on whether dynamics of these probes can provide local, site-specific information on DNA or RNA. Compared with other classes of nitroxides, R5 and R5a may have two potentially problematic issues. First, the pyrroline ring of the nitroxide is linked to the DNA 54 phosphorous atom by rotatable bonds (Figure 3.1A). Internal motions of the nitroxide, defined as rotations about these bonds, may dominate nitroxide dynamics. This may lead to uniform nitroxide spectra at different DNA sites, thus preventing studies of site- specific features. In addition, a chemically introduced phosphorothioate adopts either one of two chiral configurations (R p or S p ). Nitroxides attached to R p and S p diastereomers are sterically different, which may cause differences in their EPR spectra. Separations of R p and S p diastereomers have been demonstrated on a number of short oligonucleotides using laborious experimental procedures (26-28). Such separations have been reported only for oligonucleotides that are shorter than 15 nucleotides, and their success/failure depend heavily on sample specific features, such as the oligonucleotide sequence and the location of the phosphorothioate modification site. Due to the complexity in diastereomer separation, in many SDSL studies one may prefer to use mixed phosphorothioate diastereomers, in which an observed EPR spectrum is a sum of those obtained from either diastereomer. Therefore, it is important to establish what information on the local DNA environment can be obtained using mixed phosphorothioate diastereomers. Here, we placed R5 and R5a to multiple positions within a model dodecameric DNA duplex (Figure 3.1B), and measured the corresponding X-band EPR spectra without separating nitroxide diastereomers. Both R5 and R5a gave spectra that vary according to the location of the label (e.g., duplex center vs. termini) and the surrounding DNA sequence. The trend of spectral variations is consistent between the two probes. The results establish that R5 and R5a can be used to probe DNA local environment. This may lead to a new way for studying DNA structure and dynamics. 55 2. Chapter 3 Material and Methods 2.1. Materials All oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA). Reactive nitroxide derivatives 3-methanesulfonyloxymethyl-2,2,5,5-tetramethyl-1- oxylpyrroline (HO346, precursor for R5) and 4-bromo-3-bromomethyl-2,2,5,5- tetramethyl-1-oxylpyrroline (HO1820, precursor for R5a) were synthesized as reported previously (29, 30). 2.2. DNA labeling and purification R5 labeling was carried out as described previously (17). For R5a labeling, crude oligonucleotides containing one phosphorothioate modification (~ 7 nanomole) were treated with 1.2 mg of HO1820 in 4 L MES (1 M, pH = 5.8), 16 L water and 20 L acetonitrile. After incubation at room temperature for 24 hours, the labeled product was first purified by anion-exchange HPLC, and then desalted using a homemade G-25 Sephadex column. Details of purification procedures have been previously described (17). Desalted oligonucleotides were lyophilized, resuspended in 10 – 15 L of water and stored at –20°C. The final concentration of labeled oligonucleotides was determined by UV absorption at 260 nm. Extinction coefficients of the unmodified DNA (108,200 and 125,800 M -1 cm - 1 for the left and right strands shown in Figure 3.1B, respectively) were used, as neither the phosphorothioate modification nor the nitroxide alters absorbance at 260 nm. 56 2.3. DNA thermal denaturation DNA duplexes labeled with a single R5a were prepared in buffer A (100 mM NaCl, 50 mM Tris-HCl, pH = 7.5). The final duplex concentrations were 1 – 2 M. Melting transition curves were obtained using a DU800 UV-Vis spectrometer (Beckman Coulter, Fullerton, CA). Temperature was increased from 5°C to 75°C with a rate of 1°C/min outside the transition region and 0.5°C/min within the transition region. Absorbance at 260 nm was recorded every 0.5°C or 0.2°C accordingly. Meting curves were analyzed as previously described to obtain thermodynamic parameters of duplex formation (31). Each DNA sample was melted 4-7 times and averaged thermodynamic parameters are reported. In control studies, labeled duplexes were subjected to repeated heating/cooling cycles in which temperature was varied with the same average heating rate as in the melting experiments. After seven cycles less than 6% of R5 or R5a were found to detach from the DNA. This amount of probe detachment does not affect the measured thermodynamic parameters beyond the range of reported errors. 2.4. EPR measurements To prepare an EPR sample, approximately 1 nanomole of a spin-labeled oligonucleotide was annealed with a 10% molar excess of an unlabeled complementary strand. Annealing was carried out in buffer A with overnight incubation at room temperature. Formation of double-stranded DNA was confirmed using native gel electrophoresis (data not shown). To remove excess unannealed single-stranded DNA 57 and a small amount of detached probe, the mixture was diluted with 600 L of buffer A, and then concentrated to a final volume of 4 – 7 L at 15 C using an Ultrafree centrifugal filter (MWCO 5 kD, Millipore, Inc.). The annealed duplex was then used to prepare an EPR sample, which contained approximately 20-40 M of a labeled DNA duplex, 100 mM NaCl, 50 mM Tris-HCl (pH = 7.5), and 34% (w/w) sucrose. Finally, EPR spectra were measured with approximately 10 – 15 L of samples individually placed in a glass capillary (1.0 mm I.D.  1.2 mm O.D. Vitrocom, Inc., Mountain Lakes, NJ) that was sealed at one end. We note that a cosolvent (e.g. sucrose) was added to samples in order to minimize spectral effects due to uniform global tumbling of the model DNA duplex (Figure 3.1B, MW = 7,290 Da). Using the hydrodynamic model of Tirado and de la Torre (32), in an aqueous solution at 20°C the global tumbling of the model duplex was estimated to have rotational correlation times of 3.5 ns (    ) and 6.9 ns (   ) for rotations that are parallel and perpendicular to the DNA helical axis, respectively. This is expected to significantly average nitroxide magnetic tensors, thus largely masking site-dependent spectral variations at X-band. Presence of 34% (w/w) sucrose increases    and   to 14 ns and 28 ns, respectively. The observed EPR spectra are dictated by motions faster than 14 ns and presumably are more adept to report site-specific features. X-band EPR spectra were acquired on a Bruker EMX Spectrometer using a high sensitivity cavity (ER 4119HS, Bruker Biospin, Inc.). The incident microwave power was 2 mW, and the field modulation was 1 – 2 G at a frequency of 100 kHz. Sample 58 temperature was maintained at 5 C during spectral acquisitions using a liquid nitrogen variable temperature setup. All EPR spectra were baseline corrected and normalized to the same number of spins using software kindly provided by the Hubbell group at UCLA. 2.5. Modeling of sterically accessible nitroxide rotamer space NASNOX program was previously reported for identifying ensembles of sterically allowable R5 conformers (17, 19, 33), and was updated to allow modeling of R5a. The studies used the NMR structure of the model DNA duplex (21), with the nitroxide (R5 or R5a) modeled at either the R p or the S p phosphorothioate of a desired nucleotide. Discrete searches of torsion angles t1, t2 and t3 (Figure 3.1A) between 0° and 360° were carried out to select sterically allowable conformers. For R5, t1, t2, and t3 search steps were 5°, 30°, and 30°, respectively. For R5a, t1 was varied in a 5° step while t2 and t3 were fixed (t2 = 180°, t3 = 100° or -100°, values obtained based on MD simulations; Frushicheva, Hatmal Qin, Haworth, unpublished data). For each ensemble, a quantity S was computed according to: S     3 1 2 2 cos  where  is the angle between an individual NO vector (defined by the nitroxide nitrogen and oxygen atoms) and the average NO vector of the ensemble. 3. Chapter 3 Results 3.1. Model DNA system and nitroxide labeling 59 The model DNA used in this work is a dodecameric duplex with non-self- complementary strands (Figure 3.1B). The DNA was designated as CS, following its PDB ID 1CS2. The NMR structure of CS (Figure 3.1C) shows a near canonical B-duplex geometry, although deviations from standard B-form parameters were observed (21). Particularly, the minor groove narrows along the T8T9T10/A15A16A17 stretch and widens at the T10A11 step. Previously, we measured distances between pairs of R5 attached to the CS DNA, with the results showing good agreement between the SDSL measured distances and those predicted based on the NMR structure (19). Using the phosphorothioate scheme, nitroxides were attached, one at a time, to eight different positions within the CS duplex (Figure 3.1B). The non-symmetric nature of the CS DNA ensures that one and only one nitroxide is present in each labeled duplex. In the following text, a given site of the duplex is referred to as CSx, while a specific nitroxide labeled duplex and the associated EPR spectrum are named according to the labeling site and the nitroxide identity. For example, “CS2” refers to site 2 of the duplex (i.e., the local environment at the phosphate group of nucleotide 2), while “CS2_R5” designates a duplex with an R5 attached at the phosphate group of nucleotide 2 or its EPR spectrum. All data reported here were obtained with R5 Figure 3.2: MALDI-TOF spectrum of an R5a labeled DNA strand. Data for CS24_24T_R5a is shown. The measured mass (3901.73 Da) matches with the mass computed for the DNA strand with one R5a attached (3901.65 Da). 60 or R5a attached to mixed phosphorothioate diastereomers at each labeling site. Consistent with previous reports (17, 19), R5 labeling was found to be efficient and site-specific. Similarly, high efficiency and specificity were observed for R5a. Labeling efficiency of R5a was estimated to be > 95% based on anion-exchange HPLC. Mass spectrometry measurements showed that one and only one R5a is attached to a DNA strand (Figure 3.2). For R5a labeling, the 3-bromomethyl precursor (HO1820, Figure 3.1A) is sufficiently reactive and can be used directly without being converted to the 3- iodomethyl derivative. 3.2. DNA structure perturbation Previously, when a pair of R5 was attached to the CS duplex, only minor perturbation to the native B-form configuration was detected using CD spectroscopy and thermal denaturation measurements (19). MD simulation studies also indicated that R5 is accommodated within the groove of the helix and does not induce severe helical distortion ((33) and Hatmal, Frushicheva, Qin, Haworth, unpublished data). R5a was attached to the DNA using the same chemical scheme, and therefore is not expected to drastically perturb CS duplex based on the R5 results. To test this, thermal denaturation studies were carried out on single R5a labeled CS duplexes (Figure 3.3). The data confirm that a single R5a label has minor effects on DNA duplex stability, with the unlabeled and labeled duplexes showing small differences in the measured thermodynamic parameters of duplex formation (Table 3.1). For example, the differences 61 in the free energy of duplex formation between unlabeled and labeled duplexes (  G° 37°C ) are all less than 1.0 kcal/mol (Table 3.1). Table 3.1: Thermodynamic parameters of duplex formation for R5a labeled CS duplexes. (A) Wild-type CS duplex a Labeled position H° b (kcal/mol) S° b (cal/(mol K)) G° 37°C c (kcal/mol)  G° 37°C d (kcal/mol) None -69  2 -192  8 -9.4 - CS7 -68  5 -191  17 -9.2 0.2 CS9 -63  5 -174  15 -8.8 0.6 CS12 -67  2 -186  7 -9.2 0.2 CS14 -64  2 -176  5 -9.1 0.3 (B) Mutant CS duplex f Labeled position H° b (kcal/mol) S° b (cal/(mol K)) G° 37°C c (kcal/mol)  G° 37°C d (kcal/mol) None -75  3 -214  8 -9.1 - CS2_1A -80  1 -227  5 -9.4 -0.3 CS24_12T -78  3 -222  9 -9.0 0.1 a DNA sequence as shown in Figure 3.1A b Averages and errors were obtained based on results from multiple sets of melting experiments c G° 37°C = H° – 310K  S°. Averages of G° 37°C value from multiple melting measurements are reported, with errors estimated to be less than  0.1 kcal/mol. d  G° 37°C = G° 37°C (unlabeled) - G° 37°C (labeled) f DNA sequence modified at the duplex terminus: C1/G24 to A1/T24 The destabilizing effect is most prominent at the interior site CS9 (+0.6 kcal/mol) and 62 become smaller at the termini. We also note that at the terminal site of the mutant DNA (CS2_1A, Table 3.1B),  G° 37°C was measured to be -0.3 kcal/mol, indicating that R5a stabilizes the duplex at this site. Figure 3.3: UV melting data for a wild type CS DNA and CS14_R5a. Experimental data are shown with black circles, and simulated melting curves are shown with red lines. The fitting parameters are shown. 3.3. Site-dependent variations in R5 spectra X-band EPR spectra of R5 attached to eight sites within the CS DNA were obtained in the presence of 34% (w/w) sucrose at 5 C (Figure 3.4). All R5 spectra have a common feature – the center line is sharp and the hyperfine splitting is not observed. This suggests that R5 is undergoing fast and relatively unrestricted rotation at these sites. This is consistent with the expected large degree of freedom in R5 internal motions, where all three single bonds connecting the pyrroline ring to the DNA may rotate at the nanosecond timescale ((33) and Hatmal, Frushicheva, Qin, Haworth, unpublished data). Nonetheless, linewidth variations were observed between different R5 spectra, indicating that R5 mobility changes between different DNA sites. The normalized spectra 63 of CS2_R5 and CS14_R5 are nearly identical (Figure 3.5A), and the resulting difference spectrum is similar to those obtained in control studies where duplexes labeled at certain sites were prepared and measured multiple times (Figure 3.6). Figure 3.4: EPR spectra of R5 labeled CS DNA duplexes. 64 Figure 3.5: Comparisons of R5 spectra. 65 Figure 3.6: Reproducibility of CS 9_R5 and CS2_R5a spectra. (A) CS9_R5_1 and CS9_R5_2 samples were prepared from two different stocks of a phosphorothioate modified oligonucleotide (CS9). Their spectra were obtained 4-5 months apart. (B) CS2_R5a_1 and CS2_R5a_2 were prepared using the same stock of CS2, but different sucrose stocks. The spectra were obtained 2-2.5 years apart. This indicates that CS2 and CS14 have a very similar local environment. Interestingly, CS2_R5 and CS14_R5 show clearly narrower lines with larger amplitude compared to those at other sites (Figures 3.5B, 3.5C), with the corresponding difference spectra varying significantly from those obtained in control measurements (Figure 3.6). This indicates that CS2 and CS14, which are located at duplex 5’ termini, have higher R5 mobility as compared to sites at the duplex interior (sites 5, 7, 9, and 19) and those at the 3’ termini (sites 12 and 24). For the remaining six sites (5, 7, 9, 12, 19, and 24), slight lineshape variations can be observed (Figure 3.4). However, the differences between these sites are smaller than those between this group and CS2/CS14 (Figures 3.5B, 3.5C). We also note that EPR spectra at the 3’ terminal sites (sites 12 and 24) are nearly identical (Figure 3.5D), with R5 mobility comparable to that of the interior sites (e.g., sites 5 and 9, Figure 3.4) and clearly lower than that at the 5’ termini (sites 2 and 14) (Figure 3.5C). 66 To further quantify variations in R5 mobility, rotational correlation time ( ) was estimated from EPR spectral linewidth and amplitudes according to a formalism that treats nitroxide motions as a fast isotropic rotation (16, 34). The plot of  values at different sites (Figure 3.7) reveals nitroxide mobility variations that are consistent with conclusions drawn from direct lineshape comparisons (Figures 3.4 and 3.5). The 5’ terminal sites (CS2 and CS14) have the smallest  (2.13 ns and 2.03 ns, respectively, with errors estimated to be < 5%) and therefore the highest rate of nitroxide motions, again indicating that these two sites have higher mobility as compared to those at the interior and the 3’ termini. The  values of 3’ terminal sites (CS12 and CS24, 2.81 ns and 2.65 ns, respectively) are comparable to those of the interior sites (2.57 – 2.89 ns), indicating a similar mobility between the 3’ terminal and duplex interior sites. Finally, among interior sites, duplex centers (CS7 and CS19) show the highest  and therefore the lowest mobility. Figure 3.7: Estimated R5 rotational correlation time ( ). R5 location is indicated next to the corresponding data point. Squares and triangles represent sites belonging to the right and left DNA strands shown in Figure 3.1B, respectively. Errors in  were determined to be < 5% based on analysis of EPR spectra obtained from several R5 labeled duplexes that were prepared and measured multiple times. 67 Overall, the data reveal that R5 mobility is different at different sites, indicating site- specific couplings between R5 motions and local DNA environment. Furthermore, local environments at both 5’ terminal sites (CS2 and CS14) are distinct from the interior sites, while those at both 3’ terminal sites (CS12 and CS24) are comparable to the interior sites. Similar results were observed using R5a. 3.4. Site-dependent variations of R5a spectra X-band EPR spectra of R5 show characteristics of fast motions at all CS sites due to the large degree of freedom in nitroxide internal motions (see section 3.3). If nitroxide internal motions are reduced, one may expect to observe larger site-dependent spectral variations and better sensitivity to local DNA environment. Such a strategy, which has been successfully implemented previously in protein studies (35), was tested here by investigating R5a labeled DNA. R5a has a bromine atom at the C4 position of the nitroxide pyrroline ring (Figure 3.1A), which restricts two out of the three torsional rotations (t2 and t3, Fig. 1A) in the nitroxide internal motions according to MD simulations (Frushicheva, Hatmal, Qin, Haworth unpublished data). R5a was attached to eight positions of the CS duplex, and spectra were measured at 5°C (Figure 3.8). At each site, R5a spectrum is indeed broader than the corresponding R5 spectrum, indicating slower motions of R5a as compared to R5. With reduced internal motions, R5a is more prominently influenced by the local DNA environment, and larger spectral variations are observed between the labeled sites 68 Figure 3.8: EPR spectra of R5a labeled CS DNA duplexes. Black arrows indicate the resolved hyperfine extrema at CS7 and CS19. Red arrows indicate the presence of a second spectral component at CS2 and CS14. Spectra of CS9, 12, 14, 19, and 24 were corrected for the residual free nitroxide (< 3%). 69 (Figure 3.8). However, higher sensitivity of the probe renders lineshape analysis more complex, and simple linewidth measurements or  estimations no longer fully capture features of R5a dynamics. As a first step in analyzing site-dependent R5a motions, R5a spectra were categorized into three major groups based on differences and similarities in their lineshapes. The first group includes positions 7 and 19, which have nearly identical spectra with a split low-field peak (Figure 3.8, indicated by black arrows). Splitting of a high-field peak is also present, although harder to observe due to its intrinsically low amplitude (Figure 3.8, black arrows). These features arise from partially resolved parallel and perpendicular components of the hyperfine tensor due to incomplete averaging, and are a signature of a nitroxide undergoing anisotropic rotational diffusion. This class of lineshapes has been observed in previous studies of proteins (36) and nucleic acids (37). Group 2 includes CS5, CS9, CS12 and CS24 (Figure 3.8). These spectra show broad low-field peaks. Splittings of low and high-field peaks can also be observed, although are less prominent than those in the group 1 spectra. These features indicate that the nitroxide is undergoing anisotropic rotation, but with a higher degree of hyperfine tensor averaging and therefore higher mobility than that of group 1. Within the group 2 sites, CS12 and CS24 are located at the 3’ termini of the DNA duplex, while CS5 and CS9 are located within the duplex interior and have different adjacent nucleotides. The similarity in their spectra indicates that R5a is experiencing a similar local environment despite variations 70 in the nucleotide sequence and labeling position. This trend is consistent with the R5 results, where CS12_R5 and CS24_R5 (3’ terminal sites) give similar spectra to CS5_R5 and CS9_R5 (interior sites) (Figure 3.4). Group 3 includes CS2 and CS14, which are at the 5’ termini of the DNA duplex. Both spectra show multiple components, indicating the presence of at least two nitroxide populations with distinct modes of motion. In the CS2 spectrum, a small bump is clearly present at the shoulder of the low-field peak (Figure 3.8, red arrow), reflecting a subpopulation of a nitroxide with significantly reduced mobility. Due to the immobilized subpopulation, the center and high field lines in the CS2 spectrum are lower in their amplitudes and broader when compared to other sites. The CS14 spectrum is dominated by a high mobility nitroxide population, and has the highest degree of hyperfine tensor averaging among the R5a spectra (Figure 3.8). The immobilized subpopulation is much less visible in CS14 than that in CS2 at 5 ºC (Figure 3.8, red arrow). Consistent with R5 data, R5a indicates that 5’ terminal sites (CS2 and CS14) are different from the interior sites and the 3’ terminal sites. While R5 gives highly similar spectra at the 5’ terminal sites, R5a has distinct spectra with different proportion of spectral subcomponents. As detailed in the discussion section, the immobilized component may arise from nitroxide/DNA interactions that are captured by the R5a probe. These results, therefore, highlight the sensitivity differences between R5 and R5a. 71 3.5. DNA base mutations affect R5a spectra To further demonstrate that R5a reports on DNA local environment, the terminal C1/G24 base pair of the CS duplex was substituted with A1/T24 (Figure 3.9), and an effect on R5a spectra was studied. Two factors governed the choice of this particular mutation. First, the mutations were placed at the duplex terminus, so that only one nearest neighbor unit is affected (C1T2/A23G24). Second, within the context of one nearest neighbor unit, the C1/G24 to A1/T24 substitution gives the biggest possible change in the empirical thermodynamic parameters (∆H° (CT/GA) = -7.8 kcal/mol vs. ∆H° (AT/TA) = -7.2 kcal/mol, (38)). Assuming thermodynamic parameters are linked to DNA local environment, larger differences in thermodynamic parameters may suggest larger local variations, thus maximizing probability of observing R5a spectral changes. Figure 3.9: EPR spectra of R5a at positions 2 and 24 in the mutant (top) and wild type (bottom) CS duplexes. Arrows indicate immobilized spectral components. CS2_1A, CS24 and CS24_24T spectra were corrected for the presence of the residual free nitroxide (< 3%). 72 EPR spectra were obtained at 5°C for a single R5a attached to either position 2 or position 24 of the mutant duplex (CS2_1A and CS24_24T, respectively; Figure 3.9). The mutant CS24_24T_R5a spectrum has a narrower low-field peak as compared to that of CS24_R5a, and no splitting is observed (Figure 3.9). This reports a higher degree of hyperfine tensor averaging due to increased nitroxide mobility in the mutant. At position 2, the mutant DNA (CS2_1A_R5a) gives a multiple component spectrum, similar to that observed at the wild type CS2 site. The bump at the shoulder of the low-field peak is more pronounced in the mutant spectrum (Figure 3.9, marked by arrows), which indicates a higher fraction of an immobilized population. Overall, the mutant studies revealed that rotational diffusion of R5a is affected by variations in the nucleotide sequence at the labeling site. 3.6. Effect of a cosolvent: sucrose vs. Ficoll 70 In data presented above, EPR spectra were recorded in 34% (w/w) sucrose solution to reduce Brownian diffusion of the entire DNA molecule (see section 2.4). While this strategy has been commonly used in SDSL studies, it is not clear whether sucrose differentially affects nitroxide behavior at different DNA sites, thus interfering with the analysis of site-dependent spectral variations. To address this question, R5a spectra at three CS sites were measured in the presence of Ficoll 70 (MW = 70,000 Da) instead of sucrose. Ficoll 70 is a copolymer of sucrose with epichlorohydrin. It increases solution viscosity, but minimally affects its osmolality(39). 73 Figure 3.10: R5a spectra obtained in different cosolvents. Spectra recorded in ~30% (w/w) Ficoll 70 are shown on the left, while the corresponding spectra recorded in 34% sucrose (w/w) are shown on the right. Arrows in the same color indicate spectral features that are maintained at the same site in two cosolvents. The results reveal that spectra obtained in ~30% (w/w) Ficoll 70 maintain all the site- dependent features as those observed in sucrose. As shown in Figure 3.10, CS2_1A_R5a preserves a two component spectrum, while CS7_R5a retains a clear splitting of the low- field peak (indicated by arrows). Furthermore, in Ficoll 70, CS12_R5a yields a spectrum with a broad low-field peak and an unresolved splitting, indicating higher nitroxide mobility as compared to CS7_R5a. The same trend is observed using sucrose. We also note that all spectra measured in Ficoll 70 have narrower lines and therefore report higher 74 nitroxide mobility than the corresponding sucrose spectra. Sucrose and Ficoll are drastically different in their molecular weight and chemical composition, and likely affect macromolecular Brownian motions via different mechanisms (40). Consistency between the sucrose and Ficoll 70 data suggests that in the current study the observed spectral variations report differences in local DNA environment and are not artifacts due to the use of a particular cosolvent. 4. Chapter 3 Discussion Data reported above clearly demonstrate that X band cw-EPR spectra of both R5 and R5a may vary according to local DNA environment. This establishes that R5 and R5a can be used to examine site-specific features in DNA at the level of an individual nucleotide. In the following sections, we discuss possible probe perturbation to DNA and potential modes of nitroxide/DNA coupling that may influence the observed EPR spectra. 4.1. Perturbations due to R5 and R5a nitroxide probes A number of experiments have collectively shown that R5 and R5a, which are attached using the same chemical scheme, do not significantly alter the native conformation of the CS duplex. Thermal melting studies on R5a single-labeled (this work) and R5 double-labeled (19) DNA revealed that the probe(s) minimally affects duplex stability, with  G° 37°C < 1.0 kcal/mol in all cases studied. Circular dichroism spectroscopy measurements indicated that R5 double-labeled DNA do not alter its B- 75 form conformation (19). Distance measurements using a pair of R5 yielded results that are in good agreement with the NMR determined DNA structure (19). We conclude that within the sensitivity of these measurements, neither R5 nor R5a drastically alters the DNA duplex conformation. As extrinsic probes, R5 and R5a do impose a number of changes onto the DNA. Two most noticeable ones are presence of the pyrroline ring and neutralization of one negative charge at the phosphate group. With the labeling scheme used, there is sufficient linker flexibility to allow the pyrroline ring to adjust to the groove of the DNA. In addition, pyrroline/DNA interactions are not expected to have sufficient energy to drastically alter the native DNA structure. Instead, these interactions may give rise to unique spectral features, such as immobilized components (Figure 3.9, CS2 and CS2_1A, and see section 4.3). Effects of charge neutralization are not clear. A number of studies have examined the effect of nonsymmetric charge neutralization of the DNA backbone using alkylphosphonate modifications that are conceptually very similar to R5 and R5a. DNA containing varying lengths of consecutive or alternating alkylphosphonate modifications have been studied using gel electrophoresis assays (41), NMR (42), EPR (43), and MD simulations (44, 45). The results revealed various degrees of DNA helix bending, as well as possible changes in helical parameters and deoxyribose flexibility (41, 42, 45). However, we are aware of only one report, where solution NMR was used to examine DNA duplexes containing a single alkylphosphonate (46). The study revealed a small bending of the DNA helical axis and changes in sugar puckering and helical parameters 76 that are localized near the modification site. These results were attributed to neutralization of the phosphate charge and steric/hydrophobic effects of the alkyl moiety. Within the scope of R5 and R5a SDSL studies, further investigations are needed to examine how neutralization of a single charge perturbs native DNA conformation and affects data interpretation. 4.2. Comparison between R5 and R5a R5 and R5a differ by one chemical group (4-H vs. 4-Br at the pyrroline ring, Figure 3.1A) that primarily affects internal motions of the nitroxides. In R5, torsional rotations that dictate nitroxide internal motions (t1, t2, t3 in Figure 3.1A) are relatively unrestricted on the nanosecond timescale ((33) and Hatmal, Frushicheva, Qin, Haworth, unpublished data), resulting in a high degree of averaging of magnetic tensors and thus spectra with narrow lines (Figure 3.4). In R5a, MD simulations indicate that t2 and t3 torsional rotations are restricted, thus reducing averaging of magnetic tensors. This gives rise to broader R5a spectra with more pronounced site-dependent spectral features. Similar observations have been reported in protein SDSL studies (35). At most CS sites studied, the same trend of nitroxide mobility variations is observed between R5 and R5a, indicating that similar features of the DNA local environment are reported by these probes. For instance, both R5 and R5a show that duplex center sites (CS7, CS19) are the most restrictive in terms of nitroxide motions. At the 3’ terminal sites (CS12 and CS24), both probes give almost identical spectra, indicating that at 5 C, 77 the local environment at the 3’ termini is comparable to that of the duplex interior (CS5 and CS9) and dissimilar to that at the 5’ termini (CS2 and CS14). Furthermore, both R5 and R5a report that at the 5’ termini (CS2 and CS14) the local DNA environment is different from that at other sites. However, CS2_R5 and CS14_R5 have almost identical spectra (Figure 3.4 and 3.5A), while CS2_R5a and CS14_R5a show different proportions of immobilized subpopulations (Figure 3.8). As discussed later, the R5a spectral differences may arise from subtle changes in site-dependent DNA/nitroxide contacts. We also note that the CS14_R5a spectrum, even with the presence of the low mobility component, shows the highest overall R5a mobility compared to other sites. This is consistent with data showing that the 5’ termini have the highest R5 dynamics. While neither R5 nor R5a significantly perturbs the DNA, R5a has more restricted internal motions as compared to R5 and therefore its overall motions are more strongly coupled to local DNA environment. Probing a given site with R5 and R5a simultaneously is more informative: it may distinguish DNA structural and dynamic features that affect both probes similarly, as well as those that influence the probes differently. This aids SDSL data interpretation. 4.3. Coupling between nitroxide dynamics and DNA: modulation of nitroxide internal motions Site-dependent R5 and R5a spectral variations clearly demonstrate a coupling between DNA local environment and nitroxide dynamics. However, a detailed 78 understanding of the rules governing such coupling is required in order to use a nitroxide spectrum to deduce information about a given DNA site. DNA may modulate R5 and R5a dynamics by two modes. Each DNA site may differentially influence nitroxide internal motions. In addition, DNA motions, which encompass all dynamic modes except the uniform global tumbling, may be transferred to the pyrroline ring via motions of the phosphorous atom. In this section we consider DNA modulations of nitroxide internal motions. Possible effects due to DNA local motions are discussed in the next section. Nitroxide internal motions are dictated by DNA three-dimensional structure, which imposes restrictions on a nitroxide “rotamer” space. Two factors may dictate the rotamer space at each labeling site. One is the allowable volume that the pyrroline ring can occupy without steric collision with the DNA. The other is nitroxide/DNA interactions, such as hydrophobic contacts or hydrogen bonding, which may modify the pattern of occupancy within the sterically allowed volume. To assess the sterically allowable volume, the NASNOX program was used to generate ensembles of R5a or R5 at each CS site. Each ensemble was selected based on steric exclusion between the DNA and the nitroxide, and was characterized by a quantity S that was computed based on the angular distribution of the nitroxide N-O bond vector (see section 2.5). The S value, which ranges from 0 to1, is analogous to an order parameter. A large S indicates small directional variations thus a limited allowable volume for the nitroxide, while a small S suggests large directional variations and an expanded allowable volume. Computed S values for R5a are shown in Table 3.2. For S p diastereomers, identical S 79 values were obtained, suggesting the same steric constrictions at various CS sites. S p diastereomers direct the pyrroline ring away from the DNA and towards the solvent. This may account for the lack of site-dependent variations in S values. R5a R p diastereomers, which direct the pyrroline ringtowards the DNA major groove, exhibit small variations in S values ( 0.022 from the average, Table 3.2), again indicating similar steric constrictions. R5 modeling showed similar results, with deviations from the averaged S being less than 0.011 (data not shown). Overall, the NASNOX results indicate very similar sterically allowable volumes at each labeling site of the CS duplex. This likely stems from isostericity of Watson-Crick base pairs and regularity of the B-form DNA duplex geometry: each base-pair unit displays very similar C1’-C1’ distances and glycosidic bond orientations, and can replace one another (e.g., A/T -> C/G) without significant alterations of the phosphate-sugar backbone configuration. Since CS DNA contains all Watson-Crick base pairs, each backbone site has a similar configuration, resulting in similar sterically allowable volumes experienced by the nitroxide. However, it should be emphasized that NASNOX provides only a zero-order Labeled position S value R p S p CS2 0.537 0.496 CS5 0.554 0.495 CS7 0.576 0.496 CS9 0.546 0.495 CS12 0.510 0.495 CS14 0.513 0.498 CS19 0.546 0.495 CS24 0.522 0.494 average  standard deviation 0.538  0.022 0.496  0.001 Table 3.2: Sterically allowed conformational space of R5a as estimated by NASNOX. 80 estimation. To achieve efficiency, NASNOX uses a fixed DNA structure and employs a stepwise search algorithm without involving a force field. These simplifications may limit its accuracy. More sophisticated computation approaches, such as MD simulations, may provide more detailed understanding of how DNA affects nitroxide conformations. Figure 3.11: Proposed R5a/DNA interactions at the 5’ terminal site CS2. The models were generated using WebLab ViewerPro 3.7 (Molecular Simulations, Inc.), with R5a attached to the R p phosphorothioate of the target site in the NMR structure of the CS duplex. The 4-Br atom of R5a is shown as a dark red ball, the C2 and C5 methyls of R5a are shown as purple balls, and the C5-methyl of T2 is shown as a green ball. In the modeling, the torsion angle t1 was varied, while t2 and t3 were set based on MD simulation data, which show t2  180°; and t3 = +100° or –100°. (A) R5a modeled with t1 = –60°, t2 = 180°, and t3 = +100°. This particular t1 value gives the closest distance between the 4-Br atom of R5a and the C5-methyl of T2 without any steric collision between R5a and DNA. (B) R5a modeled with t1 = –50°, t2 = 180°, and t3 = –100°. This configuration gives the closest distance between the 2-methyl group of R5a and the C5- methyl of T2 without any steric collision. With a nearly constant sterically allowable volume along the CS duplex, spectral variations may arise from site-specific nitroxide/DNA interactions. Simple modeling using the reported NMR structure indicates that for the R p diastereomer at CS2, either the 81 2-methyl groups or the 4-bromine atom of the R5a pyrroline ring may be positioned at approximately 3 Å to the methyl group of the 3’ neighboring thymine (T2 of CS DNA) (Figure 3.11). This may give rise to interactions that significantly immobilize a subpopulation of the nitroxide, resulting in a two-component CS2_R5a spectrum (Figure 3.8). Similarly, immobilized spectral components observed in CS14_R5a (Figure 3.8) and CS2_1A_R5a (Figure 3.9) likely originate from local R5a/DNA interactions. Modeling studies predict that nitroxide/DNA interactions described above are favored only for the R p diastereomer of CS2_R5a, but not for the S p diastereomer, which will be tested in future. This analysis highlights a need to further investigate diastereomer specific behaviors, which is a common issue facing R5 and R5a. While data reported here clearly demonstrate the feasibility of probing DNA environment using mixed diastereomers, studies using individual diastereomers may expand the scope of information obtainable from nitroxide probes. In summary, different sites in the CS duplex present a similar sterically allowable space to the nitroxide, but may vary in terms of nitroxide/DNA contacts that may depend on factors such as the surrounding sequence or diastereomeric characteristics of the label. The variable contacts may partially account for the observed EPR spectral features, particularly the presence of immobilized second components. Studies are underway to elucidate the correlation between EPR spectral features and site-specific nitroxide/DNA interactions. 82 4.4. Coupling between nitroxide dynamics and DNA: correlation to DNA local motions Here we examine how R5/R5a mobility varies depending on the labeling position (duplex termini vs. interior) and the flanking nucleotide sequence. The analysis suggests a positive correlation between R5/R5a mobility and expected DNA local flexibility. This provides evidence that these probes can report on certain modes of DNA local motion. The first example comes from CS7 and CS19. Based on the labeling position (duplex center) and thermodynamic stability of the flanking sequence (-GC- dinucleotide step) (38), these two sites are expected to exhibit lower DNA local flexibility as compared to other sites. Indeed, at CS 7 and CS 19, R5/R5a spectra are similar at 5°C, and the nitroxide mobility is lower than that at other sites (Figures 3.7 and 3.8). Another example of positional dependence of nitroxide mobility comes from comparisons between 5’ terminal sites CS2, CS14 and the interior site CS5. Although these sites share the same -CT- dinucleotide sequence, a much higher R5 mobility is observed at CS2 and CS14 (Figure 3.7). Furthermore, overall R5a mobility at CS14, in spite of the presence of an immobile component, is higher than that at CS5 (Figure 3.8). This trend of nitroxide mobility may be accounted for by increased DNA local flexibility at CS2 and CS14 due to the reduction of stacking constraints at the 5’ termini. R5 and R5a mobility also changes according to the neighboring nucleotide sequence. The 3’ terminal sites (CS24 and CS12) are located across from the 5’ terminal sites (CS2 and CS14), yet R5 mobility at both 3’ termini is lower than that at the 5’ termini (Figure 3.7), and R5a mobility at CS12 and CS24 is lower than that of CS14 (Figure 3.8). In the 83 CS duplex, labels at the 5’ terminal sites are sandwiched by -CT- dinucleotide step, while those at the 3’ termini are flanked by -AG- step. Energetics of stacking at these two steps is different: gas phase ab initio calculations have shown that -AG- dinucleotide is more stable (lower stacking energy) than that of -CT- (47); calculations using Langevin dipoles also concluded that in water the order of stacking stability is pyrimidine/pyrimidine (e.g., -CT-) < purine/pyrimidine < purine/purine (e.g. -AG-) (48). The more favorably stacked sites (i.e., 3’ termini in CS DNA) likely have lower local flexibility, thus exhibiting lower nitroxide mobility. A second example of sequence dependence comes from mutation studies (Figure 3.9). Mutating the C1/G24 base pair to A1/T24 lead to a less favorably stacked dinucleotide (- AG- vs. -AT-) and a loss of one hydrogen bond at the terminal base-pair. This reduces the thermodynamic stability at site 24, giving rise to higher DNA local flexibility and consequently increased R5a mobility (Figure 3.9). As R5/R5a mobility positively correlates with expected DNA local flexibility, our data indicate that flexibility (or dynamics) at the 3’ termini of the CS duplex is similar to that of the interior sites, but noticeably lower than that at the 5’ termini. This phenomenon was reported previously, where 13 C NMR relaxation experiments revealed that order parameters of sugar C-H bond vectors at the 3’ termini of a self- complementary duplex were comparable with average order parameters at central nucleotides (49). Data obtained from R5 and R5a thus argue against a commonly used description of DNA flexibility, where duplex termini are always more dynamic than internal sites due to the lack of stacking constraints. 84 R5 and R5a assess DNA local flexibility from the perspective of the phosphodiester group, thus providing EPR probes for studying local motions at the DNA backbone. This differs from previously reported nitroxides that were attached to various base positions (5, 6, 23, 24). While DNA backbone dynamics may be important in a number of events such as DNA-protein interactions, experimental studies in this area are very limited. We are aware of work from only two groups: Redfield and co-workers examined picosecond dynamics of 31 P atoms in DNA by field-cycling NMR relaxation (50); and Drobny and co-workers investigated sequence dependent nanosecond-microsecond dynamics of deuterated 5’-methylenes using solid-state NMR (4, 51). In particular, the Drobny group has reported reduction in dynamics of deoxycytosine 5’-methylene upon cytosine methylation within certain sequences. It was proposed that the decrease in backbone flexibility is due to methylation induced base stacking enhancement (4, 51). The notion that local flexibility at the DNA backbone depends on nucleotide sequence and is correlated with base stacking stability is consistent with conclusions drawn from R5 and R5a data reported here. 5. Chapter 3 Conclusions We have demonstrated that R5 and R5a are able to report differences in local DNA environment without severely perturbing the native conformation of the parent molecule. These probes can be easily attached to arbitrary DNA sites and provide a unique view on the local environment through the phosphodiester group. Consistent with studies on other 85 nitroxides, R5 and R5a motions were found to be influenced by a number of factors, including the mode of linker motion, the location of the labeling site, and the surrounding nucleotide sequence. Further studies, such as analysis of R5/R5a spectral variations according to diastereomer identity, DNA base sequence, secondary structure elements (mispaired or modified bases, abasic sites, etc.), and environmental conditions (salt, temperature, etc.), may reveal more detailed correlations between R5 and R5a spectra and local DNA structural and dynamic features. 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(2002) Bending of DNA by asymmetric charge neutralization: All-atom energy simulations, J Am Chem Soc 124, 4838-4847. 90 46. Soliva, R., Monaco, V., Gomez-Pinto, I., Meeuwenoord, N. J., Marel, G. A., Boom, J. H., Gonzalez, C., and Orozco, M. (2001) Solution structure of a DNA duplex with a chiral alkyl phosphonate moiety, Nucleic Acids Res 29, 2973-2985. 47. Sponer, J., Jurecka, P., Marchan, I., Luque, F. J., Orozco, M., and Hobza, P. (2006) Nature of base stacking: Reference quantum-chemical stacking energies in ten unique B-DNA base-pair steps, Chem Eur J 12, 2854-2865. 48. Florian, J., Sponer, J., and Warshel, A. (1999) Thermodynamic parameters for stacking and hydrogen bonding of nucleic acid bases in aqueous solution: Ab initio/Langevin dipoles study, J Phys Chem B 103, 884-892. 49. Spielmann, H. P. (1998) Dynamics of a bis-intercalator DNA complex by 1H- detected natural abundance 13 C NMR spectroscopy, Biochemistry 37, 16863- 16876. 50. Roberts, M. F., Cui, Q., Turner, C. J., Case, D. A., and Redfield, A. G. (2004) High-resolution field-cycling NMR studies of a DNA octamer as a probe of phosphodiester dynamics and comparison with computer simulation, Biochemistry 43, 3637-3650. 51. Geahigan, K. B., Meints, G. A., Hatcher, M. E., Orban, J., and Drobny, G. P. (2000) The dynamic impact of CpG methylation in DNA, Biochemistry 39, 4939- 4946. 91 Chapter 4 A nucleotide-independent nitroxide probe reports on site- specific stereomeric environment in DNA 1. Chapter 4 Introduction Site-directed spin labeling (SDSL) is a biophysical tool that utilizes electron paramagnetic resonance (EPR) spectroscopy to monitor a chemically stable nitroxide radical covalently linked to a specific site of a macromolecule (1). One can use SDSL to obtain information about macromolecular structure and dynamics at both the local and global level. This technique requires a relatively small amount of sample (tens to hundreds of picomoles), and allows one to study high molecular weight systems under physiological conditions. It has been proven to be valuable in probing structure and dynamics of proteins, biological membranes, nucleic acids and their assemblies (2, 3). A number of methods for attaching nitroxides to DNA and RNA have been reported (3, 4). In particular, a phosphorothioate labeling scheme has been developed (5-7), where a nitroxide molecule is attached to a phosphorothioate (ps) group introduced at a defined location of the nucleic acid backbone during solid-phase chemical synthesis (Figure 4.1A). This methodology is a simple and efficient means to link a nitroxide label to a target nucleotide within an arbitrary sequence. A number of nitroxide probes, designated as the R5 series (see Chapter 2), have been attached to DNA and RNA molecules 92 utilizing the phosphorothioate labeling chemistry, and have been used to monitor RNA/RNA interactions (5), to study motions of an RNA element within a large folded ribozyme (8), to probe local structural and dynamic features in DNA (7), and to measure nanometer distances in nucleic acids (9, 10). Figure 4.1: Nitroxide labeled DNA system. (A) The phosphorothioate labeling scheme. A reaction between a phosphorothioate modified oligonucleotide and 4-bromo-3-bromomethyl-2,2,5,5-tetramethyl-1- oxylpyrroline yields an R5a labeled DNA. Chirality of the phosphorothioate linkage results in two oligonucleotide diastereomers known as R p and S p . The three torsion angles of the DNA-nitroxide linker are marked as t1(O5‟-P-S-C S ), t2(P-S-C S -C 3 ), t3(S-C S -C 3 -C 2 ). (B) Sequence and secondary structure of the CS DNA duplex. In this study, R5a was attached to phosphorothioate sites shown in green. (C) Structural models of R5a (indicated by dotted circles) attached to the R p - (left) and S p - (right) diastereomers at CS7. 93 The phosphorothioate labeling scheme relies on the ps modification, in which sulfur substitutes one of the two non-bridging oxygens in the naturally occurring phosphodiester, thus introducing a chiral center at the phosphorus atom. Using the current solid-phase synthesis scheme, a modified oligonucleotide is obtained as a mixture of two diastereomers in an approximately 50/50 proportion. The two diastereomers, designated as R p and S p (Figure 4.1A), are able to react with a nitroxide precursor. Nucleic acid systems containing mixtures of R p - and S p -nitroxides have been successfully used in many SDSL studies, including monitoring molecular interactions (5), measuring inter- spin distances (9, 10), and exploring structural and dynamic features at the labeled nucleotide (7). Systems containing pure R p - or S p -nitroxides have been explored to a much less extent, although we have previously demonstrated the feasibility of obtaining diastereopure nitroxide labeled samples with certain DNA and RNA molecules (11). Modeling studies have shown that at a given site of a parent macromolecule, the R p - and S p -nitroxides are spatially distinct (9, 12) (Figure 4.1C), and may experience a different local environment, defined as structural and dynamic features of a macromolecule at the labeling site. This may give rise to detectable differences in EPR observables. For example, in a recent study of nanosecond dynamics of an RNA element in a large ribozyme (8), it was found that R p - and S p - nitroxides gave different spectra in either the docked or the undocked state of the ribozyme, yet reported similar spectral changes upon transition between the ribozyme states. The availability of both R p and S p data provided an important support in assigning the observed EPR spectral change to RNA motions. 94 Overall, SDSL studies using pure R p - and/or S p -nitroxides may become a unique methodology for examining a given nucleic acid site in a stereo-specific manner, thus increasing the amount of information one can obtain. It may aid studies of stereo-specific interactions with the two non-bridging oxygens, particularly those related to enzymatic cleavage and ion coordination. This study explores feasibility of probing DNA local environment with R p - and S p - nitroxides by attaching an R5a probe (Figure 4.1A) to a DNA duplex in a stereo-specific fashion. The model DNA, designated as CS (Figure 4.1B), is a dodecamer with a near canonical B-form structure in solution (PDB ID 1CS2, (13)), and has been used in a number of previous SDSL studies (7, 9, 12). Particularly, X-band continuous-wave (cw) EPR spectra have been reported for R5a attached to mixtures of ps diastereomers at eight CS sites (7). These mixed-diastereomer studies established that local DNA environment differentially influences rotational motions of R5a in the 0.1 – 30 ns regime, giving rise to observable spectral variations along the DNA duplex (see Chapter 3). In this work, X-band cw-EPR spectra were obtained for pure R p -R5a and S p -R5a labeled at five different positions of the CS duplex (Figure 4.1B). At each site studied, the R p -R5a spectrum was found to differ from that of the S p -R5a, with rotational diffusions of R p -R5a being more restricted. This is attributed to the fact that in a B-form DNA duplex R p -diastereomers position the nitroxide towards the groove of the DNA, while S p - diastereomers position the nitroxide towards the solvent. Linear additions of R p -R5a and S p -R5a spectra satisfactorily recover the respective measured mixed-diastereomer spectra, supporting the notion that studies using mixtures of ps diastereomers report the 95 combined behaviors of R p - and S p -nitroxides. Furthermore, variations between different DNA sites are observed when comparing spectra within the R p - or the S p -series. Two of the five R p spectra show observable low-mobility components, which are likely due to site-specific nitroxide-DNA interactions. The S p spectra, on the other hand, seem to be less sensitive to variations in the local structural features, and primarily report on DNA backbone flexibility. These results together indicate that R p -R5a and S p -R5a provide information on the DNA local environment in a stereo- and site-specific manner. 2. Chapter 4 Materials and Methods 2.1. DNA oligonucleotides and nomenclature In the text, „CSx‟ designates site „x‟ within the CS duplex, while „CSx_R p ‟ and „CSx_S p ‟ refer to R p or S p phosphorothioate configuration at site „x‟, respectively. „CSx_R5a‟ indicates the CS duplex with R5a nitroxide attached to a diastereomeric mixture at the specific site „x‟ or the corresponding EPR spectrum; while „CSx_R p _R5a‟ and „CSx_S p _R5a‟ refer to stereoregular R5a labeled DNA duplexes or their EPR spectra. For example, „CS7_R p ‟ represents site 7 with an R p configuration and thus the local environment at the R p phosphorothioate of nucleotide 7; „CS7_R p _R5a‟ designates a duplex with an R5a attached to the R p diastereomer or the corresponding EPR spectrum. All deoxyoligonucleotides used in this work were obtained commercially from Integrated DNA Technologies (Coralville, IA). Oligonucleotide concentrations were determined by UV absorption at 260 nm as previously described (7). 96 2.2. Preparation of R p-R5a and S p-R5a oligonucleotides To attach R5a in a stereo-specific manner requires two general steps: nitroxide coupling and diastereomer separation. Nitroxide coupling was carried out by incubating a phosphorothioate-modified oligonucleotide with a reactive derivative, 4-bromo-3- bromomethyl-2,2,5,5-tetramethyl-1-oxylpyrroline (R5a precursor, kindly provided by Kálmán Hideg, University of Pécs, Hungary), following a published protocol (7). For CS2, CS9, CS12, and CS14, diastereomer separation was carried out on the unlabeled DNA, and R5a was then attached to the diastereopure oligonucleotides. For CS7, diastereomer separation was feasible after R5a coupling, which significantly shortened the preparatory procedure. R p and S p diastereomers of a given DNA oligonucleotide were separated on a DNApac PA-100 anion-exchange (AE) column (4  250 mm, Dionex Inc., Sunnyvale, CA) using a procedure modified from a published protocol (11). In each round of separation, ~ 5 nmol of an oligonucleotide was loaded onto the column and eluded at room temperature using a two-component gradient with a flow rate of 1 ml/min. The elution buffers were Buffer A: 25 mM Tris-HCl, pH 6.8; and Buffer B: 25 mM Tris-HCl, pH 6.8, 500 mM NaCl. In a typical round of HPLC separation, a DNA sample was subjected to a 15 minute segment of 0% Buffer B, followed by a 50-60 minute segment in which buffer B was increased at a rate of 1.5% per minute. Eluted samples were collected in 0.2 ml fractions using an automatic fraction collector. Following HPLC runs, respective R p and S p fractions were combined and desalted using a homemade G-25 Sephadex column. The desalted samples were subjected to additional round(s) of AE 97 HPLC separation depending on their purity. Twenty to thirty rounds of separation were required to obtain sufficient amount (4-10 nmol) of R p and S p oligonucleotides with > 98% purity. Finally, purified and desalted R p and S p oligonucleotides were lyophilized, then re-suspended in water and stored at -20ºC. 2.3. DNA thermal denaturation R5a labeled DNA duplexes were suspended in 400 µL of buffer 1 (100 mM NaCl, 50 mM Tris-HCl, pH = 7.5), with the final duplex concentrations being 1 – 2 M. Melting transition curves were obtained using a DU800 UV-Vis spectrometer (Beckman Coulter, Fullerton, CA) as described previously (14). Each DNA sample was melted 5-8 times to evaluate thermodynamic parameters of the double to single strand transition. Errors in G° determination were estimated to be  0.1 kcal/mol from multiple measurements. 2.4. EPR sample preparation and measurements To form a DNA duplex, approximately 1 nmol of an R5a labeled diastereopure oligonucleotide was annealed with a 10% molar excess of an unlabeled complementary strand in buffer 1. After an overnight incubation at room temperature, the annealing reaction mixture was diluted to 600 µL with buffer 1 and then concentrated to 5-7 L using a membrane centrifugal filter (MWCO 5 kD, Millipore Inc., Bellerica, MA), which removed unannealed single-stranded DNA and a trace amount of free spin labels. Concentrated DNA was used to prepare an EPR sample containing approximately 20 – 40 M of an R5a labeled DNA duplex suspended in buffer 1 and 34% (w/w) sucrose. 98 Sucrose was added to reduce global tumbling of the duplex and to enhance sensitivity of an X-band EPR spectrum to site-specific features of nitroxide rotational motions. A previous study has shown that sucrose does not alter key features observed in EPR spectra between different CS sites (7). EPR spectra were obtained following procedures previously described (4). Specifically, 13 – 15 L of samples were placed in glass capillaries (1.0  1.2 mm, Vitrocom, Inc., Mountain Lakes, NJ) sealed at one end. X-band EPR spectra were acquired on a Bruker EMX Spectrometer with a high sensitivity cavity (ER 4119HS, Bruker Biospin, Inc., Billerica, MA) and acquisition parameters were as follows: incident microwave power, 2 mW; modulation amplitude, 1 – 2 G; modulation frequency, 100 kHz. A liquid nitrogen variable temperature setup was used to maintain sample temperature. All EPR spectra were baseline corrected and normalized to the same number of spins using software provided by the Hubbell group at UCLA. 2.5. EPR spectra fitting Spectral fitting was carried out with the MATLAB-based EPRLL program suite (15) using the Microscopic Order Macroscopic Disorder (MOMD) model. Dimensions of the basis set were reduced via the pruning subroutine to L emx =6, L omx =5, K mx =5, M mx =4,, K mn =0, M mn =0, and p I mx =2. All spectral calculations used the same set of g and A tensor values (g xx =2.0083, g yy =2.0051, g zz =2.0022 A xx =6.9, A yy =5.7, and A zz =35,), which were determined from fitting near rigid-limit X-band spectra obtained at 0 C in 65% sucrose (w/w). The diffusion tilt angle (  D =35) and number of director orientation (n ort =20) were 99 fixed. Variable fitting parameters included: (i) and N, spherical components of the rotational diffusion rate tensor ( and N=R  / /R  , where R   and R  are the respective rate constants for rotations parallel and perpendicular to the nitroxide principal diffusion axis); (ii) c 20, the coefficient of an ordering potential , where  is an instantaneous angle relating the director to the nitroxide diffusion frame); and (iii)  (0) , the Gaussian inhomogeneous broadening factor. From the ordering potential, an order parameter, S 20 , is computed as . For each measured spectrum, the SIMPLEX minimization method was utilized to find a best-fit spectrum with the lowest root-mean-square-deviation (rmsd). An ensemble of acceptable spectra, defined as those with rmsd values within 110% of that of the best-fit spectrum, was then generated using a Monte-Carlo restart subroutine. Using the ensemble, averages and standard deviations for the fitting parameters were calculated, with the standard deviation used to represent an error for the respective parameter. 3. Chapter 4 Results 3.1. Preparation and characterization of diastereopure R5a labeled DNA In work reported here R5a was attached to pure R p and S p diastereomers at five CS sites (Figure 4.1B). Figure 4.2A shows two representative HPLC profiles demonstrating diastereomer separation of either an unlabeled (e.g., CS2) or an R5a-labeled (CS7_R5a) 100 DNA strand. All diastereomers used in this study were > 98% pure as determined by AE HPLC runs after completion of purification. We note that the diastereomer separation protocol was laborious, generally requiring 2 – 3 weeks of work for each sample (see section 2.2). In addition, the degree of chromatographic resolution of R p and S p peaks varies significantly depending on the position of ps modification and the oligonucleotide sequence, rendering diastereomer separation at certain CS sites unattainable (e.g., CS5). This is consistent with the complexity of ps diastereomer separation reported in the literature (16-18). Stereochemical configurations were assigned to chromatographic fractions based on a previous study, in which a stereo-specific nuclease digestion assay was used to determine diastereomer identity (11). As reported, without R5a attached (e.g., CS2, Figure 4.2A), the R p diastereomer elutes first, followed by the S p ; while after R5a coupling (e.g., CS7_R5a, Figure 4.2A), the elution order reverses, with S p eluting first and R p eluting later. Furthermore, for all oligonucleotides studied, the R p diastereomer was present in slight excess over S p in the mixtures supplied by the vendor, with the R p to S p ratio fluctuating between 60/40 and Figure 4.2: Separation of R p and S p diastereomers. A) Normalized AE HPLC traces are shown, corresponding to sample loading of 5-6 nmol. (B) R p vs. S p ratios determined by AE HPLC for the crude oligonucleotide mixtures supplied by the vendor. 101 50/50 (Figure 4.2B). This is consistent with other reports (19, 20), and serves as an independent control of ps configuration assignment. DNA structural perturbation due to R p -R5a and S p - R5a was assessed by thermal denaturation measurements. Differences in the standard state free energy of duplex formation between R5a labeled and wild type DNA duplexes (  Gº 37ºC ) were determined to be 0.2 – 0.7 kcal/mol (Table 4.1). At every site, differences between R p -R5a and S p -R5a  Gº 37ºC values fall in the 0 - 0.2 kcal/mol range, which is not significant compared to the error of  Gº 37ºC (  0.2 kcal/mol). These results are consistent with previous studies of R5a attached to mixed-diastereomers (7), and confirm that both R p -R5a and S p -R5a can be used to probe local environment in a DNA duplex without significantly affecting its native B-form conformation. 3.2. R5a reports different R p and S p spectra at a given DNA site Figure 4.3 shows X-band EPR spectra of R p -R5a and S p -R5a at five sites of the CS duplex obtained in the presence of 34% (w/w) sucrose at 5 C (see section 2.4). At each site, the R p -R5a spectrum is distinct from that of the S p -R5a. The largest R p and S p Labeled position  G° 37°C (a) (kcal/mol) CS2 mix (b) 0.2 CS2_R p 0.3 CS2_S p 0.2 CS9 mix (b) 0.6 CS9_R p 0.7 CS9_S p 0.5 CS12 mix (b) 0.2 CS12_R p 0.3 CS12_S p 0.1 CS14 mix (b) 0.3 CS14_R p 0.3 CS14_S p 0.3 (a)  G° 37°C = G° 37°C (unlabeled) - G° 37°C (labeled). (b) Data were previously obtained and reported (7). Table 4.1: Free energy parameters of duplex formation for R5a labeled CS duplexes. 102 spectral differences are found at CS2 and CS14 (Figure 4.3). Unlike CS2_S p _R5a, CS2_R p _R5a gives a multi-component spectrum, with a clearly visible bump at the low- field region (Figure 4.3, red arrow). This extra component indicates the presence of low- mobility nitroxide sub-population(s), which may originate from site-specific interaction(s) between R5a and DNA (see section 4.1). Affected by the immobilized sub- population(s), the CS2_R p _R5a lineshape shows amplitude reduction and linewidth increase in comparison with that of CS2_S p _R5a, suggesting overall lower nitroxide mobility at the CS2_R p diastereomer. Similar multiple-component features can be observed in CS14_R p _R5a, although the spectrum has a more dominant high-mobility nitroxide population as compared to CS2_R p _R5a (Figure 4.3). Similarly, the other three sites (CS7, CS9, and CS12) reveal clear differences between R p and S p spectra, though none of them show multiple spectral components. For example, the R p spectra have splittings in low- and high-field peaks (Figure 4.3, black arrows), which is a characteristic feature arisen from incomplete averaging of the nitroxide hyperfine magnetic tensor. This is a signature of a nitroxide undergoing a restricted mode of motion. In the S p spectra, splittings are absent at CS9 and CS12, and are barely discernable at CS7 (Figure 4.3). This indicates reduced ordering of the nitroxide motions as compared to the respective R p sites. In addition, the amplitude of EPR lines is noticeably smaller in the S p spectra, particularly at CS9 and CS12, which might suggest a lower rate of S p -R5a rotational diffusion than that of R p -R5a. 103 Figure 4.3: R p -R5a and S p -R5a spectra measured at 5ºC. Experimental spectra are shown with solid lines, and the best-fit spectra obtained from simulations are shown with black dotted lines. For the R p spectra, black arrows show the resolved hyperfine extrema, and red arrows indicate the low-mobility spectral components. Experimental spectra were corrected for residual amount of a free nitroxide (< 3%). 104 To provide a quantitative description of R p -R5a and S p -R5a dynamics, the observed spectra were simulated using the Microscopic Order Macroscopic Disorder (MOMD) model, which describes the nitroxide motions within the macromolecular environment as an anisotropic rotational diffusion constricted by an ordering potential (15, 21). Eight out of the ten R5a spectra were successfully simulated using a single population approximation (see section 2.5), and the resulting best-fit spectra match well to the corresponding measured spectra (Figure 4.3). A full-list of fitting parameters is reported in Table 4.2. For CS2_R p _R5a and CS14_R p _R5a spectra, their multiple component nature renders it much more difficult to uniquely resolve the fitting parameters, and adequate fits have not been obtained. Table 4.2: Parameters for fitting experimental R5a spectra acquired at 5 C. ( 10 7 s -1 ) R   /R  c 20 S 20  (0) (G) r.m.s.d CS2_S p 5.19 0.04 0.480.01 1.31 0.02 0.2910.005 0.12 0.10 0.00855 CS7_R p 6.94 0.30 0.990.16 2.28 0.11 0.4910.019 1.23 0.08 0.00713 CS7_S p 5.92 0.10 0.650.05 1.79 0.02 0.3980.005 0.88 0.04 0.00680 CS7 6.42 0.20 0.630.05 1.95 0.05 0.4290.009 0.89 0.08 0.00887 CS9_R p 5.85 0.19 0.490.02 1.62 0.04 0.3600.009 0.61 0.25 0.00807 CS9_S p 4.91 0.09 0.530.03 1.36 0.03 0.3020.007 0.37 0.22 0.00867 CS9 5.37 0.18 0.440.02 1.55 0.05 0.3460.011 0.63 0.30 0.00752 CS12_R p 7.68 0.16 0.620.05 1.87 0.02 0.4140.004 0.90 0.03 0.00951 CS12_S p 5.83 0.14 0.580.02 1.52 0.04 0.3400.008 0.51 0.24 0.00913 CS12 6.85 0.25 0.670.09 1.85 0.05 0.4080.011 0.93 0.07 0.00961 CS14_S p 5.95 0.12 0.330.02 1.22 0.02 0.2700.005 0.35 0.20 0.00925 Simulations yield two key parameters in describing R5a motions: (i) , which describes the rate of nitroxide rotational motions; and (ii) S 20 , which provides a measure 105 of local ordering. Figure 4.4A compares and S 20 between R p -R5a and S p -R5a at CS7, CS9 and CS12, where all diastereomer spectra have been simulated. The plots show that R p -R5a has a higher degree of ordering (larger S 20 ) and an increased diffusion rate as compared to the corresponding S p -R5a. We note that simulations treat nitroxide dynamics as a restricted Brownian rotational diffusion, and higher motional ordering does lead to an increase in diffusion rate due to reduction in the available diffusion space (22). Therefore, consistent with qualitative lineshape comparisons (i.e., linewidth, amplitude, and splitting), both S 20 and indicate that at each site R p -R5a undergoes more restrictive motions than S p -R5a. This likely originates from spatial constraints posted by the DNA (see section 4.1). A known issue in EPR simulations at a single frequency is degeneracy, in which different parameter sets may yield simulated spectra that fit comparably to a particular experimental spectrum (23). In studies reported here, correlation coefficients between variable fitting parameters were controlled to be < 0.9, which is deemed acceptable (24). Parameter uncertainties were assessed by the respective standard deviation values (Table 4.2), and were found comparable to those reported in the literature (23). These controls support the use of and S 20 to reveal relative differences in R5a dynamics between DNA sites. However, absolute values of and S 20 are likely systematically biased, as the rigid- limit g and A tensors were not accurately determined using a single X-band frequency, and details in the motional model (e.g., axially symmetric rotational diffusion, values of diffusion tilt angles) cannot be independently validated. Analyses based on the absolute 106 and S 20 values require further studies, such as those employing multiple EPR frequencies (25). Figure 4.4: Motional parameters obtained from fitting 5ºC spectra. (A) Pair-wise comparisons between R p -R5a and S p -R5a at the three sites lacking multiple components. (B) Comparisons between different sites for R p -R5a (right) and S p -R5a (left), with average and standard deviation values listed below the respective series. In summary, differences observed between diastereomer spectra at each of the five CS sites indicate that rotational dynamics of the R5a probe are differentially affected by R p and S p local DNA environment. 107 3.3. Site-specific variations in Rp-R5a and Sp-R5a spectra Variations between different DNA sites can be clearly observed within the respective series of R p -R5a or S p -R5a spectra (Figure 4.3). R p -R5a spectra can be separated into two groups, with group 1 including CS7, CS9 and CS12, and group 2 including CS2 and CS14. Spectra in group 1 show well-resolved splittings of the low- and high-field peaks (Figure 4.3, black arrows), suggesting a common mode of motion (i.e., a homogeneous nitroxide population undergoing restricted anisotropic diffusion). However, R5a mobility (i.e., rate and ordering) varies from site to site, as evident qualitatively from lineshape comparisons (Figure 4.3) and quantitatively from simulations (Figure 4.4B). Specifically, CS7_ R p _R5a shows best resolved splittings at low- and high-field regions and the largest S 20 , both reporting the highest degree of nitroxide ordering. CS12_R p _R5a has the largest line amplitudes and the narrowest center-line, and spectral simulations yield the biggest value, suggesting fast rotational dynamics of the probe. In group 2 (CS2 and CS14), spectra show no low- and high-field splitting, but clearly reveal shoulders in the low-field region (Figure 4.3, red arrows), indicating presence of heterogeneous nitroxide populations. In these spectra, their center- and low-field lines are unevenly broadened and diminished, rendering it difficult to directly compare R p -R5a mobility at CS2 and CS14 with the other R p sites. Unlike R p -R5a, all five S p -R5a spectra feature a single nitroxide population. Lineshape comparisons (Figure 4.3) and simulations (Figure 4.4B) report site-dependent variations in S p -R5a motions. For example, CS7_S p _R5a is the only S p spectrum that shows discernable splittings, and has the largest S 20 value, both reporting a high degree of 108 nitroxide ordering. CS14_S p _R5a has noticeably smaller width and larger amplitude of spectral lines, and simulations yield a combination of low ordering (small S 20 ) and fast rate (large ), indicating the highest overall nitroxide mobility within the S p series. A global comparison of all measured spectra also reveals that R p -R5a exhibits a larger degree of spectral variations between the DNA sites. Distributions of and S 20 values are broader for the R p -series as compared to that of the S p (Figure 4.4B). In addition, some R p spectra show multiple-component features, while all S p spectra are single-component (Figure 4.3). Together the data suggest that R p -R5a mobility varies more from one DNA site to the other than that of S p -R5a. 3.4. Temperature dependence of R p-R5a and S p-R5a spectra R5a spectra at sites CS2, CS7, and CS9 were obtained at other temperatures (-5C, 15C, and 25C) (Figure 4.5), and were found to vary in both stereo- and site-specific manner. The observed spectral variations generally agree with those obtained at 5 C. At all temperatures, a shoulder at the low-field peak appears in the CS2_R p spectrum (Figure 4.5A, marked by arrows), indicating presence of multiple nitroxide population(s). This multiple-component feature is absent in spectra of CS2_S p as well as CS7 and CS9. Due to the low-mobility nitroxide population(s), CS2_R p spectra have broader center lines than the corresponding CS2_S p spectra, thus appearing to report an overall lower R5a mobility. Furthermore, for CS7 and CS9, two common features are revealed by lineshape comparisons (Figure 4.5A) and spectral fittings (Figures 4.5B and 4.6; Table 4.3). First, as measured by S 20 , at a given DNA site R p -R5a undergoes more restricted motions as 109 compared to S p -R5a. Second, both R p - and S p -spectra suggest that site CS7 is characterized by more confined (larger S 20 ) and faster (higher ) nitroxide rotational diffusion than site CS9. Figure 4.5: Temperature dependence of R p -R5a and S p -R5a spectra. (A) Measured spectra with the Y- scale adjusted for each temperature set. Black arrows indicate the low-mobility spectral components. Spectra were corrected for residual amount of a free nitroxide (< 3%). (B) Plots of motional parameters obtained from fitting CS7 and CS9 spectra at different temperatures. 110 Figure 4.6: R p and S p spectra obtained at different temperatures overlaid to their best fits (dotted line). Y-scale was adjusted for each temperature set. Overall, the data indicate that the major trends observed using 5 C spectra are present at lower and higher temperatures. We note that temperature changes alter behaviors of R5a as well as that of the parent DNA molecule. Further analyses of temperature dependent data sets (e.g., S 20 and ) may aid in understanding the complex dynamical modes contributing to overall nitroxide motions. This will be pursued in the future. 111 Table 4.3: Parameters for fitting CS7 and CS9 diastereomer spectra at -5 , 15  and 25 C. ( 10 7 s -1 ) R   /R  c 20 S 20  (0) (G) r.m.s.d -5C CS7_R p 4.570.30 2.921.40 2.850.38 0.5800.059 1.210.52 0.01086 CS7_S p 4.120.15 1.610.83 2.050.20 0.4470.035 0.560.34 0.01008 CS9_R p 3.680.09 1.590.53 1.800.09 0.3990.018 0.290.27 0.01140 CS9_S p 3.77 0.01 1.180.51 1.510.11 0.3370.024 0.340.27 0.01129 15C CS7_R p 8.330.16 0.490.03 1.890.02 0.4180.003 1.090.01 0.01031 CS7_S p 7.460.10 0.390.02 1.560.01 0.3460.002 0.920.01 0.00893 CS9_R p 6.93 0.10 0.280.01 1.360.01 0.3040.003 0.260.16 0.01483 CS9_S p 6.010.06 0.300.01 1.130.01 0.2500.001 0.030.06 0.00889 25C a CS7_R p 8.470.27 0.2 1.490.02 0.3310.005 0.530.18 0.01701 CS7_S p 8.190.09 0.2 1.220.01 0.2710.002 0.260.11 0.02007 CS9_R p 7.770.04 0.2 1.060.01 0.2340.001 0.300.06 0.02311 CS9_S p 7.170.01 0.2 0.810.01 0.1780.001 0.010.02 0.01928 a Simulations had little sensitivity to R   /R  , which was fixed to 0.2 3.5. Linear combination of R p-R5a and S p-R5a spectra reproduces the mixed- diastereomer spectrum In previous studies, it has been assumed that a spectrum of R5a attached to mixtures of ps diastereomers is an average of the individual R p and S p spectra (7). To test this hypothesis, a composite spectrum at a given site was computed by adding the 5 C R p -R5a and S p -R5a spectra with coefficients corresponding to the ratio of R p /S p oligonucleotides observed in the mixtures (Figure 4.2B). At all five sites, the resulting composite spectra match well to the corresponding mixed-diastereomer spectra (Figure 4.7), reproducing all prominent spectral features such as peak splittings (e.g., CS7) and low-field bum ps (e.g., CS2 and 14). Given that the mixed-diastereomer spectra were obtained two to three years prior to those of the individual R p -R5a and S p -R5a, the degree of agreement between the 112 Figure 4.7: Comparisons between measured spectra of R5a attached to mixed-diastereomers (solid line) and those computed by weighted average of the individual R p -R5a and S p -R5a spectra (dashed line). The spectra were acquired at 5ºC. composite and measured spectra well exceeded our expectations, and unambiguously demonstrates that the mixed-diastereomer spectrum is a linear combination of the individual R p and S p spectra. Furthermore, the mixed-diastereomer spectra of CS7, CS9 and CS12 at 5 C were successfully simulated assuming that only one nitroxide population is present (Figure 4.8A and Table 4.2). The resulting fitting parameters fall in between those obtained from the individual diastereomers, and agree surprisingly well with the weighted averages of the corresponding R p and S p parameters (Figure 4.8B). We note that two diastereomers cannot interconvert without breaking and reforming a bridging P-O bond, therefore fast-exchange between the two species is unlikely at the EPR timescale. Simulation results indicate that mix-diastereomer spectra at these sites can be adequately represented using a homogenous nitroxide population, whose dynamic behavior is characterized by the weighted average of R p - R5a and S p -R5a. 113 Figure 4.8: Results of the mixed-diastereomer spectral simulations. Experimental data were obtained at 5 C. (A) Mixed-diastereomer spectra overlaid to their best-fits. (B) Comparisons of motional parameters obtained from fitting experimental spectra of mixed-diastereomers (blank), R p (gray) and S p (black). Weighted averages of R p and S p parameters (“average”, shown in striped) are also included. 4. Chapter 4 Discussion Spectral variations reported here clearly demonstrate stereo- and site-dependent coupling between R5a rotational motions and local DNA environment. Neither R p -R5a nor S p -R5a severely perturbs the native conformation of the parent DNA molecule, 114 therefore R5a can be used to probe DNA local structure and dynamics in a stereo-specific fashion. In the following text we first examine how DNA local structure may differentially affect R p -R5a and S p -R5a, giving rise to observed spectral variations. We then analyze sensitivity of R p -R5a and S p -R5a to local DNA motions, and finally discuss advantages and limitations of using mixed vs. pure diastereomers in studying DNA. 4.1. Modulations of R p-R5a and S p-R5a by DNA local structure In previous studies of R5a attached to mixed-diastereomers, the observed site-specific spectral variations were analyzed in terms of two modes of coupling between DNA local environment and nitroxide dynamics (7). First, DNA three-dimensional structure may restrain, in a site-specific manner, internal motions of R5a, which are defined as torsional rotations about bonds connecting the pyrroline ring to the phosphate of the DNA backbone (Figure 4.1A). Second, site-specific dynamical modes of DNA can be physically transmitted to the nitroxide via the motions of the phosphorus atom. Analyses presented below suggest that constraints on R5a internal motions, which arise primarily from DNA-defined allowable rotamer space and site-specific DNA-nitroxide interactions, may account for spectral variations observed between R p -R5a and S p -R5a at a given site. A common feature present at all sites under the temperatures studied is that at a given site rotational diffusion of R p -R5a is always more restricted than that of S p -R5a (Figure 4.3 and Figure 4.4). Molecular modeling clearly shows that an R p diastereomer directs the nitroxide pyrroline ring towards the major groove of the DNA helix, while an S p diastereomer directs it away from the helix and into the solvent (Figure 4.1C). In a 115 previous study (7), we have used a modeling tool (NASNOX) to generate ensembles of sterically allowable R p -R5a and S p -R5a rotamers at a given site of the CS duplex and estimated the allowable rotamer space with an S parameter, which was computed based on the angular distribution of the nitroxide N-O bond vectors (see Table 3.2 in Chapter 3). It was reported that at every CS site studied, R p -R5a has a larger S parameter than that of S p -R5a (<S> being 0.538 ± 0.022 for R p and 0.496 ± 0.001 for S p ), indicating smaller directional variations of the N-O vector and thus more restricted sterically allowable rotamer space for R p -R5a. This is in agreement with the higher degree of motional ordering (larger S 20 ) reported by R p -R5a at each DNA site (Figure 4.4). We also note that R p sites show an apparently broader distribution of S values (7), indicating larger variability in the allowable rotamer space for R p -R5a. This agrees with the broader distributions of S 20 and in the R p -series (Figure 4.4B). In addition to macromolecule imposed structural constrains, nitroxide internal motions may be affected by site-specific DNA/nitroxide interactions. It has been speculated that R p -R5a is more susceptible to these interactions, as it is located closer to the DNA (7, 12). This is now strongly supported by the observation that immobilized nitroxide subpopulations, a signature of nitroxide-DNA interactions, are observed exclusively in CS2_R p _R5a and CS14_R p _R5a spectra, but are absent in the corresponding S p -R5a spectra (Figure 4.3). Modeling studies suggest that at CS2, selected R p -R5a rotamers may interact with the methyl group of the neighboring T2 (Figures 4.9A and 4.9B), and similar interactions are also feasible at CS14. These interactions, however, are not likely for the corresponding S p -R5a, as the S p diastereomer directs the nitroxide 116 ring away from the DNA surface (Figures 4.9C and 4.9D). Interestingly, at CS9, which has a neighboring T9, an immobilized spectral component is not observed, indicating an absence of R5a/T-methyl interactions. Further studies are needed to confirm such R5a/T- methyl interactions and to explore their dependence on location (duplex interior vs. terminus) and/or surrounding nucleotide sequence. Figure 4.9: Spatial orientations of R p and S p nitroxides at CS2. Molecular models were generated based on the NMR structure using WebLab ViewerPro 3.7 (Molecular Simulations, Inc.). During modeling, torsion angles t2 and t3 were set to fixed values (t2=180  and t3=+100  or -100 ) based on MD simulations (data not shown), while t1 was varied to avoid any steric collisions between R5a and DNA. For Rp-R5a (panels (A) and (B)), the particular rotamers shown give the closest distance (indicated by the dotted line) between C5-methyl of T2 and functional groups of the R5a pyrroline ring, C4-Br (panel (A)) or C2-methyls (panel (B)). For S p -R5a (panels (C) and (D)), none of these pyrroline moieties can be positioned closer than 9Å to C5-methyl of T2, and rotamers with t1 arbitrary set to 60  are shown. 117 Taken together, R p -R5a is positioned toward the DNA major groove, its motions are more constrained and sensitive to site-dependent variations in the local structure. Consequently, R p -R5a is more adapted to report site-specific structural features, and may be valuable in probing subtle DNA conformational changes, such as those induced by ligand binding or changes of salt and solvent. 4.2. Modulation of R p-R5a and S p-R5a by DNA local motions Analyses above suggest that S p -R5a is not prone to site-specific DNA-nitroxide contacts and experiences mostly invariant rotamer space from one DNA site to another, variations in local DNA motions are therefore expected to be the main factor giving rise to S p -R5a spectral differences. Previously we have suggested that R5a mobility reports DNA flexibility that may correlate with thermodynamic stability determined by location (duplex interior vs. terminus) and flanking sequence of the labeling site (7). Within the S p -R5a series (Figure 4.3 and Figure 4.4), nitroxide rotational diffusion at CS7 is the most restricted, consistent with the expected low DNA flexibility (i.e., local DNA motion) at a site located at duplex center and flanked by a thermodynamically stable - GC- di-nucleotide (26, 27). Likewise, the highest S p -R5a mobility observed at CS14 correlates with an increased DNA flexibility presumed at a terminal site flanked by a less stable -CT- di-nucleotide. While stability of the nearest-neighbor is sufficient to account for S p -R5a data at CS7 and CS14, sequence beyond the flanking dinucleotide may also affect DNA flexibility and R5a dynamics. The two 5‟ terminal sites, CS14 and CS2, which share the same 118 trinucleotide neighboring sequence (5‟-CTA-3‟, Figure 4.1B), feature distinct S p -R5a spectra (Fig. 3). Nitroxide mobility at CS14 is clearly higher than that at CS2 (Figure 4.4B), suggesting different DNA backbone flexibility at these two sites. CS2 and CS14 are also different in their R p spectra (Figure 4.3), indicating subtle differences in the DNA groove structure. This suggests that R5a probes can report variations of local DNA structure (R p effect) and dynamics (S p effect) induced by flanking sequence(s) beyond the nearest neighbor. Previous studies of mixed diastereomers did reveal different R5a spectra at CS14 and CS2, but a detailed analysis was hampered by the multiple- component features (7). Resolving the individual R p and S p spectra clearly yields more information on local DNA environment differences at CS2 and CS14. We also note that the 3‟ terminal site CS12 features higher S 20 than the duplex interior site CS9 (Figure 4.4), indicating more restricted nitroxide motions at CS12. This is consistent with a previous observation that nitroxide rotational motions at the duplex termini may not always be less restricted as compared to duplex interior (7). CS12 and CS9 differ in both position and sequence (Figure 4.1B), which may collectively contribute to DNA thermodynamic stability in a complex fashion. Overall, while certain correlations between S p -R5a motions and DNA thermodynamic stability can be discerned from current data, an expanded database is needed for a detailed understanding of correlations between nitroxide mobility (e.g., S 20 and ) and DNA motional behaviors. As compared to S p -R5a, R p -R5a is expected to sense similar mode(s) of DNA local motions, as both use three single bonds to connect the pyrroline ring to the DNA backbone. In our studies, R p -R5a and S p -R5a report a consistent trend in nitroxide 119 ordering (S 20 (CS7) > S 20 (CS12) > S 20 (CS9); Figure 4.4B), but deviate in one of the two pair-wise comparisons of diffusion rate (R p : (CS12) > (CS7); S p : (CS12) = (CS7); Figure 4.4B). We believe this deviation arises from differences in nitroxide internal motions, which may vary from site to site for R p -R5a but are presumably constant for the entire S p -series. Studies are underway to investigate contributions of nitroxide internal motions to R p and S p spectra. Taken together, local DNA motions are likely to be the main factor contributing to variations in the S p spectra, rendering S p -R5a better suited for probing DNA local flexibility. 4.3. Mixed vs. pure diastereomers: advantages and limitations in probing DNA environment Results presented here clearly demonstrated that at each site a mixed-diastereomer spectrum is a weighted sum of individual R p and S p spectra (Figure 4.7), and therefore report collectively DNA features present at both diastereomers. At CS2 and CS14, the mix-diastereomer spectra are able to report multiple nitroxide populations. At CS7, CS9 and CS12, simulations of mixed-diastereomer spectra using a single R5a population revealed nitroxide motional parameters falling in between those of R p - and S p -R5a (Figure 4.8B). Consequently, one recovers a trend of relative nitroxide mobility ( : CS12≥CS7>CS9; S 20 : CS7>CS12>CS9) that follows those of the individual R p -R5a and S p -R5a. Overall, a mixed-diastereomer spectrum is a composite that provides information on the gross features of the DNA local environment: it reports the average amplitude and 120 rate of R5a motions at the labeling site, and reveals potential site-specific DNA-nitroxide interactions. Clearly, SDSL studies should start with the mixed-diastereomers. It will allow a fast and convenient scanning of a given oligonucleotide sequence and reveal collective features of DNA local environment at specific sites. It will also indentify sites, such as those showing multiple spectral components, where subsequent diastereopure analyses might be particularly informative. While interpreting a mixed-diastereomer spectrum may be more complicated, mixtures of R p and S p oligonucleotides are commercially available, and R5a can be easily placed at any nucleotide position within a target DNA and RNA molecule, including those of high molecular weight (9). On the other hand, preparation of diastereopure ps oligonucleotides remains very challenging, and may not be feasible for large nucleic acid molecules (> 50 nucleotides). Here diastereomer separation was achieved at five sites of the CS DNA, but was not successful at a number of other positions. This highlights a long-standing practical issue in obtaining diastereopure ps oligonucleotides. Although there are a number of reports since early 90s on preparation of diastereopure ps oligonucleotides (28-30), they were limited to small scale works, and often required a combination of diastereomer separation and/or multistep reactions. In addition, the success of diastereomer separation is rather unpredictable (17, 18), as it is strongly influenced by many factors, including oligonucleotide size, nucleotide sequence, and location of modification. As we demonstrated here the feasibility of probing DNA in a stereo-specific manner, further developments are needed to provide an efficient and facile method of diastereomer separation for a broad range of oligonucleotide systems. 121 5. Chapter 4 Conclusions Data reported here established that R p -R5a and S p -R5a, when separated, provide site- and stereo-specific information at the single nucleotide level, thus serving as unique spectroscopic probes for investigating stereomeric local environment in DNA. Furthermore, a mixed-diastereomer spectrum is shown to be a weighted average of the individual R p -R5a and S p -R5a spectra, and provides information on the gross features of the DNA local environment. While work reported here focused on lineshape analyses of R p -R5a and S p -R5a attached to a B-form DNA, future explorations of other nucleic acid structures (e.g., A-form duplexes) and EPR measurements (e.g., relaxation, inter- nitroxide distance) should be fruitful. A major obstacle remains to be a facile preparation of diastereopure ps modified oligonucleotides independent of their length, sequence and modification position. Further development in this area would greatly benefit nucleic acids studies using R5a as well as other spectroscopic probes. 122 6. Chapter 4 References 1. Altenbach, C., Flitsch, S. L., Khorana, H. G., and Hubbell, W. L. (1989) Structural studies on transmembrane proteins. 2. Spin labeling of bacteriorhodo psin mutants at unique cysteines, Biochemistry 28, 7806-7812. 2. Fanucci, G. E., and Cafiso, D. S. (2006) Recent Advances and applications of site-directed spin labeling, Curr Opin Struct Biol 16, 644-653. 3. Sowa, G. Z., and Qin, P. Z. (2008) Site-directed spin labeling studies on nucleic acid structure and dynamics, in Prog Nucleic Acids Res Mol Biol, pp 147-197. 4. Zhang, X., Cekan, P., Sigurdsson, S. T., and Qin, P. Z. (2009) Studying RNA using site-directed spin-labeling and continuous-wave electron paramagnetic resonance spectroscopy, Method Enzymol 469, 303-328. 5. Qin, P. Z., Butcher, S. E., Feigon, J., and Hubbell, W. L. (2001) Quantitative analysis of the GAAA tetraloop/receptor interaction in solution: A site-directed spin labeling study, Biochemistry 40, 6929-6936. 6. Qin, P. Z., Haworth, I. S., Cai, Q., Kusnetzow, A. K., Grant, G. P. G., Price, E. A., Sowa, G. Z., Popova, A., Herreros, B., and He, H. 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(2007) Nanometer distance measurements in RNA using site-directed spin labeling, Biophys J 93, 2110-2117. 11. Grant, G. P. G., Popova, A., and Qin, P. Z. (2008) Diastereomer characterizations of nitroxide-labeled nucleic acids, Biochem Biophys Res Commun 371, 451-455. 123 12. Price, E. A., Sutch, B. T., Cai, Q., Qin, P. Z., and Haworth, I. S. (2007) Computation of nitroxide-nitroxide distances for spin-labeled DNA duplexes, Biopolymers 87, 40-50. 13. Leporc, S., Mauffret, O., Tevanian, G., Lescot, E., Monnot, M., and Fermandjian, S. (1999) An NMR and molecular modeling analysis of d(CTACTGCTTTAG)· d(CTAAAGCAGTAG) reveals that the particular behavior of TpA ste ps is related to edge-to-edge contacts of their base-pairs in the major groove, Nucleic Acids Res 27, 4759-4767. 14. Qin, P. Z., Jennifer, I., and Oki, A. (2006) A model system for investigating lineshape/structure correlations in RNA site-directed spin labeling, Biochem Biophys Res Commun 343, 117-124. 15. Earle, K. A., and Budil, D. E. (2006) Calculating slow-motion ESR spectra of spin-labeled polymers, in Advanced ESR Methods in Polymer Reaserch (Schlick, S., Ed.), pp 53-83, John Wiley and Sons, New York. 16. Kanehara, H., Mizuguchi, M., and Makino, K. (1996) Isolation of oligodeoxynucleoside phosphorothioate diastereomers by the combination of DEAE ion-exchange and reversed-phase chromatography, Nucleosides Nucleotides 15, 399-406. 17. Thorogood, H., Grasby, J. A., and Connolly, B. A. (1996) Influence of the phosphate backbone on the recognition and hydrolysis of DNA by the EcoRV restriction endonuclease, J Biol Chem 271, 8855-8862. 18. Subach, F. B., Miuller, S., Tashlitskiĭ, V. N., PIatrauskene, O. V., and Gromova, E. S. (2003) [Preparation of DNA-duplexes, containing internucleotide thiophosphate grou ps in various positions of the recognition site for the EcoRII restriction endonuclease] [Article in Russian], Bioorg Khim. 29, 623-631. 19. Wilk, A., and Stec, W. (1995) Analysis of oligo(deoxynucleoside phosphorothioate)s and their diastereomeric composition, Nucleic Acids Res 23, 530-534. 20. Cheruvallath, Z. S., Sasmor, H., Cole, D. L., and Ravikumar, V. T. (2000) Influence of diastereomeric ratios of deoxyribonucleoside phosphoramidites on the synthesis of phosphorothioate oligonucleotides, Nucleosides Nucleotides Nucleic Acids 19, 533-543. 21. Meirovitch, E., Nayeem, A., and Freed, J. H. (1984) Analysis of protein-lipid interactions based on model simulations of electron spin resonance spectra, J Phys Chem B 88, 3454-3465. 124 22. Martínez, M. C., and García de la Torre, J. (1987) Brownian dynamics simulation of restricted rotational diffusion, Biophys J 52, 303-310. 23. Columbus, L., Kalai, T., Jeko, J., Hideg, K., and Hubbell, W. L. (2001) Molecular motion of spin-labeled side chains in a-helices: analysis by variation of side chain structure, Biochemistry 40, 3828-3846. 24. Budil, D. E., Lee, S., Saxena, S., and Freed, J. H. (1996) Nonlinear-least-squares analysis of slow-motion EPR spectra in one and two dimensions using a modified Levenberg-Marquardt algorithm, J Mag Res A 120, 155-189. 25. Zhang, Z., Fleissner, M. R., Tipikin, D. S., Liang, Z., Moscicki, J. K., Earle, K. A., Hubbell, W. L., and Freed, J. H. (2010) Multifrequency Electron Spin Resonance study of the dynamics of spin labeled T4 lysozyme, J Phys Chem B 114, 5503-5521. 26. SantaLucia, J. (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics, Proc Nat Acad Sci 95, 1460-1465. 27. Florian, J., Sponer, J., and Warshel, A. (1999) Thermodynamic parameters for stacking and hydrogen bonding of nucleic acid bases in aqueous solution: Ab Initio/Langevin Dipoles study, J Phys Chem B 103, 884-892. 28. Fidanza, J. A., Ozaki, H., and McLaughlin, L. W. (1992) Site-specific labeling of DNA sequences containing phosphorothioate diesters, J Am Chem Soc 114, 5509- 5517. 29. Nawrot, B., Rebowska, B., Cieslinska, K., and Stec, W. J. (2005) New approach to the synthesis of oligodeoxyribonucleotides modified with phosphorothioates of predetermined sense of P-chirality, Tetrahedron Letters 46, 6641-6644. 30. Oka, N., Yamamoto, M., Sato, T., and Wada, T. (2008) Solid-Phase synthesis of stereoregular oodeoxyribonucleoside phosphorothioates using bicyclic oxazaphospholidine derivatives as monomer units, J Am Chem Soc 130, 16031- 16037. 125 Chapter 5 Nitroxide sensing DNA micro-environment: effect of a single nucleotide substitution 1. Chapter 5 Introduction Functionalization of a macromolecule with a nitroxide probe is a first step in Site Directed Spin Labeling (SDSL), an EPR-based technique that is commonly used to study structure, dynamics and functions of biological molecules. In particular, continuous-wave (cw) EPR spectra measured at X-band (~ 9.4 GHz) frequency are dictated by reorientational dynamics of a nitroxide probe in the nanosecond regime. As described in Chapter 1, nitroxide rotational motions are coupled to structural and dynamic features at the labeling site of a macromolecule (here referred as local environment). Importantly, mechanism of such coupling depends on the particular method of nitroxide attachment and determines sensitivity of cw-EPR spectra to secondary and tertiary structure, conformational changes, local and global motions of a parent macromolecule. Every labeling scheme used requires detailed understanding of nitroxide dynamic behavior, however, due to intrinsic complexity, modes of nitroxide/macromolecule coupling and their effect on EPR spectra often remain unclear. In SDSL, the most well understood probe up to date is a cysteine reactive MTSL nitroxide for protein studies, as its 126 structural and dynamic coupling to protein molecules was investigated in depth by experiments (EPR, X-ray) (1-4) and MD simulations (5-7). R5-series of nitroxides, introduced in Chapter 2 of this dissertation, have been developed for nucleic acids applications. In particular, R5 and R5a nitroxides within this series were proved to be useful in probing DNA local environment (8, 9), monitoring RNA/RNA interactions (10) , studying motions of an RNA element within a large folded ribozyme (11), and measuring nanometer distances (12, 13). These applications require better mechanistic understanding of coupling modes between DNA/RNA molecules of interest and R5/ R5a nitroxides. R5 and R5a are attached to either R p or S p diastereomer of a phosphorothioate (ps) that is chemically introduced at the specific nucleotide of a nucleic acid molecule. Using a model dodecameric DNA duplex (CS, Figure 5.1A), with a near canonical B-form structure, it was established that R5 and R5a nitroxides are coupled to DNA local structural and dynamic properties in a site- and stereo-specific manner (8, 9). As described in Chapters 4 of this dissertation, R p and S p nitroxides are structurally distinct, giving rise to different diastereomer spectra at the same DNA site. R p places the nitroxide toward the major grove, making it sensitive to variation in the DNA local structure. Whereas S p nitroxide is solvent-exposed and least affected by the structural factor, being a better reporter of local DNA motions. In comparison with S p , R p diastereomer imposes larger sterical constraints on the nitroxide rotational motions and, in some cases, favors site-specific DNA/nitroxide interactions. Although the origin of these interactions is not quite clear, they are believed 127 to be related to oligonucleotide sequence. In CS DNA, 5-methyl (5-CH 3 ) group of the thymine, located 3’ from the labeling site was suggested to interact with the nitroxide moiety (8, 9, 14). Previously reported MD simulation data proposed that interactions of an R p nitroxide attached to site 7 of the CS DNA with 5-CH 3 of nucleotide 8 (dT 8 ) (Figure 5.1A) alters rotational dynamics of the probe. Additionally, similar interactions may have resulted in a distinct population of the slow-mobility nitroxide, observed in multiple-component R p -R5a spectra at the 5’ terminal sites CS2 and CS14 (9). Figure 5.1: Nitroxide labeled CS oligonucleotides and diastereomer separation. (A) CS DNA constructs used in the study. (B) R p and S p nitroxide labeled DNA separated using AE HPLC. Expanding our understanding of the nitroxide rotational coupling to local features of the DNA environment, X-band cw-EPR measurements were used to explore how presence of 5-CH 3 adjacent to the labeling site affects rotational dynamics of R p and S p nitroxides. As a first step, R5 and R5a mobility was monitored at site 7 of the wild type 128 (CS T 8 ) and mutant CS duplex (CS U 8 ) ,where the deoxythymidine nucleotide 3’ to the labeling site (dT 8 ) was mutated to deoxyuridine (dU 8 ), replacing 5-CH 3 with 5-H (Figure 5.1A). Obtained spectra indicate that the single nucleotide mutation alters nanosecond dynamics of the R p -R5a, but produces no detectable changes for S p -R5a and R p /S p -R5 nitroxides. These results are in qualitative agreement with MD simulations data ((14) and Hatmal, Frushicheva, Haworth, Qin - unpublished). 2. Chapter 5 Materials and Methods 2.1. Materials and abbreviations Wild type and mutant CS deoxyoligonucleotides used in this study were commercially obtained from Integrated DNA Technologies (Coralville, IA). Nitroxide labeling reagents 3-methanesulfonyloxymethyl-2,2,5,5-tetramethyl-1-oxylpyrroline (R5 precursor) and 4-bromo-3-bromomethyl-2,2,5,5-tetramethyl-1-oxylpyrroline (R5a precursor) were kindly provided by Kálmán Hideg (University of Pécs, Hungary). In this work, the spin-labeled DNA and the corresponding EPR spectrum are designated using CS T 8 and CS U 8 abbreviations for the wild type and mutant DNA respectively, identity of a ps configuration (R p or S p ) and a nitroxide probe (R5 or R5a). For example, CS T 8 _R p _R5a and CS U 8 _R p _R5a indicate wild type and mutant CS DNA with R5a nitroxide attached to R p stereoisomer of nucleotide 7, respectively. 129 2.2. Nitroxide labeling and purification R5 and R5a labeling procedures were previously described (8, 15). Coupling of a ps- modified oligonucleotide with a nitroxide precursor was followed by separation of R p and S p diastereomers. For wild type and mutant CS DNA, R p and S p separation was carried out using an anion-exchange (AE) HPLC with gradient elution according to the published protocol (9). To obtain 2-3 nmole of a distereopure nitroxide labeled oligonucleotide, three rounds of AE HPLC separation were required with a single DNA load of ~7 nmole. The resulting R p and S p fractions were combined, desalted using a homemade G-25 Sephadex column, lyophilized and stored at -20ºC. Purity of the individual R p and S p oligonucleotides were determined to be more than 98% as controlled by an additional step of AE HPLC after completion. R p and S p configurations were assigned to chromatographic fractions according to the previous study (16). Concentrations of CS oligonucleotides were determined by UV absorption at 260 nm. 2.3. EPR sample preparation and measurements To prepare an EPR sample, a diastereopure nitroxide labeled oligonucleotide (~1 nmol) was incubated with 10% molar excess of the complementary strand in buffer 1 (100 mM NaCl, 50 mM Tris-HCl, pH = 7.5) overnight at room temperature. To remove residual amount of unattached nitroxide, the reaction mixture was diluted with buffer 1 up to 600 L volume and then concentrated to 7 L using 5 kDa Millipore (Bellerica, MA) centrifugal filters. Finally, an EPR sample (10-15 L) contained 20-40 M of concentrated DNA suspended in buffer 1, with sucrose (34 % w/w) added to reduce the 130 rate of DNA global tumbling. DNA samples were loaded into glass capillaries (1.0  1.2 mm, Vitrocom, Inc., Mountain Lakes, NJ), sealed on one end. cw-EPR spectra were collected at ~9.34 GHz microwave frequency on a Bruker EMX spectrometer with a high sensitivity cavity (ER 4119HS, Bruker Biospin, Inc., Billerica, MA). Acquisition parameters were 2 mW incident microwave power and 1-2 G field modulation amplitude at 100 kHz, which was optimized to avoid spectral distortion. Temperature during EPR measurements was maintained using a liquid nitrogen temperature controller. All EPR spectra were baseline corrected and normalized to the same area using software provided by Hubbell group. When required, EPR spectra were corrected for residual amount of a free tumbling nitroxide (<3%) by manual subtraction. 2.4. EPR spectra fitting Experimentally obtained R5a spectra were simulated using EPRLL program, based on Microscopic Order Macroscopic Disorder (MOMD) model (17), which describes motions of a nitroxide attached to a macromolecule as an anisotropic rotational diffusion constricted by an ordering potential. Details of the spectral fitting procedure are specified in the previous work (9). Briefly, A and g magnetic tensor parameters (g xx =2.0083, g yy =2.0051, g zz =2.0022 A xx =6.9, A yy =5.7, and A zz =35,) were used as input values and kept constant during the fitting procedure. Diffusion tilt angle (  D =35) was fixed. To obtain an adequate fit to an experimental spectrum the following parameters were varied: rotational diffusion tensor components and N ( and N=R  / /R  , where R   and R  are the rate constants for rotations parallel and perpendicular to the nitroxide 131 principal diffusion axis); the coefficient of an ordering potential c 20 ( , where  is an instantaneous angle between the director and the nitroxide diffusion axes); and Gaussian inhomogeneous broadening factor  (0) . Each spectrum was repeatedly fitted using SIMPLEX minimization algorithm and an average and standard deviation of each fitting parameter were calculated as described (9). 3. Chapter 5 Results R5 and R5a nitroxides were attached to site CS7 of a wild type and mutant DNA, with nucleotide 8 being dT 8 and dU 8 respectively (Figure 5.1A). This mutation doesn’t perturb the canonical hydrogen bonding pattern with adenine, but eliminates 5-methyl group, allowing effect of 5-CH 3 on the nitroxide spectra to be studied directly. Successful separation of R p and S p isomers for both R5 and R5a labeled CS oligonucleotides (Figure 5.1B), allowed the results of base mutation to be monitored stereospecifically. Figures 5.2 and 5.3 show X-band EPR spectra obtained at 25 C using R5 and R5a nitroxides respectively. For R5, the corresponding R p and S p spectra are identical between the wild type and mutant DNA. For R5a, dT 8 to dU 8 substitution has no effect on S p -R5a, but induce a noticeable increase in the amplitude of R p -R5a spectral lines. This suggests higher mobility of R p -R5a nitroxide upon 5-CH 3 to 5-H substitution. R5a rotational dynamics was subsequently analyzed using spectral simulations. Experimental spectra of R5a at various temperatures were successfully simulated using MOMD model assuming presence of a single nitroxide population. At all the 132 Figure 5.2: Comparisons between cw-EPR spectra obtained for R5 labeled wild type and mutant DNA duplexes. Measurements were done at 25 C. temperatures studied, the resulting best-fit spectra match well to the corresponding measured spectra (Figures 5.4A and 5.5), with a full list of simulation parameters provided in Table 5.1. Spectral simulations yield two key parameters which describe R5a motions: the rate of nitroxide rotational diffusion ( ), and the degree of nitroxide 133 ordering (c 20 ). At 25C, dT 8 to dU 8 mutation has negligible effect on and c 20 values for S p -R5a. Figure 5.3: Comparisons between cw-EPR spectra obtained for R5a labeled wild type and mutant DNA duplexes. Measurements were done at 25 C. For R p -R5a, mutation is found to increase the diffusion rate (larger ) and reduce the degree of nitroxide ordering (smaller c 20 ) above the parameter errors (Figures 5.4B). Similar differences in the rate ( ), and amplitude (c 20 ) of R p -R5a motions between the wild type and mutant DNA are observed at 5 and 15C (Figures 5.5). These results are 134 in complete agreement with conclusions drawn from direct comparisons of experimental spectra (Figures 5.2 and 5.3). Figure 5.4: Simulations results for R5a spectra measured at 25 C using wild type and mutant DNA. (A) Overlays of experimental (solid line) and simulated (dotted line) R5a spectra. (B) Motional parameters obtained from R5a spectral fittings. 135 Figure 5.5: Simulations results for R p -R5a spectra experimentally obtained at (A) 5 C and (B) 15 C. 136 Table 5.1: Parameters obtained from simulations of R5a spectra measured at different temperatures. ( 10 7 s -1 ) R   /R  c 20  (0) (G) r.m.s.d 5C CS T 8 _R p 6.940.30 0.990.16 2.280.11 1.23 0.08 0.00713 CS U 8 _R p 7.440.17 0.760.06 2.090.04 1.10 0.04 0.00807 15C CS T 8 _R p 8.330.16 0.490.03 1.890.02 1.090.01 0.01031 CS U 8 _R p 8.760.19 0.390.03 1.740.02 0.950.03 0.01130 25C a CS T 8 _R p 8.470.27 0.2 1.490.02 0.530.18 0.01701 CS U 8 _R p 9.290.15 0.2 1.340.01 0.340.08 0.02564 CS T 8 _S p 8.190.09 0.2 1.220.01 0.260.11 0.02007 CS U 8 _S p 8.330.13 0.2 1.230.01 0.350.10 0.01725 a Simulations had little sensitivity to R   /R  , which was fixed to 0.2 In summary, cw-EPR data reveal that S p -R5 and S p -R5a nitroxides are not influenced by 5-CH 3 of the thymine nucleotide, located 3’ from the labeling site. Whereas dT 8 to dU 8 mutation has no detectable effect on rotational dynamics of R p -R5, an overall increase in R p -R5a mobility is observed. This finding emphasizes sensitivity difference to local DNA environment between R5 and R5a probes. 4. Chapter 5 Discussion As discussed in Chapter 4 of this work, ps stereochemistry is an important determinant of nitroxide rotational dynamics. It was speculated that S p nitroxide is solvent exposed and is not susceptible to establish site-specific contacts with DNA (9). 5- CH 3 has no detectable effect on S p spectra, suggesting that dT 8 to dU 8 mutation induces no changes in local flexibility of the DNA backbone. Considering that DNA motions equally contribute to S p and R p spectra, most likely, 5-CH 3 modulates R p -R5a rotational dynamics via internal motional mode (i.e., rotational oscillations of the DNA-nitroxide 137 linker). We note that nitroxide internal motions can be modulated by a macromolecule using structural constrains and/or site-specific interactions. If 5-CH 3 constrains R p -R5a motions, dT 8 to dU 8 mutation should result in a lowering of nitroxide order and a decrease of the diffusion rate usually associated with release of steric constrains (9, 18). In case of 5-CH3/ R p -R5a interactions, reduction in R p -R5a ordering should be accompanied by an increase in motional rate. Although current data may not rule out a possibility that 5-CH 3 of dT 8 simultaneously poses a structural barrier to R p -R5a diffusion and interacts with the probe, the larger value obtained in the mutant suggests that non- specific 5-CH 3 /nitroxide contacts may be an important determinant of the observed spectroscopic effect. The data reported here indicate that rotational dynamics of R p -R5 are not affected by DNA mutation. MD simulations reported in our laboratory show that torsional oscillations around R5 linker are significantly less restricted as compared to R5a, due to absence of a sterically bulky 4-Br substituent (Hatmal, Frushicheva, Haworth, Qin – unpublished). According to these data, 4-Br reduces amount of the attainable nitroxide rotamers, making R p -R5a presumably more sensitive to structural variations in the DNA major grove. Most likely, dT 8 interacting R p -R5 rotamers are either weakly populated compared to R p -R5a or sterical constrains due to 5-CH 3 have negligible effect on an already large ensemble of allowable R p -R5 rotamers. Either of these effects may account for the lack in detectable spectral changes between CS T 8 _R p _R5 and CS U 8 _R p _R5. Modern computational approaches, such as MD simulations comprise a powerful tool for examining molecular behavior in atomic details, and may provide information 138 about different mechanisms of dynamic coupling between R5/ R5a nitroxides and DNA. However, direct correlations between experimental and computational results may not be always straightforward due to, for example, data convergence and force-field problems. Analyses of the recent MD simulations (Hatmal, Frushicheva, Haworth, Qin – unpublished) on CS T 8 and CS U 8 duplexes carrying single R5 or R5a at nucleotide 7, show that these data qualitatively capture experimentally observed dT 8 /dU 8 -dependent spectral changes. Namely, dT 8 to dU 8 mutation alters distribution of R p -R5a rotamers, and, within the error, has no effect on R p -R5a and S p -R5/R5a distributions. More quantitative analyses and further refinement of these data will help to understand how 5- CH 3 modulates R p -R5a dynamic behavior via structural restrains and/or site-specific interactions. Interactions of R p -R5a nitroxide with a thymine nucleotide located 3’ from the nitroxide position may not be limited to site CS7. Similar contacts with the thymine 5- CH 3 , have been proposed to immobilize part of the nitroxide population, causing distinct component/s observed in R p -R5a spectra at CS2 and CS14 (9). Unlike CS7, interactions at these 5’ terminal sites slow down an exchange between interacting (immobile) and non-interacting (mobile) nitroxide species, giving rise to clearly visible features in their X-band spectra. Obtaining R p -R5a spectra at larger EPR frequencies could be useful in studying DNA/nitroxide contacts. High-frequency spectra are particularly sensitive to faster nitroxide motions, and may help to ‘immobilize’ interacting with DNA R p -R5a rotamers, resulting in an additional spectral component/s which may not be resolved at the X-band. This can be pursued in the future using CS T 8 _R p _R5a and CS U 8 _R p _R5a. 139 5. Chapter 5 Conclusions Stereo-specific spectroscopic effect of a single thymine to uracil base substitution was demonstrated using X-band cw-EPR measurements. 5-methyl group of the thymine, located 3’ from the labeling site, modulates rotational motions of R p -R5a, but has no detectable effect on S p -R5a, S p -R5a and R p -R5 nitroxides attached to site CS7 of a canonical DNA duplex. While first correlations between these results and MD simulations data have been obtained, future studies combining experimental and theoretical approaches will help to gain more in-depth understanding of R5 and R5a dynamic coupling to sequence-dependent structural and dynamic DNA features. Further investigations of the proposed site-dependent DNA-nitroxide interactions will enable use of R p -R5a/R5 nitroxides to probe local conformational changes associated with ligand or protein binding, effects of salt and solvent in large biologically relevant DNA molecules. 140 6. Chapter 5 References 1. Fleissner, M., Cascio, D., and Hubbell, W. (2009) Structural origin of weakly ordered nitroxide motion in spin-labeled proteins., Protein Sci 18, 893-908. 2. Guo, Z., Cascio, D., Hideg, K., and Hubbell, W. (2008) Structural determinants of nitroxide motion in spin-labeled proteins: solvent-exposed sites in helix B of T4 lysozyme, Protein Sci 17, 228-239. 3. Langen, R., Oh, K., Cascio, D., and Hubbell, W. (2000) Crystal structures of spin labeled T4 lysozyme mutants: implications for the interpretation of EPR spectra in terms of structure, Biochemistry 39, 8396-8405. 4. Mchaourab, H., Lietzow, M., Hideg, K., and Hubbell, W. (1996) Motion of spin- labeled side chains in T4 lysozyme. Correlation with protein structure and dynamics, Biochemistry 35, 7692-7704. 5. Pistolesi, S., Ferro, E., Santucci, A., Basosi, R., Trabalzini, L., and Pogni, R. (2006) Molecular motion of spin labeled side chains in the C-terminal domain of RGL2 protein: a SDSL-EPR and MD study, Biophys Chem 123, 49-57. 6. Sezer, D., Freed, J., and Roux, B. (2008) Parametrization, molecular dynamics simulation, and calculation of electron spin resonance spectra of a nitroxide spin label on a polyalanine alpha-helix, J Phys Chem B 112, 5755-5767. 7. Tombolato, F., Ferrarini, A., and Freed, J. (2006) Modeling the effects of structure and dynamics of the nitroxide side chain on the ESR spectra of spin- labeled proteins, J Phys Chem B 110, 26260-26271. 8. Popova, A. M., Kalai, T., Hideg, K., and Qin, P. Z. (2009) Site-specific DNA structural and dynamic features revealed by nucleotide-independent nitroxide probes, Biochemistry 48, 8540-8550. 9. Popova, A. M., and Qin, P. Z. (2010) A nucleotide-independent nitroxide probe reports on site-specific stereomeric environment in DNA, Biophys J 99, 2180- 2189. 10. Qin, P. Z., Butcher, S. E., Feigon, J., and Hubbell, W. L. (2001) Quantitative analysis of the isolated GAAA tetraloop/receptor interaction in solution: a site- directed spin labeling study, Biochemistry 40, 6929-6936. 11. Grant, G. P., Boyd, N., Herschlag, D., and Qin, P. Z. (2009) Motions of the substrate recognition duplex in a group I intron assessed by site-directed spin labeling, J Am Chem Soc 131, 3136-3137. 141 12. Cai, Q., Kusnetzow, A. K., Hubbell, W. L., Haworth, I. S., Gacho, G. P., Van Eps, N., Hideg, K., Chambers, E. J., and Qin, P. Z. (2006) Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence- independent nitroxide probe, Nucleic Acids Res 34, 4722-4730. 13. Cai, Q., Kusnetzow, A. K., Hideg, K., Price, E. A., Haworth, I. S., and Qin, P. Z. (2007) Nanometer distance measurements in RNA using site-directed spin labeling, Biophys J 93, 2110-2117. 14. Price, E. A., Sutch, B. T., Cai, Q., Qin, P. Z., and Haworth, I. S. (2007) Computation of nitroxide-nitroxide distances in spin-labeled DNA duplexes, Biopolymers 87, 40-50. 15. Qin, P. Z., Haworth, I. S., Cai, Q., Kusnetzow, A. K., Grant, G. P., Price, E. A., Sowa, G. Z., Popova, A., Herreros, B., and He, H. (2007) Measuring nanometer distances in nucleic acids using a sequence-independent nitroxide probe, Nat Protoc 2, 2354-2365. 16. Grant, G. P. G., Popova, A., and Qin, P. Z. (2008) Diastereomer characterizations of nitroxide-labeled nucleic acids, Biochem Biophys Res Commun 371, 451-455. 17. Earle, K. A., and Budil, D. E. (2006) Calculating slow-motion ESR spectra of spin-labeled polymers, In Polymer Reasearch : Advanced ESR Methods (Schlick, S., Ed.), pp 53-83, John Wiley and Sons, New York. 18. Martinez, M. C., and Garcia de la Torre, J. (1987) Brownian dynamics simulation of restricted rotational diffusion, Biophys J 52, 303-310. 142 Chapter 6 Studying biologically relevant DNA with a nucleotide- independent nitroxide probe 1. Chapter 6 Introduction Physical properties of DNA, such as helix geometry, flexibility, water and ion structures, and electrostatics critically impact all aspects of biological functions involving gene expression, maintenance, and regulation. Sequence-dependent deviations from the ideal B-form behavior are prevalent, and play essential roles in DNA recognition by proteins and small molecules. However, current knowledge on sequence-dependent properties has not yet met our needs for understanding and manipulating biological functions, and it remains essential to characterize conformational and dynamic heterogeneity of free and bound DNA. Here, an EPR-based site-directed spin labeling (SDSL) methodology is applied to probe local sequence-dependent structural and dynamic properties in DNA duplexes under physiological conditions. In particular, cw-EPR spectra are sensitive to rotational dynamics of a nitroxide probe, linked to the specific site of a parent macromolecule (Chapter 1). Nitroxide mobility is modulated by secondary and tertiary structure, conformational changes, intermolecular interactions, and macromolecule motions, 143 allowing one to interrogate structural and dynamic behavior of a biological molecule (1, 2). Chapter 2 describes an R5-series of phosphorothioate (ps) reactive nitroxide probes for SDSL studies of nucleic acids. R5-nitroxides being covalently linked to the phosphodiester backbone are largely solvent-exposed and thus may be useful in monitoring localized motions in nucleic acid systems. Studies reported in chapters 4 and 5, investigate R5a, a 4-bromo substituted nitroxide in the R5-series, using a model B- form DNA duplex. It was concluded, that R5a sensitivity to DNA local structural and dynamic features (here referred as local environment) is coupled to a diastereomeric state of the phosphorothioate (R p or S p ) (3). While separation of R p and S p remains challenging, mixed-diastereomers enable efficient scanning of any oligonucleotide sequence. Mixtures of ps diastereomers can be easily utilized in revealing the gross features of the local environment and may help to identify the sites where subsequent analysis using pure diastereomers is informative. Here, R5a nitroxide scanning was coupled with X-band cw-EPR measurements to probe local environment in biologically relevant DNA. In particular, three systems were studied: one is a human bcl-2 DNA sequence, related to a chromosomal abnormality common in human cancer (4); other two DNA molecules contain a stretch of consecutive guanine (G) and cytosine (C) nucleotides (designated as G/C DNA) often found in genomic sequences and involved in DNA methylation. R5a was attached to several positions within these DNA molecules and range of spectral variability was analyzed. Importantly, structural and dynamic properties of the bcl-2 and G/C DNA sequences 144 were previously investigated using means of chemical probing or NMR spectroscopy (5, 6). Results of R5a scanning may potentially complement information obtained using these techniques, providing a valuable insight on sequence-dependent flexibility and structural heterogeneity in bcl-2 and G/C DNA. 2. Chapter 6 Materials and Methods All ps-modified deoxyoligonucleotides and non-modified complementary DNA were purchased from IDT (Coralville, IA). Phosphorothioate reactive precursors for R5a and R5 were provided by the laboratory of Kálmán Hideg (University of Pécs, Hungary). DNA spin-labeling and HPLC purification were carried out according to the published protocols (7, 8) An anion-exchange HPLC gradient, used to separate nitroxide labeled DNA, was adjusted according to the oligonucleotide size (8). All the data reported here were obtained using mixtures of ps diastereomers. Oligonucleotides containing regions of self-complementarity: G 3 C 3 , (GC) 3 and HhaI sequences (referred as G/C DNA here) were characterized using MALDI-TOF MS and native gel electrophoresis before and after nitroxide labeling. Details of MS experiments are described in Chapter 2. Formation of a regular duplex for G/C DNA was confirmed using a native polyacrylamide gel. First, complementary DNA strands were annealed in a 1:1 molar ratio, by heating at 95 C in 100 mM NaCl, 50 mM Tris-HCl (pH=7.5) for 2 minutes with subsequent incubation at room temperature (~ 2 hours). Electrophoretic mobilities of the annealed dsDNA (~ 5 L of 20 M) and of the corresponding ssDNA (~ 5 L of 40 M) were monitored using 20% PAGE. The gel and the running buffer 145 contained 100 mM NaCl and 0.1 M Tris-Borate (pH=7.5). Temperature of the electophoresis experiments was maintained using a 4 C circulating water bath. Single- and double-stranded 24-mer DNA were used as external standards of gel mobility. DNA bands were visualized with ethidium bromide staining. For cw-EPR measurements, a nitroxide labeled DNA strand (~0.3 nmole) and its complement (in 10% molar excess) were annealed in 100 mM NaCl, 50 mM Tris-HCl (pH=7.5) using a procedure described above. Each 10-15 L EPR sample contained 34% (w/w) sucrose. Bruker EMX spectrometer (Bruker BioSpin, Inc., Billerica, MA) was used to acquire X-band cw-EPR spectra as reported (7). Measurements were done at room temperature or using a liquid nitrogen temperature controller. All EPR spectra were processed using a standard baseline correction and normalization routine. 3. Chapter 6 Results and Discussion 3.1. Probing DNA local environment at bcl-2 major break point region This work focuses on probing structural and dynamic features in a DNA sequence within a human bcl-2 gene at chromosome 18. Bcl-2 is involved in a most-common chromosomal translocation in human cancer t(14;18), occurring in more than 95% of all follicular lymphomas (4). The t(14;18) translocation juxtaposes bcl-2 gene, located on chromosome 18 to the D and J intron regions of the immunoglobuline heavy chain (IgH) locus on chromosome 14 (Figure 6.1). Specific to B-cells, t(14;18) causes an overexpression of an anti-apoptotic protein bcl-2 and thereby lymphoma. While the mechanism of the double-strand DNA break at the bcl-2 gene is unclear, it was shown 146 that ~75% of all breaks occur in a small 150 bp region, designated as the major break point region (Mbr). Three hotspots (peaks 1 through 3, Figure 6.1) of breakage, approximately 15-20 bp in length have been identified (4). Based on chemical probing and other experiments, Lieber et al. proposed that DNA in the peak 1 and peak 3 regions adopts a non-B conformation (5, 9, 10). Presumably, the non-B DNA is recognized and cleaved by the RAG complex, which normally functions in the process of V(D)J recombination of IgH (4, 11). Overall, presence of alternative DNA structure/s in the bcl- 2 Mbr appears to be the basis for an abnormal double-strand break in chromosome 18, preceding t(14;18) translocation. Figure 6.1: A scheme of t(14;18) chromosomal translocation. 147 Previously, a DNA sequence within the bcl-2 Mbr has been probed using a bisulfite modification assay (9-11). Bisulfite converts unstacked cytosines to uracils, with a frequency of conversion being subsequently measured by DNA sequencing. Notably, peak 1 and peak 3 regions demonstrate abnormally high bisulfite reactivity (Figure 6.2) (11), indicating a link between chemical reactivity and chromosomal fragility. In addition, the data suggest that a reactivity pattern, for example the one observed at the peak 1 region (Figure 6.2), correlates with amount of consecutive cytosines (11). Figure 6.2: Bisulfite reactivity pattern at the bcl-2 major break point region (Mbr). The results were obtained for a 528-bp DNA by Raghavan et al. (11). Here, a DNA sequence near the Mbr peak 1 (red line) is shown. Black bars indicate cytosine bisulfite reactivity for the top and bottom strands. Each bar represents chemical conversion of the respective cytosine in one DNA molecule. Amount of DNA molecules analyzed is shown at the lower right corner for the top and bottom strands. A part of the DNA sequence in blue represents the bcl-2 44-bp construct used in EPR studies. 148 In spite of its use, the basis for bisulfite reactivity in a double-stranded DNA remains unclear. The original proposal was that frequent modifications are related to presence of non-B form structures (11). This included a triplex model, where in the peak 1 region the bottom strand is annealed to an up- or downstream duplex (9, 10). The top strand remains unpaired, becoming extremely vulnerable to bisulfite modification and enzymatic cleavage. In the most recent study, Lieber et al. suggested that bisulfite distinguishes DNA regions adopting an intermediate between A-form and B-form conformation (designated as A/B-form), characteristic for poly-C (i.e., containing stretches of consecutive cytosine nucleotides) sequences (5). Another hypothesis, which was not ruled out, is a presence of rapid melting and reannealing, or DNA breathing, in the regions of high bisulfite reactivity. Overall, bisulfite probing, being a technically simple and easily applicable approach, offers indirect information about structural and dynamic properties of a DNA molecule. EPR-based measurements are sensitive to structure and dynamics of a macromolecule at the labeling site and may help to complement the results of chemical probing experiments. In this study, R5a nitroxide was attached to several sites of the 44-nt long DNA, derived from the bcl-2 Mbr sequence (Figures 6.2 and 6.3). Nitroxide rotational dynamics have been monitored using X-band cw-EPR measurements at room temperature. The resulting R5a spectral lineshapes were divided into three groups (Figure 6.3). Spectra at sites 25, 40, 5’ and 24’ have visible splittings at the low- and high-field lines, suggesting a nitroxide undergoing restricted anisotropic diffusion. In a second group, bcl- 149 2_31 spectrum shows no resolved spectral splittings and features sharper low- and high- field lines, compared to spectra in the first group, overall indicating larger R5a mobility. The third group is comprised of a bcl-2_30’ spectrum which shows a clear bump at the shoulder of the low-field line. This spectral feature indicates presence of multiple nitroxide populations with higher and lower mobilities. In the previously described work (Chapters 3 and 4), low mobility components in R5a spectra are linked to interactions between the nitroxide moiety and a neighboring thymine located 3’ of the labeling site (3’-T) (3, 7). However, bcl-2_30’ lacks 3’-T and molecular origin of nitroxide immobilization should be further investigated. Figure 6.3: cw-EPR spectra of a single R5a attached to different sites of 44-nt bcl-2 DNA. Data were recorded at room temperature. Each star indicates a site of nitroxide modification. Figure 6.4 provides a more detailed comparison between the spectra obtained at sites bcl-2_31 and bcl-2_30’, located directly across the helix. The spectra were measured 150 using two nitroxides, R5 and R5a, at room temperature. Similar to R5a, bcl-2_30’_R5 features a multicomponent spectrum (Figure 6.4A), though here proportion of the immobilized component is substantially reduced. Higher internal flexibility of R5 nitroxide may account for this observation (see Chapter 5 for more discussion). Compared to bcl-2_30’, bcl-2_31 spectra report larger amplitude of the low-field lines (Figures 6.4A and 6.4B), with an absence of spectral splittings (Figure 6.4B). These suggest that site 31 is characterized by higher nitroxide mobility (R5 or R5a) than site 30’. Figure 6.4: Pairwise comparisons between R5 (A) and R5a (B) bcl-2 spectra, obtained at room temperature. Arrows indicate low-mobility spectral components. 151 In addition, bcl_2 spectra were obtained at conditions, which according to a biochemical characterization may stabilize formation of a triplex structure (100 mM NaCl, 50 mM Tris-HCl (pH=7.5), 10 mM MgCl 2 and 0.1 mM spermidine) (9, 10). Expected, that formation of a DNA triplex should affect global tumbling of the molecule as well as local nitroxide fluctuations, modulating the resulting EPR spectra. However, no spectral changes were reported by either R5a or R5 (data not shown). In summary, R5a and R5 results suggest that DNA structural and dynamic features vary between the different sites of bcl-2 Mbr. Future work may help to determine a molecular origin of the observed spectral variations. First, an effect of local and long- range base sequence on the obtained R5a spectra can be studied. Second, extending DNA duplex used here beyond the peak 1 region may help to further test a non-B DNA hypothesis. While EPR results obtained here may not be directly correlated with available bisulfate reactivity data, future studies will allow us to better define sensitivity of these techniques to DNA structure and dynamic behavior. In addition, inter-nitroxide distance measurement may help to obtain information about conformational state/s at bcl-2 Mbr. Distance measurements between a pair of R5 nitroxides were established by Cai et al. (8, 12) and can be used to probe distribution of duplex width along the bcl-2 sequence as well as its global structure. These experiments will allow testing different hypotheses regarding the nature of the non-B form structure (i.e., triplex model vs. A/B-form model, (5, 9)). Overall, cw-EPR and distance mapping may be effectively combined to probe unique structural and dynamic properties at bcl-2 Mbr and study the role of nucleotide sequence in chromosomal fragility. 152 3.2. Probing DNA local environment in GGGCCC and GCGCGC sequences Stretches of consecutive G, or alternating G and C nucleotides (here referred as G/C DNA) have been found in many important DNA, including telomeres, promoter and transcription factor binding sites, and restriction endonuclease sequences. Over the past two decades, evidences of unique physical properties of G/C DNA have been found. For example, molecules containing a stretch of three or more consecutive G (or C) nucleotides (i.e., G-tract) were crystallized in an A-form or an intermediate between A- and B-forms (13-15). Although conformational states of the G-tract containing DNA are not well characterized in solution, they tend to adopt an A-form structure under dehydration conditions easier than sequences of random nucleotide composition. Using gel electrophoresis and X-ray crystallography, it has been demonstrated that divalent ions induce bending of a helical axis in GGCC and GGGCCC containing oligonucleotides (16-18). Distinct electrostatic properties of a G-tract have been analyzed using electrostatic surface potential calculations and MD simulations (19, 20). Finally, NMR experiments measuring rates of imino proton exchange, suggest that G-C base pair in a G/C sequence has much shorter lifetime than an isolated G-C pair outside of a G/C region (21). G/C sequences can be often found in genomic DNA. In particular, major break point region (Mbr) of the human bcl-2 gene features islands of consecutive cytosines. These poly-C stretches are located near the hotspots of a chromosomal break (peak 1 and peak 3 regions), which is related to human cancer (see section 3.1 for details). It was suggested 153 that distinct structural and dynamic properties of G/C sequences may be linked to the observed chromosomal abnormality (5, 11). Bcl-2 Mbr was extensively studied using bisulfite modification assay (9-11). In the recent work, using model DNA duplexes, containing stretches of consecutive and alternating G and C nucleotides, bisulfite reactivity was correlated to the length of an uninterrupted poly-C sequence (Figure 6.5) (5). Using modeling and electrostatic calculations the authors concluded that the basis of high reactivity lies in structural and electrostatic properties of the G-tract. Work reported in this section aims to complement the results of bisulfite probing experiments, using cw-EPR measurements. Figure 6.5: Results of bisulfite probing for DNA duplexes, containing (A) GGGCCC and (B) GCGCGC sequences, as reported by Tsai et al. (5). Each circle represents cytosine conversion in one DNA molecule. Overall, 64 DNA molecules were analyzed. Data for the bottom DNA strands are not shown. 154 The first important step was to establish a correlation between the data obtained using bisulfite modification and R5a scanning experiments. Based on the work by Tsai et al. (5), two DNA constructs containing a patch of consecutive (G 3 C 3 , GGGCCC) or alternating ((GC) 3 , GCGCGC) G and C nucleotides were chosen for nitroxide functionalization (Figure 6.6A). Cytosines in the G 3 C 3 sequence are about five times more reactive toward bisulfite than cytosines in the (GC) 3 (Figure 6.5). One may expect that structural and dynamic origin of different chemical reactivity in G 3 C 3 and (GC) 3 will give rise to different R5a mobility. Figure 6.6: G/C DNA constructs and their characterization. (A) Two 24-bp DNA were used, G 3 C 3 , with consecutive, and (GC) 3 with alternating G and C nucleotides (in red). G 3 C 3 and (GC) 3 contain self- complementary sequence regions, indicated in purple. Black stars show sites of ps modification. (B) Successful formation of G 3 C 3 and (GC) 3 DNA heteroduplexes demonstrated using native PAGE. Results were obtained using ps-modified oligonucleotides. R5a labeling doesn’t interfere with duplex formation (data not shown). 155 24-nt long G 3 C 3 and (GC) 3 oligonucleotides contain 12-nt self-complementary regions (in purple), leading to formation of a homoduplex with retarded mobility on a native gel (Figure 6.6B, lanes 3 and 4). In spite of this, annealing with a complementary oligonucleotide yielded a thermodynamically more stable heteroduplex (Figure 6.6B, lanes 1 and 2). Conclusively, presence of self-complementary regions in G 3 C 3 and (GC) 3 DNA doesn’t interfere with formation of a regular heteroduplex structure. G 3 C 3 and (GC) 3 were R5a-labeled at the central -GC- dinucleotide (Figure 6.6A) and EPR measurements were carried out at various temperatures (Figure 6.7). The resulting spectra show little or no difference between R5a mobility at G 3 C 3 and (GC) 3 . The lineshapes obtained feature splittings at the low- and high-field lines and are similar to spectra observed in the bcl-2 studies (Figure 6.3, sites 25, 40, 5’ and 24’). Figure 6.7: Pairwise comparisons between R5a spectra of G 3 C 3 and (GC) 3 . 156 Presence of alternative DNA structures resolved by PAGE, such as homoduplexes and possibly tetramers (Figure 6.6), most likely don’t interfere with EPR results, since annealing of an R5a labeled G 3 C 3 strand with its complementary sequence in 1:3 molar ratio had an effect on distribution of the coexisting DNA species, but not on the observed EPR spectrum (data not shown). Unlike bisulfite probing, R5a reports identical DNA environment at the labeling site of G 3 C 3 and (GC) 3 sequences. Most likely, bisulfite reactivity and R5a mobility are modulated by different structural and dynamic properties of G/C DNA, though more studies may be needed to testify this conclusion. 3.3. Probing DNA local environment at the restriction site of HhaI A palindromic DNA sequence containing alternating G and C nucleotides serves as a signal recognized by the HhaI restriction-modification system. Restriction-modification system is a defense mechanism, used by prokaryotes to protect themselves against foreign DNA. In HhaI, it involves restriction endonuclease and associated methyltranferase which bind GCG↓C sequence, with arrow indicating a cleavage site and the underlined cytosine indicating a methylation target. Cytosine methylation at GCGC serves to protect bacterial DNA from destructive action of its own endonuclease and thus distinguish an invading genetic material. The basis for DNA recognition by sequence-specific binding proteins has been a subject of investigation for many decades. It has been proposed that some proteins like endonucleases use an indirect readout mechanism, by establishing non-specific contacts 157 with the DNA sugar-phosphate backbone (22). This emphasizes that sequence-specific structural and dynamic properties of the DNA backbone might play an important role in localization of the protein binding site before full sequence interrogation. Conformational dynamics of the sugar-phosphate backbone, which might be involved in recognition of GCGC by HhaI restriction-modification enzymes, was previously investigated using solid-state and solution NMR (6). For solid-state NMR measurements, either 5’- or 2’-methylene groups were deuterated in a site-specific manner, and local dynamics was monitored using deuterium lineshape analysis (sensitive to motions in s- ms range) and spin-lattice relaxation (sensitive to motions in ns- s range) (23, 24). In solution, 13 C nuclei relaxation parameters of C1’, C2’ and C3’ provided information about local fluctuations of the furanose ring on the ps-ns timescale (25). Interestingly, results obtained from these measurements may not be directly correlated with each other, suggesting that complex dynamic modes are necessary to describe structural fluctuations of the DNA backbone. For example, using deuterium lineshape analysis, it was suggested that C 6 and C 8 cytosines in G 5 C 6 G 7 C 8 are characterized by different mobility of the C5’ methylenes, however mobility reported at the C2’ positions of the C 6 and C 8 appear to be identical (23, 24). Similarly, local dynamics of the furanose ring were interrogated by spin-lattice relaxation measurements of the 13 C nuclei in solution and D nuclei in hydrated solid-state (26). Significantly larger variability in the values of relaxation parameters along the GCGC sequence was obtained using D as compared to 13 C nuclei measurements, suggesting that different motional modes of the furanose moiety contribute to two types of NMR experiments. Overall, solution and solid-state NMR 158 methodologies combined provided a more detailed picture of local backbone flexibility at the HhaI recognition site on the timescales ranging from pico- to microseconds. To our knowledge, HhaI restriction site is the only DNA sequence, where local dynamics of the base and sugar-phosphate backbone have been investigated to a large degree (6, 21, 23-26). In line with these experiments, we use R5a EPR lineshape measurements to probe rotational motions at the phosphate group of the DNA backbone. X-band cw-EPR lineshapes are modulated by motions in the nanosecond regime (0.1-30 ns), intermediate between motional timescales of the solid and solution state NMR experiments. Thus, EPR measurements, providing information about dynamics at the DNA phosphate group in the 0.1-30 ns time window, may complement the results obtained with other spectroscopic techniques and thus improve our understanding of sequence-specific dynamic behavior in DNA. R5a nitroxide was attached, one at a time, to four sites within a GCGC region of the 24-bp DNA, derived from the construct used by Drobny and co-workers (6). The original self-complementary sequence (Figure 6.8A, in purple) was extended with six nucleotides at 5’ and 3’ ends. This was done to prevent formation of a homoduplex DNA containing two nitroxide molecules. Annealing of R5a labeled and non-modified complementary strands yielded a regular heteroduplex DNA, as confirmed using PAGE (data not shown). Obtained EPR spectra show little differences between the sites studied (Figures 6.8B and 6.8C), indicating a similar local environment sensed by R5a along the GCGC region. Methylation of the target cytosine at the restriction site in the opposite DNA strand has no apparent effect on the R5a spectra (Figure 6.9). Additionally, comparisons between 159 HhaI spectra and R5a spectra obtained using G 3 C 3 or (GC) 3 constructs (all are 24-mers) (Figure 6.8C) suggest that G/C sequences may be characterized with comparable nitroxide mobility. Figure 6.8: Results of R5a scanning at HhaI restriction site. (A) 24-bp DNA construct used for cw-EPR studies, with GCGC highlighted in red. Stars indicate sites of ps modification and R5a labeling. Self- complementary sequence regions are shown with a purple box. (B) HhaI-R5a spectra acquired at 15 C. (C) Pairwise comparisons between R5a spectra obtained using G/C DNA. 15 C data are shown. 160 Figure 6.9: Effect of cytosine methylation on R5a spectra. Spectra with a regular (HhaI) and 5- methylated cytosine (HhaI-Met) in the complementary (bottom) DNA strand are compared. 37 C data are shown, as they were obtained within a short period of time. Relatively small variations in nitroxide spectra, observed at the GCGC sites are overall consistent with the results of solution NMR 13 C relaxation experiments (25). One may suggest that ps-ns fluctuations of the phosphate and sugar moieties, which modulate R5a lineshapes and 13 C relaxation, respectively are coupled, though more studies are needed to test this hypothesis. At the same time, solid-state NMR studies report more pronounced site-dependent differences in local backbone flexibility at the slower motional timescales (ns-ms) (23, 24, 26). 161 In summary, R5a data obtained in this study may add to a better characterization of local dynamics at the GCGC sequence. For that, quantitative analysis of HhaI_R5a spectra is required and may be pursued in the future. 4. Chapter 6 Conclusions R5-nitroxides were for the first time used to probe local structural and dynamic features in biologically relevant DNA sequences. Sequence-dependent dynamic behavior of DNA is poorly understood, however it may provide a clue on mechanisms of protein- DNA interactions, causes of diseases and their potential treatment. EPR-based methods using R5-nitroxides have a great potential for monitoring rotational fluctuations of the phosphodiester backbone, probing local structural features and conformational heterogeneity in large DNA molecules. While more work is needed to understand the modes of DNA-nitroxide coupling, results reported here expand our knowledge of R5a sequence-dependent behavior. Studying bcl-2 and G/C DNA with R5a is a first attempt to bridge results of R5a probing and results provided by other methods like chemical reactivity or NMR. Clearly, direct correlations may be complicated, as an outcome of these experiments is a product of many different factors such as DNA structural properties, dynamical modes and their timescales, data analysis and interpretation. Work reported here is a part of a global effort in developing new methodologies and, importantly, defining their limitations and capabilities to study structure, dynamics, and functionality of biological molecules. 162 5. Chapter 6 References 1. Fanucci, G., and Cafiso, D. (2006) Recent advances and applications of site- directed spin labeling, Curr Opin Struct Biol 16, 644-653. 2. Fajer, P. C. (2001) Electron Paramagnetic Resonance (EPR) and Spin-Labeling, In Encyclopedia of Life Sciences, pp 1-5, John Wiley & Sons, London. 3. Popova, A. M., and Qin, P. Z. (2010) A nucleotide-independent nitroxide probe reports on site-specific stereomeric environment in DNA, Biophys J 99, 2180- 2189. 4. Raghavan, S. C., and Lieber, M. R. (2006) DNA structures at chromosomal translocation sites, Bioessays 28, 480-494. 5. Tsai, A. G., Engelhart, A. E., Hatmal, M. M., Houston, S. I., Hud, N. V., Haworth, I. S., and Lieber, M. R. 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(1997) Strained DNA is kinked by low concentrations of Zn 2+ , Proc Natl Acad Sci U S A 94, 10565-10570. 19. Hud, N. V., and Plavec, J. (2003) A unified model for the origin of DNA sequence-directed curvature, Biopolymers 69, 144-158. 20. Poner, J., Florian, J., Ng, H. L., Poner, J. E., and Packova, N. (2000) Local conformational variations observed in B-DNA crystals do not improve base stacking: computational analysis of base stacking in a d(CATGGGCCCATG) 2 B<-->A intermediate crystal structure, Nucleic Acids Res 28, 4893-4902. 21. Dornberger, U., Leijon, M., and Fritzsche, H. (1999) High base pair opening rates in tracts of GC base pairs, J Biol Chem 274, 6957-6962. 164 22. Little, E. J., Babic, A. C., and Horton, N. C. (2008) Early interrogation and recognition of DNA sequence by indirect readout, Structure 16, 1828-1837. 23. Pederson, K., Meints, G. A., Shajani, Z., Miller, P. A., and Drobny, G. P. (2008) Backbone dynamics in the DNA HhaI protein binding site, J Am Chem Soc 130, 9072-9079. 24. Meints, G. A., Miller, P. A., Pederson, K., Shajani, Z., and Drobny, G. (2008) Solid-state nuclear magnetic resonance spectroscopy studies of furanose ring dynamics in the DNA HhaI binding site, J Am Chem Soc 130, 7305-7314. 25. Shajani, Z., and Varani, G. (2008) 13 C relaxation studies of the DNA target sequence for hhai methyltransferase reveal unique motional properties, Biochemistry 47, 7617-7625. 26. Echodu, D., Goobes, G., Shajani, Z., Pederson, K., Meints, G., Varani, G., and Drobny, G. (2008) Furanose dynamics in the HhaI methyltransferase target DNA studied by solution and solid-state NMR relaxation, J Phys Chem B 112, 13934- 13944. 165 Chapter 7 Future work Studies reported here have set up a framework for using the phosphorothioate labeling scheme, and particularly R5a nitroxide, to probe local structural features and nanosecond motions in DNA. Most of the other nitroxides, developed for nucleic acid applications, rely on modification of a base, instead, R5a may be useful in obtaining information on intrinsic flexibility of the DNA backbone, its link to DNA energetics and sequence-dependent functions. This will help to explain the role of DNA backbone motions in protein-DNA recognition, involved in gene regulation, DNA repair and transcriptional control. The major limitation of the backbone labeling scheme is a complicated and time- consuming preparation of diastereopure nitroxide labeled oligonucleotides. Although one may effectively scan a DNA sequence and obtain information about local DNA environment using mixtures of phosphorothioate diastereomers, interpretation of the data may be complicated. Overall, further developments in the area of phosphorothioate stereoselectivity will benefit the use of R5-series of nitroxides in nucleic acids studies. Rotational coupling of R5a to DNA structure and dynamics is an important subject for investigation. Integrated approaches using experiments and computational modeling, such as MD simulations can be fruitful in revealing how, for example, systematic 166 changes in DNA local structure, such as A- to B-form transitions, presence of mismatches and modified bases, may affect nitroxide mobility. This work can be further extended to study structurally more diverse and complex RNA molecules. Currently, information from cw-EPR measurements remains to be unique, as it may not be directly correlated with the results obtained from other experimental techniques, such as NMR or chemical probing. Thus, R5a data can neither be validated nor integrated in the broad picture of DNA dynamics ranging from picoseconds to seconds. 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Abstract (if available)

Abstract In site-directed spin labeling, a covalently attached nitroxide probe containing a chemically stable unpaired electron is utilized to obtain information on the local environment of the parent macromolecule. Studies presented in this dissertation examine feasibility of probing local DNA structural and dynamic features using a class of nitroxides that are linked to chemically substituted phosphorothioate positions at the DNA backbone (R5-series). Two members of this family, designated as R5 and R5a, were attached to multiple sites of a dodecameric DNA duplex without severely perturbing the native B-form conformation. Measured X-band electron paramagnetic resonance (EPR) spectra, which report on nitroxide rotational motions, were found to vary depending on the location of the label and the identity of a phosphorothioate diastereomer (Rp or Sp). Spectral simulations and molecular modeling have been used to define basic principles for correlating observed variation in EPR spectra with site-specific structural and dynamic features in DNA. Overall, these studies advance our understanding of coupling between DNA and R5/R5a, which may ultimately enable the use of nucleotide-independent probes to obtain quantitative description of sequence-specific properties in large biologically relevant DNA molecules.

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Asset Metadata

Creator Popova, Anna M. (author)

Core Title Sensing sequence-specific DNA micro-environment with nucleotide-independent nitroxides

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 04/11/2011

Defense Date 03/23/2011

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag DNA structure and dynamics,Electron Paramagnetic Resonance (EPR),nitroxide label,OAI-PMH Harvest,Site-Directed Spin Labeling (SDSL),spectroscopy

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Qin, Peter Z. (committee chair), Bradforth, Stephen E. (committee member), Langen, Ralf (committee member)

Creator Email for_a@mail.ru,popova@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3729

Unique identifier UC1148649

Identifier etd-Popova-4512 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-446593 (legacy record id),usctheses-m3729 (legacy record id)

Legacy Identifier etd-Popova-4512.pdf

Dmrecord 446593

Document Type Dissertation

Rights Popova, Anna M.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu