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Stability in Nucleic Acid Hairpins

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Stability in Nucleic Acid Hairpins The Pennsylvania State University The Graduate School Eberly College of Science STABILITY IN NUCLEIC ACID HAIRPINS: 1. MOLECULAR DETERMINANTS OF COOPERATIVITY AND 2. LINKAGE BETWEEN PROTON BINDING AND FOLDING A Thesis in Chemistry by Ellen M. Moody Copyright 2004 Ellen M. Moody Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2004 The thesis of Ellen M. Moody was reviewed and approved* by the following: Philip C. Bevilacqua Associate Professor of Chemistry Thesis Advisor Chair of Committee Christopher Falzone Senior Lecturer of Chemistry Juliette T. J. Lecomte Associate Professor of Chemistry James G. Ferry Stanley Person Professor and Director Center for Microbial Structural Biology Ayusman Sen Professor of Chemistry Head of the Department *Signatures are on file in the Graduate School. ABSTRACT Nucleic acids are central to a wide range of biological processes. The structures as well as the sequence of these biopolymers can carry genetic information, perform catalysis, and are recognized by other components of the cell. Most nucleic acids exist as a combination of simple secondary structures that assemble into much larger, complex structures. The studies contained in this thesis use DNA and RNA hairpin loops as a model system to investigate expandability of loops, cooperativity of interactions, and proton binding. Thermodynamic and phylogenetic analysis have revealed stable DNA and RNA tri- and tetra-loop motifs. In this thesis, in vitro, combinatorial selection approaches have been used to isolate 4 stable DNA tetraloop motifs: d(cGNNAg), d(cGNABg), d(cCNNGg) and d(gCNNGc), where N = A, C, G, or T and B = C, G or T. These motifs have been characterized by thermal melting, circular dichroism, and nucleotide analogue substitution. The d(cGNABg) motif has biological relevance, as seen by Habig and Loeb, who found that this hairpin motif negatively regulates priming during reverse transcription in avian hepatitis B virus (HBV). Expansions of stable loops are known to occur and selected d(cGNNAg) and d(cGNABg) motifs are shown to be an expansion of the known stable d(cGNAg) hairpin loop. Analysis of the d(cGNABg) and d(cGNAg) loops using C3 spacers indicates that these loops can be expanded at any position expect 5' of the first loop nucleotide. C3 spacers provide the backbone length of an additional nucleotide without the possible interactions of a base. Insertion of C3 spacers at this position appears to disrupt a loop- closing base pair interaction, which was found to be common in certain stable DNA and iii RNA hairpin loops. Incorporation of C3 spacers interrupts the loop-closing basepair interaction and allows them to be studied in combination with other interactions. Cooperativity among three interactions (2 loop-loop hydrogen bonds and the loop-closing basepair interaction) in the stable d(cGNAg) triloop was investigated by single, double and triple mutant analysis. Cooperativity in folding is an important aspect in nucleic acids and helps to explain the structural and energetic basis of stability in certain structures. These three interactions were found to be highly non-additive with indirect coupling, meaning that all three interactions were necessary to retain the integrity of the loop. The d(cGNABg) tetraloop was also characterized and through NMR analysis and functional group mutations, it was categorized as an expanded d(cGNAg) triloop. Through additional studies, the expanded triloop, similar to the parent triloop, was found to be highly non-additive with indirect coupling. The ability of a loop to be expanded upon a stable core with retention of structure and coupling allows these hairpins to have enhanced sequence diversity. A related RNA tetraloop, r(cGAAAg), whose structure has been solved, also contains a sheared GA basepair and loop-closing basepair interaction, along with 5 additional interactions. Single, double and triple mutant analyses revealed that the RNA was showed greater additivity than its DNA counterpart and that the coupling was direct. This suggests that this common RNA loop motif may be more adaptable to mutations without loss in stability than its DNA counterpart. These differences are rationalized in terms of molecular features of RNA and DNA. The structures of nucleic acids are also affected by cation binding. A thermodynamic framework for proton binding to nucleic acids was developed and tested iv with experiments and data simulation. Proton binding to nucleic acids facilitates folding, and vice versa, and allows for general acid/base catalysis as well as electrostatic catalysis. Investigation focused on the protonation of N1 of adenosine (pKa of free base + = 3.5), which can form an A ● C basepair. The pKa of this base needs to be shifted ~3.5 units to have an appreciable amount in the protonated form at neutral pH. Oligonucleotides were designed that contained possible A+● C basepairs with optimal nearest-neighbor partners and the pKa of N1 of A was found to be shifted to neutrality. 31 This pKa was measured by P NMR on a phosphorothioate labeled sample, which allowed for the monitoring of a single phosphorus resonance. The simulations that were developed for the thermodynamic framework gave new insight into the pH dependences of nucleic acids. Simulated data showed that multiple pKas in the unfolded state could combine to give an apparent pKa shifted towards neutrality and that the degree of both acid and alkaline denaturation is dependent on the sequence of the oligonucleotide. In addition, equations derived from this framework were used to fit ∆G versus pH values from thermal melting experiments and gave good fits to the data. The structures of nucleic acids contribute to function in biology. Stable sequences are likely to facilitate folding, and expansions of stable sequences could give greater functional diversity. Cooperativity was found to be important in the folding of these stable sequences, and stability was also increased upon proton binding. Combining cooperativity of structure formation and proton binding opens the possibility of creating highly shifted pKas, which could introduce new functions to DNA and RNA. v Table of Contents List of Figures…….……………………………………………………………………..xiv List of Tables and Schemes……………………………………………………………xviii List of Equations…………………………………………………………………………xx List of Abbreviations…………………………………………………………………..xxiv Acknowledgements……………………………………………………………………..xxv Ch. 1: Introduction………………………………………………………………………1 1.1 General Background on RNA and DNA………………………………….1 1.2 Important Physical Interactions in Nucleic Acids………………………...3 1.2.1 Electrostatics………………………………………………………3 1.2.2 Hydrogen Bonding………………………………………………...5 1.2.3 Stacking……………………………………………………………5 1.2.4 Shape………………………………………………………………6 1.3 Model Systems and Nearest Neighbor Model…………………………….7 1.4 Methods for Identification and Characterization………………………….8 1.4.1 In vitro Selection (SELEX) Methodologies……………………….9 1.4.2 Background and History of TGGE………………………………10 1.4.3 Identification of Stable Hairpin Loops…………………………..11 1.4.4 Modifications to Bases and Backbone…………………………...12 1.4.5 UV Melting………………………………………………………14 1.4.6 Propagation of Errors……………...……………………………..15 vi 1.5 Topics Addressed in this Thesis…………………………………………16 1.5.1 Expandability of Hairpins………………………………………..16 1.5.2 Cooperativity of Interactions in DNA and RNA………………...17 1.5.3 pKa Shifting in Nucleic Acids……………………………………21 1.6 Format of this Thesis…………………………………………………….24 1.7 References………………………………………………………………..26 Ch. 2: Selection for Thermodynamically Stable DNA Tetraloops Using Temperature Gradient Gel Electrophoresis Reveals Four Motifs: d(cGNNAg), d(cGNABg), d(cCNNGg), and d(gCNNGc)…………………….42 2.1 Abstract…………………………………………………………………..42 2.2 Introduction……………………………………………………………...43 2.3 Material and Methods…………………………………………………...46 2.3.1 Preparation of DNA……………………………………………...46 2.3.2 Temperature Gradient Gel Electrophoresis……………………...47 2.3.3 Selection Procedure……………………………………………...48 2.3.4 UV Melting Experiments………………………………………...49 2.3.5 Circular Dichroism……………………………………………….50 2.4 Results……………………………………………………………………51 2.4.1 Design and Optimization of the DNA Tetraloop Library………..51 2.4.2 Selection of Stable DNA Tetraloops……………………………..52 2.4.3 Identification of Stable DNA Hairpins…………………………..54 vii 2.4.4 Thermodynamic Characterization of d(cGNNAg) and d(cGCABg) Minihairpins……………………………………………………...55 2.4.5 Thermodynamic Characterization of d(cCNNGg) and d(gCNNGc) Minihairpins……………………………………………………...58 2.4.6 Characterization of Minihairpins by CD Spectroscopy………….60 2.5 Discussion………………………………………………………………..62 2.6 Additional Studies………………………………………………………..66 2.6.1 PCR Optimization for RNA Tetraloop Studies………………….67 2.6.2 Selections for Penultimate Basepairs…………………………….68 2.6.3 Concatenation of Multiple Sequences in a Single Clone………...69 2.7 Acknowledgements………………………………………………………72 2.8 References………………………………………………………………..73 Ch. 3: Thermodynamic Coupling of the Loop and Stem in Unusually Stable DNA Hairpins Closed by CG Base Pairs…………………………………………….94 3.1 Abstract…………………………………………………………………..94 3.2 Introduction………………………………………………………………95 3.3 Material and Methods……………………………………………………96 3.4 Results……………………………………………………………………97 3.5 Discussion………………………………………………………………..99 3.6 Acknowledgments…………………………………………………...….100 3.7 References………………..……………………………………………..101 viii Ch. 4: Folding of a Stable DNA Motif Involves a Highly Cooperative Network of Interactions…………………………………………………………………….109 4.1 Abstract…………………………………………………………………109
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