PROBING RNA STRUCTURE AND RNA-LIGAND COMPLEXES WITH CHEMICAL PROBES

By ANDREW MOORE

1 LARGE-SCALE NATIVE PREPARATION OF IN VITRO TRANSCRIBED RNA 2 ASSEMBLY OF COMPLEX RNAS BY SPLINTED LIGATION 3 METHODS OF SITE-SPECIFIC LABELING OF RNA WITH FLUORESCENT DYES 4 FLUOROPHORE LABELING TO MONITOR TRNA DYNAMICS 5 USE OF DEOXYRIBOZYMES IN RNA RESEARCH 6 STRATEGIES IN RNA CRYSTALLOGRAPHY 7 COMPARATIVE GEL ELECTROPHORESIS ANALYSIS OF HELICAL JUNCTIONS IN RNA 8 THE STRUCTURE AND FOLDING OF BRANCHED RNA ANALYZED BY FLUORESCENCE RESONANCE ENERGY TRANSFER 9 ANALYSIS OF RNA FOLDING BY NATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS 10 USING ANALYTICAL ULTRACENTRIFUGATION (AUC) TO MEASURE GLOBAL CONFORMATIONAL CHANGES ACCOMPANYING EQUILIBRIUM TERTIARY FOLDING OF RNA MOLECULES 11 USE OF SMALL ANGLE X-RAY SCATTERING (SAXS) OF CHARACTERIZE CONFORMATIONAL STATES OF FUNCTIONAL RNAS1 12 TIME-RESOLVED X-RAY SCATTERING AND RNA FOLDING CONTENTS 13 2-AMINOPURINE AS A PROBE OF RNA CONFORMATIONAL TRANSITIONS 14 FLUORESCENCE POLARIZATION ANISOTROPY TO MEASURE RNA DYNAMICS 15 STUDYING RNA USING SITE-DIRECTED SPIN-LABELING AND CONTINUOUS-WAVE ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY 16 MAPPING GLOBAL FOLDS OF OLIGONUCLEOTIDES BY PULSED ELECTRON–ELECTRON DOUBLE RESONANCE 17 LASER-INDUCED TEMPERATURE JUMP INFRARED MEASUREMENTS OF RNA FOLDING 18 PROBING NUCLEIC ACID–ION INTERACTIONS WITH BUFFER EXCHANGE-ATOMIC EMISSION SPECTROSCOPY 19 USING ANOMALOUS SMALL ANGLE X-RAY SCATTERING TO PROBE THE ION ATMOSPHERE AROUND NUCLEIC ACIDS 20 SIMULATIONS OF RNA INTERACTIONS WITH MONOVALENT IONS 21 ION–RNA INTERACTIONS: THERMODYNAMIC ANALYSIS OF THE EFFECTS OF MONO- AND DIVALENT IONS ON RNA CONFORMATIONAL EQUILIBRIA 22 PREDICTING ELECTROSTATIC FORCES IN RNA FOLDING C H A P T E R O N E LARGE-SCALE NATIVE PREPARATION OF IN VITRO TRANSCRIBED RNA 1 INTRODUCTION 2 NATIVE PURIFICATION OF RNA: AFFINITY CHROMATOGRAPHY METHOD 2 1 MATERIALS 2 2 PREPARATION OF HMM PROTEIN 2 3 PREPARATION OF DNA TEMPLATES BY PCR 2 4 TRANSCRIPTION AND PURIFICATION OF RNA 3 NATIVE PURIFICATION OF RNA: ANION-EXCHANGE CHROMATOGRAPHY 3 1 MATERIALS 3 2 CLONING OF THE PLASMID DNA TEMPLATE 3 3 CELL CULTURE AND PLASMID PURIFICATION 3 4 IN VITRO RNA TRANSCRIPTION 3 5 WEAK ANION-EXCHANGE FPLC ACKNOWLEDGMENTS REFERENCES ABSTRACT BIOPHYSICAL STUDIES OF RNA REQUIRE CONCENTRATED SAMPLES THAT ARE CHEMICALLY AND STRUCTURALLY HOMOGENEOUS HISTORICALLY, THE MOST WIDELY USED METHODS FOR PREPARING THESE SAMPLES INVOLVE IN VITRO TRANSCRIPTION, DENATURATION OF THE RNA, PURIFICATION BASED ON SIZE, AND SUBSEQUENT REFOLDING THESE METHODS ARE USEFUL BUT ARE INHERENTLY SLOW AND DO NOT GUARANTEE THAT THE RNA IS PROPERLY FOLDED POSSIBLE MIS-FOLDING IS OF PARTICULAR CONCERN WITH LARGE, COMPLEXLY FOLDED RNAS TO ADDRESS THESE PROBLEMS, WE HAVE DEVELOPED METHODS FOR PURIFYING IN VITRO TRANSCRIBED RNAS IN THEIR NATIVE, FOLDED STATES THESE METHODS ALSO HAVE THE ADVANTAGE OF BEING RAPID AND READILY SCALED TO VIRTUALLY ANY SIZE RNA OR TRANSCRIPTION AMOUNT TWO METHODS ARE PRESENTED: THE FIRST IS AN AFFINITY