IR AND RAMAN SPECTROSCOPY
CHAPTER 1: ENCAPSULATION OF FLUORESCENTLY LABELED RNAS INTO SURFACE-TETHERED VESICLES FOR SINGLE-MOLECULE FRET STUDIES IN TIRF 1 INTRODUCTION 2 MATERIALS 21 LIPID VESICLE PREPARATION 22 MICROFLUIDIC CHAMBER AND SURFACE PASSIVATION 23 REAGENTS FOR RNA LABELING, ENCAPSULATION, AND IMAGING 3 METHODS 31 LIPID CAKE PREPARATION 32 SITE-SPECIFIC LABELING OF LONG RNAS 33 RNA ENCAPSULATION INTO PHOSPHOLIPID VESICLES 34 VESICLE AND DYE CHARACTERIZATION 35 MICROFLUIDIC CHAMBER PREPARATION INCLUDING SURFACE PASSIVATION FOR PRISM-BASED TIRFM 36 SMFRET EXPERIMENT 37 SINGLE-MOLECULE VIDEO (SMV) PROCESSING AND DATA ANALYSIS 4 NOTES REFERENCES CHAPTER 2: PREPARATION OF SNAPF-BEADS FOR COLOCALIZATION SINGLE-MOLECULE SPECTROSCOPY (COSMOS) OF RNA-PROTEIN COMPLEXES 21 EXPRESSION AND PURIFICATION OF SNAPF-PROTEIN 22 IMMOBILIZATION OF SNAPF-PROTEIN ON AGAROSE BEADS 23 DEPLETION OF SNAP-SUBSTRATES USING SNAPF-BEADS 31 EXPRESSION AND PURIFICATION OF SNAPF-PROTEIN 32 IMMOBILIZATION OF SNAPF-PROTEIN ON AGAROSE BEADS 33 DEPLETION OF SNAP-SUBSTRATES USING SNAPF-BEADS CHAPTER 3: COMPARATIVE COLOCALIZATION SINGLE-MOLECULE SPECTROSCOPY (COSMOS) WITH MULTIPLE RNA SPECIES 21 DEPOSITING AND IMAGING MULTIPLE RNA SPECIES ON A SINGLE SLIDE FOR COSMOS 22 DATA ANALYSIS FOR COSMOS EXPERIMENTS WITH MULTIPLE RNAS 31 DEPOSITING AND IMAGING MULTIPLE RNA SPECIES ON A SINGLE SLIDE FOR COSMOS 32 DATA ANALYSIS FOR COSMOS EXPERIMENTS WITH MULTIPLE RNAS CHAPTER 4: DYNAMIC LIGHT SCATTERING ANALYSIS ON RNA ASSOCIATED TO PROTEINS 31 SAMPLE PREPARATION 32 DESIGNING THE EXPERIMENT 321 BASICS 322 THE EXPERIMENT CHAPTER 5: BIOINFORMATIC APPLICATION OF FLUORESCENCE-BASED IN VIVO RNA REGIONAL ACCESSIBILITY DATA TO IDENTIFY NOVEL SRNA TARG 21 IRS3 GENERAL MATERIALS AND REAGENTS 22 IRS3 ASRNA PROBE INSERTION 23 IRS3 EXPERIMENTATION 24 COMPUTATIONAL PREDICTION OF MRNA TARGETS OF SRNAS 25 BIOINFORMATIC FILTRATION OF COMPUTATIONALLY-PREDICTED MRNA TARGETS 26 GENERAL MATERIALS AND REAGENTS FOR ELECTROPHORETIC MOBILITY SHIFT ASSAYS (EMSAS) 27 IN VITRO TRANSCRIPTION (IVT)-COMPATIBLE PCR AMPLIFICATION OF SRNAS AND MRNAS 28 IVT OF SRNAS AND MRNAS 29 EMSA 31 IRS3 EXPERIMENTAL DESIGN AND EXECUTION 311 SELECTION OF REGIONS WITHIN RNA OF INTEREST FOR ACCESSIBILITY PROBING 312 ASRNA PROBE INSERTION 313 IRS3 EXPERIMENTATION 314 DATA ANALYSIS 315 IDENTIFICATION OF LIKELY FUNCTIONAL REGIONS TO MAKE A HOT REGION MATRIX 32 INTARNA PREDICTION OF SRNA TARGETS 321 COMPUTATIONAL TARGET PREDICTION USING INTARNA WEB SERVER 322 COMPUTATIONAL TARGET PREDICTION USING LOCALLY INSTALLED INTARNA 33 BIOINFORMATIC FILTERING OF MRNA TARGETS BASED ON HOT REGIONS IDENTIFIED VIA IRS3 331 CREATING A ``HOT REGION MATRIX´´ 332 FORMATTING INTARNA TARGET PREDICTIONS 333 FILTERING TARGET PREDICTIONS BY OVERLAP WITH HOT REGIONS TO OBTAIN MRNA CANDIDATES FOR EXPERIMENTAL CONFIRMATION 334 FILTERING MRNA CANDIDATES AND SELECT REPRESENTATIVE SEQUENCES FOR EXPERIMENTAL CONFIRMATION BY TRANSCRIPTOMICS 34 EXPERIMENTAL VALIDATION OF PREDICTED AND FILTERED RNA: RNA INTERACTIONS VIA EMSA 341 IVT-COMPATIBLE AMPLIFICATION OF RNA OF INTERESTS AND MRNA TARGET CANDIDATES 342 IVT OF POTENTIAL MRNA TARGETS FOR EMSA ANALYSIS 343 IVT OF SRNAS (OR RNAS OF INTEREST) 344 EMSAS TO CONFIRM DIRECT RNA OF INTEREST: MRNA INTERACTIONS CHAPTER 6: SURFACE PLASMON RESONANCE FOR INVESTIGATING MOLECULAR INTERACTIONS WITH RNA 21 PREPARATION OF RNA MOLECULES AND COLE1 ROP PROTEIN 22 INTERACTION STUDIES 31 PREPARATION OF THE INSTRUMENT 32 PREPARATION OF THE SENSOR CHIP 33 PREPARATION AND IMMOBILIZATION OF THE BIOTINYLATED TARGETS 34 PREPARATION OF THE RNA AND PROTEIN SAMPLES 35 BINDING OF THE PROTEIN ROP TO THE RNA-RNA COMPLEXES 36 ANALYSIS OF THE SENSORGRAMS CHAPTER 7: RNA FOLDING AND UNFOLDING UNDER FORCE: SINGLE-MOLECULE EXPERIMENTS AND THEIR ANALYSIS 21 SOFTWARE 31 DATA ANALYSIS 311 DATA ANALYSIS PROCEDURE (SEE NOTE 2) 32 INTERPRETATION OF HMM ANALYSIS USING KNOWN OR PREDICTED RNA 3D STRUCTURE CHAPTER 8: MAPPING OF POSTTRANSCRIPTIONAL TRNA MODIFICATIONS BY TWO-DIMENSIONAL GEL ELECTROPHORESIS MASS SPECTROMETRY 21 INSTRUMENTS AND EQUIPMENT 22 TWO-DIMENSIONAL GEL ELECTROPHORESIS 23 IN-GEL DIGESTION OF SEPARATED TRNA 24 NANO-LIQUID CHROMATOGRAPHY 31 TWO-DIMENSIONAL GEL ELECTROPHORESIS (SEE NOTE 1) 311 FIRST DIMENSION (DENATURING CONDITIONS) 312 SECOND DIMENSION (SEMI-DENATURING CONDITION) 32 IN-GEL RNASE DIGESTION 33 RNA DIGEST PRODUCT DESALTING 34 LC-MS/MS 341 NANO-LIQUID CHROMATOGRAPHY 342 MASS SPECTROMETRY ANALYSIS 35 DATA ANALYSIS CHAPTER 9: NATIVE ELECTROSPRAY IONIZATION MASS SPECTROMETRY OF RNA-LIGAND COMPLEXES 22 REAGENTS 23 REAGENT SETUP 31 RNA PREPARATION FOR NATIVE ESI-MS 311 OLIGORIBONUCLEOTIDE PURIFICATION AND RNA-RNA/LIGAND FORMATION 312 OLIGORIBONUCLEOTIDE CONCENTRATION 313 SAMPLE PREPARATION FOR NATIVE ESI-MS 32 MASS SPECTROMETRY ANALYSIS 33 DATA ANALYSIS 331 CHARGE STATE DETERMINATION OF THE PEAKS 332 MW CALCULATION FROM ESI SPECTRA CHAPTER 10: APPLICATION OF FTIR SPECTROSCOPY TO ANALYZE RNA STRUCTURE ABBREVIATIONS 21 ENZYMES, BUFFERS, AND CHEMICALS 22 FTIR SPECTROSCOPY 31 IN VITRO TRANSCRIPTION FOR FTIR STUDIES 311 DESIGN OF THE PRIMERS 312 IN VITRO TRANSCRIPTION 313 DNA TEMPLATE DIGESTION 314 RNA PURIFICATION 315 RNA FOLDING 32 RNA CHEMICAL SYNTHESIS 33 INFRARED SPECTRA ACQUISITION 331 FTIR SETUPS 332 RNA SEQUENCE ANALYSIS IN D2O SOLUTION 333 RNA SEQUENCES ANALYZED WITH AN ATR 34 SPECTRAL MANIPULATION (BASELINE SUBTRACTION, BASELINE DRIFT CORRECTION) 35 RNA SPECTRUM ANALYSIS 351 OUT-OF-PLANE BASE VIBRATION SPECTRAL DOMAIN 352 IN-PLANE BASE VIBRATION SPECTRAL DOMAIN 353 SUGAR CONFORMATION SPECTRAL DOMAIN 354 PHOSPHATE SPECTRAL DOMAIN 36 SPECIFIC CASE OF PROTEIN/RNA COMPLEXES 361 ANALYSIS OF THE RNA-COMPLEXED PROTEIN SECONDARY STRUCTURE 362 USING OMNIC: THE MANIPULATION OF THE SPECTRUM CAN BE DONE FOLLOWING THE STEPS BELOW CHAPTER 11: APPLICATION OF SYNCHROTRON RADIATION CIRCULAR DICHROISM FOR RNA STRUCTURAL ANALYSIS 21 CHEMICALS 22 SRCD SPECTROSCOPY 31 IN VITRO TRANSCRIPTION OF RNA 32 BUFFERS COMPATIBLE WITH SRCD ANALYSIS 33 RNA SAMPLE LOADING 34 SRCD SETUP AND DATA ACQUISITION 35 SPECTRAL DATA TREATMENT 36 RNA SPECTRA ANALYSIS 37 MELTING CURVES 371 THERMAL SCAN ACQUISITION 372 INTERPRETATION OF THERMAL SCANS 38 SPECIFIC CASE OF PROTEIN/RNA COMPLEXES CHAPTER 12: APPLICATION OF NIR RAMAN SPECTROSCOPY TO PROBE THE FLEXIBILITY OF RNA STRUCTURE 22 RAMAN SPECTROMETER 31 PREPARATION OF VIROIDS FOR RAMAN EXPERIMENTS 311 ASBVD RNA TRANSCRIPTION 312 ASBVD RNA PREPARATION FOR RAMAN EXPERIMENTS 32 CONDITIONS FOR THE ANALYSIS OF SELF-CLEAVED VIROIDS 33 BASIC THEORY OF RAMAN INELASTIC SCATTERING 34 NIR RAMAN INSTRUMENT DESIGN 35 ASSIGNMENTS OF RAMAN MARKERS 36 RAMAN SPECTRA OF VIROIDS 37 QUANTIFICATION OF RAMAN MARKERS 38 EFFECTS OF HIGH TEMPERATURES ON RNA SPECTRA 39 D2O EFFECTS 310 EFFECTS DUE TO SELF-CLEAVAGE ACTIVITY CHAPTER 13: SMALL-ANGLE NEUTRON SCATTERING OF RNA-PROTEIN COMPLEXES 21 NEUTRON SOURCES AND INSTRUMENTS 22 SAMPLE CHECKLIST FOR SANS EXPERIMENTS 23 DETAILED RECOMMENDATIONS ON SAMPLE QUALITY 24 DETAILED RECOMMENDATIONS ON SAMPLE QUANTITY 25 SAMPLE PREPARATION AND DEUTERATION 26 SAMPLE FOR LIGAND TITRATION SANS 31 SCHEMATIC REPRESENTATION OF A SANS EXPERIMENT 32 INSTRUMENTAL SETUP FOR RNA-PROTEIN COMPLEXES 33 BASIC SANS THEORY, SOLVENT CONTRAST VARIATION, AND DEUTERATION 34 RAW DATA REDUCTION 35 BASIC DATA ANALYSIS AND MODEL-FREE PARAMETERS 36 SOPHISTICATED DATA ANALYSIS: LOW-RESOLUTION AB INITIO ENVELOPES 37 SOPHISTICATED DATA ANALYSIS II: COMBINED APPROACHES USING SANS AND NMR DATA 38 CONCLUDING REMARKS AND PERSPECTIVES CHAPTER 14: STRUCTURAL ANALYSIS OF RNA BY SMALL-ANGLE X-RAY SCATTERING 21 CLONING OF CASSETTES 22 PURIFICATION OF RNA ON POLYACRYLAMIDE GELS AND BAND VISUALIZATION 23 AUTOCATALYTIC CLEAVAGE OF RNA WITH RIBOZYME 24 RNA ELECTROELUTION 25 RNA PURIFICATION BY CHROMATOGRAPHY 26 HPLC-SEC CHROMATOGRAPHY 27 DATA PROCESSING AND ANALYSIS 31 RNA PRODUCTION BY IN VITRO TRANSCRIPTION (SEE FIG 2 AND NOTE 1) 311 RNA CLONING USING THE CASSETTE TECHNIQUE 312 PHOSPHORYLATION OF OLIGONUCLEOTIDES 313 HYBRIDIZATION OF PHOSPHORYLATED OLIGONUCLEOTIDES 314 LIGATION OF THE RNA GENE INTO PUC 118 VECTOR 315 BACTERIAL TRANSFORMATION AND SEARCH FOR POSITIVE CLONES 316 IN VITRO T7 TRANSCRIPTION 317 RNA CLONING USING THE TRANSZYME STRATEGY 318 TRANSCRIPTION AND AUTOCATALYTIC CLEAVAGE 32 RNA PURIFICATION (FIG 4) 321 RNA PURIFICATION ON GEL (SEE NOTE 3) 322 RNA EXTRACTION BY ELECTROELUTION USING BIOTRAP DEVICE 323 RNA EXTRACTION BY ELECTROELUTION IN A DIALYSIS BAG 324 RNA PURIFICATION BY CHROMATOGRAPHY (SEE NOTE 4) 325 RNA QUANTIFICATION AND QUALITY CONTROL (SEE NOTE 5) 33 SEC-SAXS EXPERIMENT 331 COLUMN AND SAMPLE PREPARATION (SEE NOTE 6) 332 TEST ELUTION (OPTIONAL) 333 DATA ACQUISITION 334 DATA PROCESSING 34 DATA ANALYSIS 35 SAXS-BASED RNA MODELING (SEE NOTE 11) CHAPTER 15: SITE-SPECIFIC SPIN LABELING OF RNA FOR NMR AND EPR STRUCTURAL STUDIES 21 SYNTHESIS OF RNAS BY IN VITRO TRANSCRIPTION 22 LIGATION OF RNA SEGMENTS 23 ANALYTICAL ELECTROPHORESIS OF RNA 24 PURIFICATIONS OF RNA 25 SPIN LABELING OF RNA 26 NMR STUDIES OF RNA 27 EPR STUDIES OF RNA 31 SYNTHESIS OF RNA SEGMENTS BY IN VITRO TRANSCRIPTION USING T7 RNA POLYMERASE 311 OPTIMIZATION OF IN VITRO TRANSCRIPTION CONDITIONS 312 PREPARATIVE TRANSCRIPTIONS 313 PURIFICATION OF RNA TRANSCRIPTS 32 LIGATION OF RNA SEGMENTS 321 HYBRIDIZATION OF RNAS TO THE DNA SPLINT 322 LIGATION OF RNA FRAGMENTS 33 SITE-SPECIFIC SPIN LABELING OF RNA 34 NMR EXPERIMENTS 35 EPR EXPERIMENTS 351 PREPARATION OF RNA SAMPLES 352 SPIN LABELING EFFICIENCY VIA NITROXIDE STANDARDS CHAPTER 16: ANALYSIS OF THE HIV-1 GENOMIC RNA DIMERIZATION INITIATION SITE BINDING TO AMINOGLYCOSIDE ANTIBIOTICS USING ISOTHER 21 INSTRUMENTATION 22 INSTRUMENT ACCESSORIES 23 SAMPLE PREPARATION 32 DESIGNING A TYPICAL ITC EXPERIMENT 321 EXPERIMENTAL CONSIDERATIONS 322 INSTRUMENT PARAMETERS 33 PERFORMING THE ITC EXPERIMENT 34 ITC DATA ANALYSIS CHAPTER 17: USE OF FLUORESCENT NUCLEOTIDES TO MAP RNA-BINDING SITES ON PROTEIN SURFACE 22 FLUORESCENCE SPECTROSCOPY 31 DETERMINATION OF APPARENT DISSOCIATION CONSTANT KD USING INTRINSIC PROTEIN FLUORESCENCE 32 DETERMINATION OF APPARENT DISSOCIATION CONSTANT USING FLUORESCENCE ANISOTROPY OF THE LABELED NUCLEOTIDE 33 OBTAINING RIBONUCLEOTIDE-PROTEIN CRYSTALS BY CO-CRYSTALLIZATION 34 OBTAINING RIBONUCLEOTIDE-PROTEIN CRYSTALS BY SOAKING OF THE PROTEIN CRYSTALS IN RIBONUCLEOTIDE SOLUTION 35 DETERMINATION OF THE RIBONUCLEOTIDE POSITION IN THE EXPERIMENTAL ELECTRON DENSITY CHAPTER 18: FLUORESCENT OLIGONUCLEOTIDE PROBES FOR THE QUANTIFICATION OF RNA BY REAL-TIME QPCR 11 CONTEXT 12 REAL-TIME QPCR QUANTIFICATION PRINCIPLE 13 REAL-TIME QPCR DETECTION CHEMISTRIES 14 DESIGN OF FLUORESCENT PROBES FOR THE QUANTIFICATION OF RNAS BY QPCR 21 BIOINFORMATICS TOOLS NEEDED PRIOR TO DESIGN 22 BIOINFORMATICS TOOLS NEEDED FOR THE DESIGN 23 OTHER BIOINFORMATICS TOOLS 31 EXAMPLE OF DESIGN IN EUKARYOTES: HYDROLYSIS PROBE ASSAY FOR THE DETECTION OF A SPECIFIC PANEL OF HOMO SAPIENS CYP3A5 MRNA 32 ELABORATION OF AN AMPLIFICATION STRATEGY TO ACHIEVE QPCR ASSAY SPECIFICITY TO QUANTIFY THE RNA OF INTEREST 33 RETRIEVAL OF RNA SEQUENCE OF INTEREST 34 RETRIEVAL OF RNA SEQUENCES FROM HOMOLOGOUS GENES 35 DESIGN OF THE OLIGONUCLEOTIDE PRIMERS AND PROBE FOR THE ASSAY 36 ORDERING THE OLIGONUCLEOTIDE PRIMERS AND PROBE CHAPTER 19: PROBING RNA-PROTEIN INTERACTIONS AND RNA COMPACTION BY SEDIMENTATION VELOCITY ANALYTICAL ULTRACENTRIFUGATION 21 RNA TRANSCRIPTION 22 PROTEIN EXPRESSION AND PURIFICATION 23 BUFFERS FOR RNA AND PROTEINS 24 ANALYTICAL ULTRACENTRIFUGATION 25 SOFTWARE FOR AUC RUN SETUP, DATA ACQUISITION, AND DATA ANALYSIS 31 RNA SAMPLE PREPARATION 32 PROTEIN SAMPLE PREPARATION 33 SETTING UP EQUILIBRIUM COUNTERION/PROTEIN-MEDIATED RNA COMPACTION REACTIONS 34 SETTING UP REACTIONS TO PROBE EQUILIBRIUM CONFORMATIONAL CHANGE OF AN RNA UPON LIGAND/PROTEIN BINDING 35 AUC CELL ASSEMBLY AND LOADING SAMPLES INTO THE CELLS 36 LOADING AUC CELLS INTO THE ROTOR 37 EXPERIMENTAL DESIGN 38 SETTING UP A SEDIMENTATION VELOCITY RUN AND DATA ACQUISITION 39 ANALYZING SV DATA TO OBTAIN HYDRODYNAMIC PROPERTIES 391 STEPS COMMON TO MWL AND SWL DATA ANALYSIS 392 STEPS SPECIFIC TO MWL ANALYSIS (RNA-PROTEIN INTERACTIONS) 393 STEPS FOR SWL ANALYSIS (COUNTERION OR LIGAND-INDUCED RNA COMPACTION) CHAPTER 20: RNA NANOSTRUCTURE MOLECULAR IMAGING 21 BUFFERS AND SOLUTIONS FOR IN VITRO TRANSCRIPTION 22 TEM IMAGING 23 AFM IMAGING 31 IN VITRO TRANSCRIPTION FOR AFM AND TEM ANALYSIS 33 TEM POSITIVE STAINING 331 PREPARATION OF CARBON-COATED GRIDS 332 ACTIVATION OF THE CARBON-COATED GRIDS BY GLOW DISCHARGE 333 SPREADING AND STAINING 34 AFM IMAGING CHAPTER 21: GRAD-CRYO-EM: TOOL TO ISOLATE TRANSLATION INITIATION COMPLEXES FROM RABBIT RETICULOCYTE LYSATE SUITABLE FOR STRUCT 21 CELL EXTRACTS, BUFFERS, SOLUTIONS, AND REAGENTS 22 ANTIBIOTICS AND TRANSLATION INHIBITORS 23 INSTRUMENT AND ACCESSORIES 31 MRNA TRANSCRIPTION AND CAPPING 32 LINEAR SUCROSE GRADIENT PREPARATION 33 ?-GLOBIN/48SIC FORMATION AND ISOLATION FROM SUCROSE GRADIENT 331 THE EXPERIMENT CHAPTER 22: APPLICATION OF NMR SPECTROSCOPY TO DETERMINE SMALL RNA STRUCTURE 22 NMR SPECTROSCOPY 31 IN VITRO TRANSCRIPTION FOR NMR STUDIES 33 NMR RESONANCE ASSIGNMENT OF RNA 34 STRUCTURE CALCULATION 35 SPECIFIC CASE OF MODIFIED RNA