Single Molecule Tools, Part A: Fluorescence Based Approaches

by Walter

• ISBN13: 9780123749543
• ISBN10: 0123749549
• Edition: 1st
• Format: Hardcover
• Copyright: 2010-06-14
• Publisher: Elsevier Science

Summary

Single molecule tools have begun to revolutionize the molecular sciences, from biophysics to chemistry to cell biology. They hold the promise to be able to directly observe previously unseen molecular heterogeneities, quantitatively dissect complex reaction kinetics, ultimately miniaturize enzyme assays, image components of spatially distributed samples, probe the mechanical properties of single molecules in their native environment, and "just look at the thing" as anticipated by the visionary Richard Feynman already half a century ago. This volume captures a snapshot of this vibrant, rapidly expanding field, presenting articles from pioneers in the field intended to guide both the newcomer and the expert through the intricacies of getting single molecule tools.* Includes time-tested core methods and new innovations applicable to any researcher employing single molecule tools * Methods included are useful to both established researchers and newcomers to the field * Relevant background and reference information given for procedures can be used as a guide to developing protocols in a number of disciplines

Table of Contents

Contributors	p. xiii
Preface	p. xxi
Volume in Series	p. xxiii
Star Polymer Surface Passivation for Single-Molecule Detection	p. 1
Introduction	p. 2
Surface Grafting of PEO and Protein Repellence	p. 2
The NCO-sP(EO-stat-PO) System	p. 4
Preparation of sP(EO-stat-PO)-Coated Substrates for Single-Molecule Experiments	p. 6
Analysis of Protein Structure and Function on NCO-sP(EO-stat-PO) Surfaces	p. 11
Acknowledgments	p. 16
References	p. 16
Azide-Specific Labeling of Biomolecules by Staudinger-Bertozzi Ligation: Phosphine Derivatives of Fluorescent Probes Suitable for Single-Molecule Fluorescence Spectroscopy	p. 19
Introduction	p. 20
Materials and Methods	p. 21
Acknowledgments	p. 28
References	p. 28
Preparation of Fluorescent Pre-mRNA Substrates for an smFRET Study of Pre-mRNA Splicing in Yeast	p. 31
Introduction	p. 32
Identification of a Yeast Pre-mRNA with a Small Intron that is Spliced Efficiently In Vitro	p. 32
Synthetic Fluorescent Ubc4 Pre-mRNA	p. 33
Do the Dyes Affect the Efficiency of Splicing?	p. 37
Mutant Pre-mRNAs	p. 37
Tethering the Pre-mRNA to the Microscope Slide	p. 38
Summary and Conclusion	p. 39
Acknowledgments	p. 40
References	p. 40
Nanovesicle Trapping for Studying Weak Protein Interactions by Single-Molecule FRET	p. 41
Introduction	p. 42
Nanovesicle Trapping Approach	p. 44
smFRET Measurements of Weak Protein-Protein Interactions	p. 47
Single-Molecule Kinetic Analysis of Three-State Protein-Protein Interactions	p. 54
Further Developments	p. 57
Concluding Remarks	p. 58
Acknowledgments	p. 59
References	p. 59
Droplet Confinement and Fluorescence Measurement of Single Molecules	p. 61
Introduction	p. 62
Methods for Droplet Generation	p. 65
Methods for Droplet Manipulation	p. 69
Droplet Coalescence and Mixing	p. 73
Experimental Considerations for Single Fluorophore Detection	p. 73
Single-Molecule Measurements in Droplets	p. 79
Future Prospects	p. 82
Acknowledgments	p. 83
References	p. 84
Single-Molecule Fluorescence Spectroscopy Using Phospholipid Bilayer Nanodiscs	p. 89
Introduction	p. 90
Nanodiscs and HDL Particles	p. 91
Single-Molecule Techniques and Applications to Membrane Proteins	p. 95
Cytochrome P450 3A4 and Its Allosteric Behavior	p. 96
Image Filtering by Singular-Value Decomposition	p. 102
Islet Amyloid Polypeptide Binding to Nanodiscs	p. 106
¿-Synuclein Conformations on Nanodiscs	p. 109
Summary	p. 112
Acknowledgments	p. 112
References	p. 112
Single-Molecule Spectroscopy Using Microfluidic Platforms	p. 119
Introduction	p. 120
Microchip Fabrication	p. 121
Instrumentation for Fluorescence Detection	p. 123
Detergent-Assisted Microchannel Electrophoresis	p. 124
Fluorescence Correlation Spectroscopy	p. 127
Acknowledgments	p. 131
References	p. 131
Detection of Protein-Protein Interactions in the Live Cell Plasma Membrane by Quantifying Prey Redistribution upon Bait Micropatterning	p. 133
Introduction	p. 134
Methodological Requirements	p. 136
The Micropatterning Technique	p. 137
Experimental Design	p. 139
Procedure	p. 140
Interpretation of Results	p. 145
Figures of Merit	p. 147
Conclusions	p. 148
Acknowledgments	p. 149
References	p. 149
Analysis of Complex Single-Molecule FRET Time Trajectories	p. 153
Introduction	p. 154
Analysis of Simple Trajectories	p. 156
Analysis of Complex Trajectories	p. 160
Post-HMM Processing and Data Visualization	p. 168
Acknowledgment	p. 176
References	p. 176
Single-Molecule Fluorescence Studies of Intrinsically Disordered Proteins	p. 179
Introduction	p. 180
Single-Molecule Fluorescence Methods	p. 181
Site-Specific Labeling of Intrinsically Disordered Proteins	p. 187
Examples of SMF Characterization of IDP Structure and Dynamics	p. 190
Concluding Remarks	p. 198
Acknowledgments	p. 200
References	p. 200
Measuring the Energetic Coupling of Tertiary Contacts in RNA Folding using Single Molecule Fluorescence Resonance Energy Transfer	p. 205
Introduction	p. 206
Thermodynamic Cooperativity Overview	p. 207
Measuring Folding Equilibrium in RNA	p. 209
Designing an smFRET Experiment to Measure Cooperativity	p. 210
Additional Comments	p. 217
Acknowledgments	p. 219
References	p. 219
A Highly Purified, Fluorescently Labeled In Vitro Translation System for Single-Molecule Studies of Protein Synthesis	p. 221
Introduction	p. 222
A Highly Purified, Escherichia coli-Based In Vitro Translation System	p. 225
Biochemical Assays	p. 233
Preparation of Fluorescently Labeled Translation Components	p. 242
Conclusions and Future Perspectives	p. 253
Acknowledgments	p. 254
References	p. 255
Watching Individual Proteins Acting on Single Molecules of DNA	p. 261
Introduction	p. 262
Preparation of DNA Substrates	p. 265
Preparation of Fluorescent Proteins	p. 268
Instrument	p. 270
Single-Molecule Imaging of Proteins on DNA	p. 280
Data Analysis Methods	p. 287
Acknowledgments	p. 289
References	p. 289
DNA Curtains for High-Throughput Single-Molecule Optical Imaging	p. 29
Introduction	p. 294
Total Internal Reflection Fluorescence Microscopy	p. 294
DNA Curtains	p. 297
Visualizing Protein-DNA Interactions	p. 307
Conclusions and Future Directions	p. 314
Acknowledgments	p. 314
References	p. 314
Scanning FCS for the Characterization of Protein Dynamics in Live Cells	p. 317
Introduction	p. 318
Implementation	p. 320
Data Analysis	p. 327
Applications	p. 334
Conclusion	p. 341
References	p. 342
Observing Protein Interactions and Their Stoichiometry in Living Cells by Brightness Analysis of Fluorescence Fluctuation Experiments	p. 345
Introduction	p. 346
Brightness Classification of Fluorescent Molecules	p. 347
Brightness Measurements in Cells	p. 354
Acknowledgments	p. 361
References	p. 361
Detection of Individual Endogenous RNA Transcripts In Situ Using Multiple Singly Labeled Probes	p. 365
Introduction	p. 366
Design and Synthesis of Fluorescent Oligonucleotide Probe Sets	p. 369
Preparation of Samples for In Situ Hybridization	p. 373
Hybridization	p. 377
Imaging	p. 381
Image Analysis	p. 384
Acknowledgments	p. 386
References	p. 386
Single mRNA Tracking in Live Cells	p. 387
Introduction	p. 388
Significance of Tracking mRNA	p. 389
Labeling mRNA in Living Cells	p. 391
Imaging mRNA Movements	p. 394
Analyzing mRNA Motions	p. 396
Conclusions	p. 402
Acknowledgments	p. 403
References	p. 403
Single-Molecule Sequencing: Sequence Methods to Enable Accurate Quantitation	p. 407
Introduction	p. 408
Basic Principles of Single-Molecule Sequencing	p. 409
Preparation of Genomic DNA for Single-Molecule Sequencing	p. 410
Bacterial Genome Sequencing	p. 416
Human Genome Sequencing and Quantitation	p. 418
Chromatin Immunoprecipitation Studies	p. 421
Digital Gene Expression for Transcriptome Quantitation	p. 423
Summary	p. 428
Acknowledgments	p. 428
References	p. 430
Real-Time DNA Sequencing from Single Polymerase Molecules	p. 431
Introduction	p. 432
Principle of Single-Molecule, Real-Time DNA Sequencing	p. 433
Components of SMRT Sequencing	p. 435
Single-Molecule DNA Polymerase Dynamics	p. 446
Conclusions	p. 451
Acknowledgments	p. 452
References	p. 452
Author index	p. 457
Subject Index	p. 467
Table of Contents provided by Ingram. All Rights Reserved.