Single Molecule Tools: Fluorescence Based Approaches

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  • Edition: 1st
  • Format: Hardcover
  • Copyright: 2010-06-01

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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

Contributorsp. xiii
Prefacep. xxi
Volume in Seriesp. xxiii
Star Polymer Surface Passivation for Single-Molecule Detectionp. 1
Introductionp. 2
Surface Grafting of PEO and Protein Repellencep. 2
The NCO-sP(EO-stat-PO) Systemp. 4
Preparation of sP(EO-stat-PO)-Coated Substrates for Single-Molecule Experimentsp. 6
Analysis of Protein Structure and Function on NCO-sP(EO-stat-PO) Surfacesp. 11
Acknowledgmentsp. 16
Referencesp. 16
Azide-Specific Labeling of Biomolecules by Staudinger-Bertozzi Ligation: Phosphine Derivatives of Fluorescent Probes Suitable for Single-Molecule Fluorescence Spectroscopyp. 19
Introductionp. 20
Materials and Methodsp. 21
Acknowledgmentsp. 28
Referencesp. 28
Preparation of Fluorescent Pre-mRNA Substrates for an smFRET Study of Pre-mRNA Splicing in Yeastp. 31
Introductionp. 32
Identification of a Yeast Pre-mRNA with a Small Intron that is Spliced Efficiently In Vitrop. 32
Synthetic Fluorescent Ubc4 Pre-mRNAp. 33
Do the Dyes Affect the Efficiency of Splicing?p. 37
Mutant Pre-mRNAsp. 37
Tethering the Pre-mRNA to the Microscope Slidep. 38
Summary and Conclusionp. 39
Acknowledgmentsp. 40
Referencesp. 40
Nanovesicle Trapping for Studying Weak Protein Interactions by Single-Molecule FRETp. 41
Introductionp. 42
Nanovesicle Trapping Approachp. 44
smFRET Measurements of Weak Protein-Protein Interactionsp. 47
Single-Molecule Kinetic Analysis of Three-State Protein-Protein Interactionsp. 54
Further Developmentsp. 57
Concluding Remarksp. 58
Acknowledgmentsp. 59
Referencesp. 59
Droplet Confinement and Fluorescence Measurement of Single Moleculesp. 61
Introductionp. 62
Methods for Droplet Generationp. 65
Methods for Droplet Manipulationp. 69
Droplet Coalescence and Mixingp. 73
Experimental Considerations for Single Fluorophore Detectionp. 73
Single-Molecule Measurements in Dropletsp. 79
Future Prospectsp. 82
Acknowledgmentsp. 83
Referencesp. 84
Single-Molecule Fluorescence Spectroscopy Using Phospholipid Bilayer Nanodiscsp. 89
Introductionp. 90
Nanodiscs and HDL Particlesp. 91
Single-Molecule Techniques and Applications to Membrane Proteinsp. 95
Cytochrome P450 3A4 and Its Allosteric Behaviorp. 96
Image Filtering by Singular-Value Decompositionp. 102
Islet Amyloid Polypeptide Binding to Nanodiscsp. 106
-Synuclein Conformations on Nanodiscsp. 109
Summaryp. 112
Acknowledgmentsp. 112
Referencesp. 112
Single-Molecule Spectroscopy Using Microfluidic Platformsp. 119
Introductionp. 120
Microchip Fabricationp. 121
Instrumentation for Fluorescence Detectionp. 123
Detergent-Assisted Microchannel Electrophoresisp. 124
Fluorescence Correlation Spectroscopyp. 127
Acknowledgmentsp. 131
Referencesp. 131
Detection of Protein-Protein Interactions in the Live Cell Plasma Membrane by Quantifying Prey Redistribution upon Bait Micropatterningp. 133
Introductionp. 134
Methodological Requirementsp. 136
The Micropatterning Techniquep. 137
Experimental Designp. 139
Procedurep. 140
Interpretation of Resultsp. 145
Figures of Meritp. 147
Conclusionsp. 148
Acknowledgmentsp. 149
Referencesp. 149
Analysis of Complex Single-Molecule FRET Time Trajectoriesp. 153
Introductionp. 154
Analysis of Simple Trajectoriesp. 156
Analysis of Complex Trajectoriesp. 160
Post-HMM Processing and Data Visualizationp. 168
Acknowledgmentp. 176
Referencesp. 176
Single-Molecule Fluorescence Studies of Intrinsically Disordered Proteinsp. 179
Introductionp. 180
Single-Molecule Fluorescence Methodsp. 181
Site-Specific Labeling of Intrinsically Disordered Proteinsp. 187
Examples of SMF Characterization of IDP Structure and Dynamicsp. 190
Concluding Remarksp. 198
Acknowledgmentsp. 200
Referencesp. 200
Measuring the Energetic Coupling of Tertiary Contacts in RNA Folding using Single Molecule Fluorescence Resonance Energy Transferp. 205
Introductionp. 206
Thermodynamic Cooperativity Overviewp. 207
Measuring Folding Equilibrium in RNAp. 209
Designing an smFRET Experiment to Measure Cooperativityp. 210
Additional Commentsp. 217
Acknowledgmentsp. 219
Referencesp. 219
A Highly Purified, Fluorescently Labeled In Vitro Translation System for Single-Molecule Studies of Protein Synthesisp. 221
Introductionp. 222
A Highly Purified, Escherichia coli-Based In Vitro Translation Systemp. 225
Biochemical Assaysp. 233
Preparation of Fluorescently Labeled Translation Componentsp. 242
Conclusions and Future Perspectivesp. 253
Acknowledgmentsp. 254
Referencesp. 255
Watching Individual Proteins Acting on Single Molecules of DNAp. 261
Introductionp. 262
Preparation of DNA Substratesp. 265
Preparation of Fluorescent Proteinsp. 268
Instrumentp. 270
Single-Molecule Imaging of Proteins on DNAp. 280
Data Analysis Methodsp. 287
Acknowledgmentsp. 289
Referencesp. 289
DNA Curtains for High-Throughput Single-Molecule Optical Imagingp. 29
Introductionp. 294
Total Internal Reflection Fluorescence Microscopyp. 294
DNA Curtainsp. 297
Visualizing Protein-DNA Interactionsp. 307
Conclusions and Future Directionsp. 314
Acknowledgmentsp. 314
Referencesp. 314
Scanning FCS for the Characterization of Protein Dynamics in Live Cellsp. 317
Introductionp. 318
Implementationp. 320
Data Analysisp. 327
Applicationsp. 334
Conclusionp. 341
Referencesp. 342
Observing Protein Interactions and Their Stoichiometry in Living Cells by Brightness Analysis of Fluorescence Fluctuation Experimentsp. 345
Introductionp. 346
Brightness Classification of Fluorescent Moleculesp. 347
Brightness Measurements in Cellsp. 354
Acknowledgmentsp. 361
Referencesp. 361
Detection of Individual Endogenous RNA Transcripts In Situ Using Multiple Singly Labeled Probesp. 365
Introductionp. 366
Design and Synthesis of Fluorescent Oligonucleotide Probe Setsp. 369
Preparation of Samples for In Situ Hybridizationp. 373
Hybridizationp. 377
Imagingp. 381
Image Analysisp. 384
Acknowledgmentsp. 386
Referencesp. 386
Single mRNA Tracking in Live Cellsp. 387
Introductionp. 388
Significance of Tracking mRNAp. 389
Labeling mRNA in Living Cellsp. 391
Imaging mRNA Movementsp. 394
Analyzing mRNA Motionsp. 396
Conclusionsp. 402
Acknowledgmentsp. 403
Referencesp. 403
Single-Molecule Sequencing: Sequence Methods to Enable Accurate Quantitationp. 407
Introductionp. 408
Basic Principles of Single-Molecule Sequencingp. 409
Preparation of Genomic DNA for Single-Molecule Sequencingp. 410
Bacterial Genome Sequencingp. 416
Human Genome Sequencing and Quantitationp. 418
Chromatin Immunoprecipitation Studiesp. 421
Digital Gene Expression for Transcriptome Quantitationp. 423
Summaryp. 428
Acknowledgmentsp. 428
Referencesp. 430
Real-Time DNA Sequencing from Single Polymerase Moleculesp. 431
Introductionp. 432
Principle of Single-Molecule, Real-Time DNA Sequencingp. 433
Components of SMRT Sequencingp. 435
Single-Molecule DNA Polymerase Dynamicsp. 446
Conclusionsp. 451
Acknowledgmentsp. 452
Referencesp. 452
Author indexp. 457
Subject Indexp. 467
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