Single Molecule Tools, Part A: Fluorescence Based Approaches

1. Auflage 2010
ISBN: 978-0-08-095927-6
Verlag: Elsevier Science & Techn.
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, 528 Seiten, Web PDF

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

Autoren/Hrsg.

Inhaltsverzeichnis

1;Front Cover;1
2;Methods in Enzymology;4
3;Copyright Page;5
4;Contents;6
5;Contributors;14
6;Preface;22
7;Volumes in Series;24
8;Chapter 1: Star Polymer Surface Passivation for Single-Molecule Detection;52
8.1;1. Introduction;53
8.2;2. Surface Grafting of PEO and Protein Repellence;53
8.3;3. The NCO-sP(EO-stat-PO) System;55
8.4;4. Preparation of sP(EO-stat-PO)-Coated Substrates for Single-Molecule Experiments;57
8.5;5. Analysis of Protein Structure and Function on NCO-sP(EO-stat-PO) Surfaces;62
8.6;Acknowledgments;67
8.7;References;67
9;Chapter 2: Azide-Specific Labeling of Biomolecules by Staudinger-Bertozzi Ligation: Phosphine Derivatives of Fluorescent Probes Suitable for Single-Molecule Fluorescence Spectroscopy;70
9.1;1. Introduction;71
9.2;2. Materials and Methods;72
9.2.1;2.1. Materials;72
9.2.2;2.2. General methods;73
9.2.3;2.3. Synthesis of Alexa488-phosphine (Fig. 2.1);74
9.2.4;2.4. Synthesis of Cy3B-phosphine (Fig. 2.2);76
9.2.5;2.5. Synthesis of Alexa647-phosphine20 Å (Fig. 2.3A);76
9.2.6;2.6. Synthesis of Alexa647-phosphine24 Å (Fig. 2.3B);77
9.2.7;2.7. Azide-specific labeling;77
9.2.8;2.8. Quantitation of labeling efficiency;78
9.2.9;2.9. Quantitation of labeling specificity;78
9.3;Acknowledgments;79
9.4;References;79
10;Chapter 3: Preparation of Fluorescent Pre-mRNA Substrates for an smFRET Study of Pre-mRNA Splicing in Yeast;82
10.1;1. Introduction;83
10.2;2. Identification of a Yeast Pre-mRNA with a Small Intron that is Spliced Efficiently In Vitro;83
10.3;3. Synthetic Fluorescent Ubc4 Pre-mRNA;84
10.4;4. Do the Dyes Affect the Efficiency of Splicing?;88
10.5;5. Mutant Pre-mRNAs;88
10.6;6. Tethering the Pre-mRNA to the Microscope Slide;89
10.7;7. Summary and Conclusion;90
10.8;Acknowledgments;91
10.9;References;91
11;Chapter 4: Nanovesicle Trapping for Studying Weak Protein Interactions by Single-Molecule FRET;92
11.1;1. Introduction;93
11.2;2. Nanovesicle Trapping Approach;95
11.2.1;2.1. Lipid selection;96
11.2.2;2.2. Lipid nanovesicle preparation and protein trapping;97
11.3;3. smFRET Measurements of Weak Protein-Protein Interactions;98
11.3.1;3.1. Surface immobilization of nanovesicles;98
11.3.2;3.2. Control experiments;100
11.3.3;3.3. Application to weak interactions between intracellular copper transporters;102
11.4;4. Single-Molecule Kinetic Analysis of Three-State Protein-Protein Interactions;105
11.5;5. Further Developments;108
11.6;6. Concluding Remarks;109
11.7;Acknowledgments;110
11.8;References;110
12;Chapter 5: Droplet Confinement and Fluorescence Measurement of Single Molecules;112
12.1;1. Introduction;113
12.2;2. Methods for Droplet Generation;116
12.2.1;2.1. Emulsification;116
12.2.2;2.2. Injection;117
12.2.3;2.3. Microfluidics;119
12.3;3. Methods for Droplet Manipulation;120
12.3.1;3.1. Optical manipulation;120
12.3.2;3.2. Lab-on-chip methods for droplet manipulation;122
12.4;4. Droplet Coalescence and Mixing;124
12.5;5. Experimental Considerations for Single Fluorophore Detection;124
12.5.1;5.1. Protocol for aligning the apparatus;127
12.5.2;5.2. Protocol for preparation of emulsion samples;128
12.5.3;5.3. Protocol for droplet injection;128
12.6;6. Single-Molecule Measurements in Droplets;130
12.7;7. Future Prospects;133
12.8;Acknowledgments;134
12.9;References;135
13;Chapter 6: Single-Molecule Fluorescence Spectroscopy Using Phospholipid Bilayer Nanodiscs;140
13.1;1. Introduction;141
13.2;2. Nanodiscs and HDL Particles;142
13.2.1;2.1. POPC–MSP1D1 discs;143
13.3;3. Single-Molecule Techniques and Applications to Membrane Proteins;146
13.4;4. Cytochrome P450 3A4 and Its Allosteric Behavior;147
13.4.1;4.1. Incorporation of CYP3A4 in Nanodiscs;150
13.4.2;4.2. Surface attachment of CYP3A4–Nanodiscs;151
13.5;5. Image Filtering by Singular-Value Decomposition;153
13.5.1;5.1. SVD-based image filtering pseudocode;155
13.6;6. Islet Amyloid Polypeptide Binding to Nanodiscs;157
13.6.1;6.1. FCS measurement of rIAPP binding Nanodiscs;158
13.7;7. a-Synuclein Conformations on Nanodiscs;160
13.7.1;7.1. smFRET measurement of aS bound to Nan;161
13.8;8. Summary;163
13.9;Acknowledgments;163
13.10;References;163
14;Chapter 7: Single-Molecule Spectroscopy Using Microfluidic Platforms;170
14.1;1. Introduction;171
14.2;2. Microchip Fabrication;172
14.2.1;2.1. Design drawing and photomask printing;172
14.2.2;2.2. Molding master fabrication;173
14.2.3;2.3. Fabrication of PDMS chip;173
14.3;3. Instrumentation for Fluorescence Detection;174
14.4;4. Detergent-Assisted Microchannel Electrophoresis;175
14.4.1;4.1. Preparation of separation buffer and sample solutions;176
14.4.2;4.2. Microchip electrophoresis with electrokinetic injection;176
14.5;5. Fluorescence Correlation Spectroscopy;178
14.6;Acknowledgments;182
14.7;References;182
15;Chapter 8: Detection of Protein-Protein Interactions in the Live Cell Plasma Membrane by Quantifying Prey Redistribution upon Bait Micropatterning;184
15.1;1. Introduction;185
15.2;2. Methodological Requirements;187
15.2.1;2.1. Detection of weak interactions;187
15.2.2;2.2. Quantification;187
15.2.3;2.3. Applicability to living cells;187
15.2.4;2.4. Applicability to plasma membrane proteins;188
15.2.5;2.5. Dynamic range/sensitivity;188
15.2.6;2.6. False negatives/false positives;188
15.2.7;2.7. High throughput capabilities;188
15.3;3. The Micropatterning Technique;188
15.4;4. Experimental Design;190
15.4.1;4.1. Cellular expression system;190
15.4.2;4.2. Capture ligand;190
15.4.3;4.3. Instrumentation;190
15.4.4;4.4. Chip production;190
15.5;5. Procedure;191
15.5.1;5.1. Microcontact printing;192
15.5.2;5.2. Incubation of cells onto the micropatterned surface;193
15.5.3;5.3. Microscopy;194
15.5.4;5.4. Data analysis;195
15.6;6. Interpretation of Results;196
15.7;7. Figures of Merit;198
15.8;8. Conclusions;199
15.9;Acknowledgments;200
15.10;References;200
16;Chapter 9: Analysis of Complex Single-Molecule FRET Time Trajectories;204
16.1;1. Introduction;205
16.2;2. Analysis of Simple Trajectories;207
16.2.1;2.1. Selection of trajectories for analysis;208
16.2.2;2.2. Analysis of FRET state distribution;209
16.2.3;2.3. Kinetic analysis with thresholding algorithms;209
16.3;3. Analysis of Complex Trajectories;211
16.3.1;3.1. Hidden Markov analysis of complex FRET trajectories;211
16.3.2;3.2. Overview of HMM software available for smFRET analysis;214
16.3.3;3.3. Preprocessing trajectories for analysis by HMM;215
16.3.4;3.4. Selecting the appropriate number of FRET states;218
16.4;4. Post-HMM Processing and Data Visualization;219
16.4.1;4.1. Local detection of correlation based on HMM;219
16.4.2;4.2. Data condensation and visualization;220
16.4.3;4.3. Applications to single-molecule studies of yeast pre-mRNA splicing;222
16.4.4;4.4. Summary of a detailed strategy for analysis of complex trajectories with QuB;225
16.5;Acknowledgments;227
16.6;References;227
17;Chapter 10: Single-Molecule Fluorescence Studies of Intrinsically Disordered Proteins;230
17.1;1. Introduction;231
17.2;2. Single-Molecule Fluorescence Methods;232
17.2.1;2.1. Single-molecule fluorescence resonance energy transfer;233
17.2.2;2.2. Dual-color single-molecule coincidence;236
17.2.3;2.3. Fluorescence correlation spectroscopy;237
17.3;3. Site-Specific Labeling of Intrinsically Disordered Proteins;238
17.3.1;3.1. Common chemistries for protein labeling with fluorescent dyes;238
17.3.2;3.2. Advanced techniques for protein dual-labeling for smFRET;240
17.4;4. Examples of SMF Characterization of IDP Structure and Dynamics;241
17.4.1;4.1. Application 1: Structural properties and dynamics of the prion determiningregion of the yeast prion protein Sup35;242
17.4.2;4.2. Application 2: Denaturant-induced expansion of the a-synuclein disordered state;244
17.4.3;4.3. Application 3: Coupled binding and folding of a-synuclein;246
17.5;5. Concluding Remarks;249
17.6;Acknowledgments;251
17.7;References;251
18;Chapter 11: Measuring the Energetic Coupling of Tertiary Contacts in RNA Folding using Single Molecule Fluorescence Resonance;256
18.1;1. Introduction;257
18.2;2. Thermodynamic Cooperativity Overview;258
18.3;3. Measuring Folding Equilibrium in RNA;260
18.4;4. Designing an smFRET Experiment to Measure Cooperativity;261
18.4.1;4.1. Identification of tertiary contacts;261
18.4.2;4.2. Knocking out tertiary contacts;263
18.4.3;4.3. Designing single molecule constructs;263
18.4.4;4.4. Validating a new single-molecule construct;266
18.4.5;4.5. Measurement of cooperativity;268
18.5;5. Additional Comments;268
18.6;Acknowledgments;270
18.7;References;270
19;Chapter 12: A Highly Purified, Fluorescently Labeled In Vitro Translation System for Single-Molecule Studies of Protein Synthesis;272
19.1;1. Introduction;273
19.2;2. A Highly Purified, Escherichia coli-Based In Vitro Translation System;276
19.2.1;2.1. Tris–polymix buffer system;276
19.2.2;2.2. Preparation and purification of ribosomes and ribosomal subunits;277
19.2.3;2.3. Preparation of mRNAs;277
19.2.4;2.4. Preparation and purification of fMet-tRNAfMet, Phe-tRNAPhe, and Lys-tRNALys;278
19.2.5;2.5. Preparation and purification of translation factors;279
19.3;3. Biochemical Assays;284
19.3.1;3.1. Initiation assays;284
19.3.2;3.2. Elongation assays;288
19.3.3;3.3. Termination assays;290
19.3.4;3.4. Ribosome recycling assays;292
19.4;4. Preparation of Fluorescently Labeled Translation Components;293
19.4.1;4.1. Phylogenetic analysis/structural modeling;294
19.4.2;4.2. Ribosome labeling;295
19.4.3;4.3. tRNA labeling;300
19.4.4;4.4. Translation factor labeling;303
19.5;5. Conclusions and Future Perspectives;304
19.6;Acknowledgments;305
19.7;References;306
20;Chapter 13: Watching Individual Proteins Acting on Single Molecules of DNA;312
20.1;1. Introduction;313
20.2;2. Preparation of DNA Substrates;316
20.2.1;2.1. Preparation of biotinylated lambda DNA ;316
20.2.2;2.2. Preparation of DNA–bead complexes;316
20.2.3;2.3. Preparation of DNA–bead complexes end-labeled with Cy3-labeled antibody;317
20.3;3. Preparation of Fluorescent Proteins;319
20.3.1;3.1. RecBCD labeled with a fluorescent nanoparticle (RecBCD–nanoparticle);319
20.3.2;3.2. Rad54/Tid1 labeled with a fluorescent antibody (FITC–Rad54/Tid1);319
20.3.3;3.3. Chemically modified fluorescent RecA or Rad51 proteins (RecAFAM/Rad51FAM);320
20.4;4. Instrument;321
20.4.1;4.1. Flow cell design;321
20.4.2;4.2. Flow cell fabrication;322
20.4.3;4.3. Microscope with laser trap and microfluidic system;324
20.4.4;4.4. Temperature determination and control;327
20.5;5. Single-Molecule Imaging of Proteins on DNA;331
20.5.1;5.1. Unwinding of DNA by a single RecBCD enzyme;332
20.5.2;5.2. Direct observation of RecBCD–nanoparticle translocation;332
20.5.3;5.3. Rad54/Tid1 translocation;334
20.5.4;5.4. Real-time Rad51 assembly;335
20.5.5;5.5. Real-time Rad51 disassembly;336
20.5.6;5.6. Visualization of RecAFAM/RecA-RFP/Rad51FAM filament formation;337
20.6;6. Data Analysis Methods;338
20.6.1;6.1. Two-dimensional Gaussian fitting;339
20.6.2;6.2. Automatic DNA length measurement;339
20.7;Acknowledgments;340
20.8;References;340
21;Chapter 14: DNA Curtains for High-Throughput Single-Molecule Optical Imaging;344
21.1;1. Introduction;345
21.2;2. Total Internal Reflection Fluorescence Microscopy;345
21.2.1;2.1. General description of TIRFM;346
21.2.2;2.2. Building a simple prism-type TIRFM;346
21.2.3;2.3. Flowcells and injection system;346
21.3;3. DNA Curtains;348
21.3.1;3.1. DNA curtains as a method for aligning thousands of DNA molecules;348
21.3.2;3.2. Manually etched diffusion barriers;349
21.3.3;3.3. Nanofabricated linear diffusion barriers;349
21.3.4;3.4. More complex barrier patterns;354
21.3.5;3.5. Trouble-shooting;357
21.4;4. Visualizing Protein-DNA Interactions;358
21.4.1;4.1. Quantum dots as a general fluorescent labeling strategy;358
21.4.2;4.2. Visualizing ATP-dependent DNA translocation;359
21.4.3;4.3. Using DNA curtains to image nucleosomes;361
21.4.4;4.4. Diffusion of MMR along DNA;361
21.5;5. Conclusions and Future Directions;365
21.6;Acknowledgments;365
21.7;References;365
22;Chapter 15: Scanning FCS for the Characterization of Protein Dynamics in Live Cells;368
22.1;1. Introduction;369
22.2;2. Implementation;371
22.2.1;2.1. Scan paths;373
22.2.2;2.2. Calibration of the scan path;377
22.3;3. Data Analysis;378
22.3.1;3.1. Calculation of correlation curves;378
22.3.2;3.2. Corrections;381
22.3.3;3.3. Data fitting;383
22.4;4. Applications;385
22.4.1;4.1. sFCS in Caenorhabditis elegans embryo;385
22.4.2;4.2. Small-circle sFCS;386
22.4.3;4.3. sFCS with a perpendicular scan path to measure in unstable membranes;388
22.4.4;4.4. Dual-focus sFCS;390
22.4.5;4.5. Dual-color sFCS;391
22.5;5. Conclusion;392
22.6;References;393
23;Chapter 16: Observing Protein Interactions and Their Stoichiometry in Living Cells by Brightness Analysis of Fluorescence Fluctuation Experiments;396
23.1;1. Introduction;397
23.2;2. Brightness Classification of Fluorescent Molecules;398
23.2.1;2.1. Single brightness state;398
23.2.2;2.2. Two brightness states;400
23.2.3;2.3. Fluorescent proteins;405
23.3;3. Brightness Measurements in Cells;405
23.3.1;3.1. Brightness titration;405
23.3.2;3.2. Control and calibration experiments;407
23.3.3;3.3. Cell selection and measurement;408
23.4;Acknowledgments;412
23.5;References;412
24;Chapter 17: Detection of Individual Endogenous RNA Transcripts In Situ Using Multiple Singly Labeled Probes;416
24.1;1. Introduction;417
24.2;2. Design and Synthesis of Fluorescent Oligonucleotide Probe Sets;420
24.2.1;2.1. Design;420
24.2.2;2.2. Synthesis and purification;420
24.3;3. Preparation of Samples for In Situ Hybridization;424
24.3.1;3.1. Fixation solutions;424
24.3.2;3.2. Fixation protocols;426
24.4;4. Hybridization;428
24.4.1;4.1. Hybridization solutions;429
24.4.2;4.2. Hybridization protocols;430
24.5;5. Imaging;432
24.5.1;5.1. Microscopy equipment;432
24.6;6. Image Analysis;435
24.7;Acknowledgments;437
24.8;References;437
25;Chapter 18: Single mRNA Tracking in Live Cells;438
25.1;1. Introduction;439
25.2;2. Significance of Tracking mRNA;440
25.3;3. Labeling mRNA in Living Cells;442
25.3.1;3.1. Selection of probes for SPT;442
25.3.2;3.2. The MS2-GFP system;443
25.3.3;3.3. Minimizing photobleaching and phototoxicity;444
25.4;4. Imaging mRNA Movements;445
25.4.1;4.1. Experimental considerations;445
25.4.2;4.2. Instrumentation;446
25.4.3;4.3. 3D tracking;447
25.5;5. Analyzing mRNA Motions;447
25.5.1;5.1. Localization algorithms;448
25.5.2;5.2. Tracking algorithms;449
25.5.3;5.3. Categories of single particle motion;450
25.5.4;5.4. Interpretation of mRNA tracking data;452
25.6;6. Conclusions;453
25.7;Acknowledgments;454
25.8;References;454
26;Chapter 19: Single-Molecule Sequencing: Sequence Methods to Enable Accurate Quantitation;458
26.1;1. Introduction;459
26.2;2. Basic Principles of Single-Molecule Sequencing;460
26.3;3. Preparation of Genomic DNA for Single-Molecule Sequencing;461
26.3.1;3.1. DNA fragmentation and quantitation;462
26.3.2;3.2. Poly-A tailing;466
26.3.3;3.3. 3prime end blocking;466
26.4;4. Bacterial Genome Sequencing;467
26.4.1;4.1. Preparation and sequencing of bacterial DNA;467
26.4.2;4.2. Assessment of coverage and lack of bias;468
26.5;5. Human Genome Sequencing and Quantitation;469
26.5.1;5.1. Copy number variation;471
26.6;6. Chromatin Immunoprecipitation Studies;472
26.6.1;6.1. Preparation of ChIP DNA;473
26.6.2;6.2. ChIP DNA poly-A tailing;474
26.6.3;6.3. ChIP DNA 3prime blocking;474
26.7;7. Digital Gene Expression for Transcriptome Quantitation;474
26.7.1;7.1. Methodology for single-molecule sequencing digital gene expression;475
26.7.2;7.2. Demonstration of DGE counting reproducibility;479
26.8;8. Summary;479
26.9;Acknowledgments;479
26.10;References;481
27;Chapter 20: Real-Time DNA Sequencing from Single Polymerase Molecules;482
27.1;1. Introduction;483
27.2;2. Principle of Single-Molecule, Real-Time DNA Sequencing;484
27.3;3. Components of SMRT Sequencing;486
27.3.1;3.1. Zero-mode waveguides for observation volume confinement;486
27.3.2;3.2. ZMW surface derivatization for targeted enzyme immobilization;486
27.3.3;3.3. Phospholinked nucleotides for uninterrupted DNA polymerization;488
27.3.4;3.4. DNA polymerase—the sequencing ‘‘engine’’;491
27.3.5;3.5. Instrument for highly parallel monitoring of sequencing reactions;492
27.3.6;3.6. DNA sequencing assay example;494
27.3.7;3.7. Data analysis;495
27.4;4. Single-Molecule DNA Polymerase Dynamics;497
27.4.1;4.1. Determination of single-molecule kinetic parameters;497
27.4.2;4.2. DNA polymerase pausing;498
27.5;5. Conclusions;502
27.6;Acknowledgments;503
27.7;References;503
28;Author Index;508
29;Subject Index;518
30;Color Plate;528