Single-Molecule Tools for Bioanalysis

278 Pages·2022·24.122 MB·English

by  Shuo Huang

Single-Molecule
Tools for Bioanalysis

Single-Molecule

Tools for

Bioanalysis

edited by
Shuo Huang
Published by
Jenny Stanford Publishing Pte. Ltd.
Level 34, Centennial Tower
3 Temasek Avenue
Singapore 039190
Email: [email protected]
Web: www.jennystanford.com
British Library Cataloguing‑in‑Publication Data
A catalogue record for this book is available from the British Library.
Single-Molecule Tools for Bioanalysis
Copyright © 2022 Jenny Stanford Publishing Pte. Ltd.
All rights reserved. This book, or parts thereof, may not be reproduced in any
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ISBN 978-981-4800-44-0 (Hardcover)
ISBN 978-1-003-18913-8 (eBook)
Contents
Preface

Acknowledgements

ix
1. Single-Molecule Analysis by Biological Nanopores x1i

Yuqin Wang and Shuo Huang

1.1 Introduction 2

1.1.1 Single-Molecule Bioph ysics and Nanopore 2

1.1.2 Nanop ore Methods 4

1.1.3 Biological Nanopores
6
1.2 Methodology 7

1.2.1 Preparation and Engineering of

Biological Nanopores 7

1.2.2 The Instrument and the Device 8

1.2.3 The Electrochemistry Mechanism 10

1.2.4 The Nanopore Measurement 11

1.2.5 Measu rement Noise and Bandwidth 12

1.2.6 Data Analysis 12

1.3 Appli c ations 13

1.3.1 DNA Sensing and Sequencing
13
1.3.2 Efforts toward Protein Sequencing 17

1.3.3 Sensing of Small Molecules and

Single-Molecule C hemistry Intermediates 19

1.3.4 Single-Molecule Enzymology 22
2. 1O.4p ticSaulm Twmeaerzye arsn fdo Pr rMoaspneipcutsla tion of Single Molecules 2453

Guangtao Song and Yan Zeng

2.1 Introduction 44
2.2 Optical Trapping Theory 45
vi Contents

2.2.1 Rayleigh Optics Approxim
ation 46
2.2.2 Ray Optics Approximati on 47

2.2.3 Electromagnetic Theory

(MDSA Appro x imation) 47

2.3 Optical Tweezers Instr u mentation 49

2.3.1 Optical Setup 49

2.3.2 Trapping Laser 50

2.3.3 Beam Steering Module 51

2.3.4 Trapping Objectives 52

2.3.5 Position Detection 53

2.4 Force Calibration in Optical Tweezers 54

2.4.1 Viscous Drag Force Calibration 54

2.4.2 Brownian Motion Calibra
tion 55
2.4.3 Direct Measurement 55

2.5 Combined Optical Trapping and

Single-Molecule Fluorescence Spectr
oscopy 56
2.6 Nanophotonic Optical Tweezers 57

2.7 Appli c ations of Optical Tweezers in

Singl e -Molecule Manipulation
59
2.7.1 M echanical Properties of DNA 59

2.7.2 Folding and Structural Dynamics of

Proteins and Nucle i c Acids 60

2.7.3 Dynamics of Molecular Motors 63
3. 2S.i8n glSeu-Mmomleacruyl ea nBdio Pseernsspinegc tbivye Fluorescence Resonance
65
Energy Transfer 79

Ying Lu, Jianbing Ma, and Ming Li

3.1 Introduction 80

3.2 Imple mentation of smFRET 81

3.2.1 Optical Setup 81

3.2.2 Fluorophore Labeling 84

3.2.3 Surface Modificat ion 86

3.2.4 Photo-Protection Strategy 86

3.3 Appli c ations of smFRET 87

3.3.1 Structural Dynamics of Nucleic Acids 87

3.3.2 Protein Structural Dyna m ics 89

3.3.3 Biomolecular Interactions 91

3.4 New D evelopments of smFRET 93

3.4.1 Multicolor smFRET 93

3.4.2 Strategies to Break Concentrations Barrier 95
3.4.3 SmFRET under Forces 96
Contents vii

3.4.4 Surface-Induced Fl u orescence Attenuation 98

3.4.5 Quenchers-in-a-Liposome FRET 99
4. 3D.5N A SOurmigmamari ya sa nSidn gPleer-sMpoelcetcivuele Biosensors 110221

Travis A. Meyer, Qinyi Lu, Kristin Weiss, and Yonggang Ke

4.1 Introduction
122
4.2 Protein and Nucleic Acid Detection 126

4.3 Analysis of Biomolecu lar Interactions

and A ctivity 134

4.3.1 Protein Activity 141

4.3.2 Alternative Nucleic Acid Conforma
tions 146
4.4 Control and Visualization of Chemical Reactions 151

4.5 Photonic Techniques for Biotechnological

Applications 154
5. 4S.i6n glSeu-Mmomleacruyl ea nMda Fnuiptuurlaet Pioenr sbpye Mctaivgense tic Tweezers 116703

Zilong Guo and Hu Chen

5.1 Introduction 174

5.2 Princ i ples and Technical Details of

Magn etic Tweezers 176

5.2.1 Force Generation 177

5.2.2 Force Calibration 178

5.2.3 Torque Generation and Measurement 180

5.2.4 Extension Measurements 181

5.2.5 Data Analysis
183
5.3 Applications of Magnetic Tweezers 184

5.3.1 DNA Elasticity and Conformational

Transition 184

5.3.2 DNA Topoisomerase 186

5.3.3 DNA and RNA Helicase 187

5.3.4 DNA–Protein Interactions 189

5.3.5 Protein Folding and Unfolding 191

5.3.6 Protein–Protein In teractions 193

5.3.7 Mechanical Manipulation of Cells 194

5.4 Emer g ing Developments
195
5.4.1 Freely-Orbiting Magnetic Tweezers 195

5.4.2 Combination of Magnetic Tweezers

with Fluorescence 196

5.4.3 Fast Dynamics Studied by Electromagnets 197
5.5 Summary and Perspectives 197
viii Contents
6. Long-Time Recording of Single-Molecule Dynamics in

Solution by Anti-Brownian Trapping 213

Quan Wang, Elif Karasu, and Hugh Wilson

6.1 Intro d uction 214

6.2 Princ i ples of Anti-Brownian Trapping
217
6.2.1 Fundamentals 217

6.2.2 A Brief History of the Development of

the Technique 217

6.2.3 Essential Components of an ABEL Trap
220
6.3 Selected Applications of the ABEL Trap 229

6.3.1 Reaching Ultimate Limit: Trapping S
ingle
Organic Fluorophores in Solution 230

6.3.2 Dissecting Pigment Organization of

Single Biliproteins in Solution 231

6.3.3 Sensing Biomolecular Interactions by

Single-Molecule Transport 234
Index

6.4 Summary and Perspective 236
257
Preface
Single-molecule biophysics research is a highly interdisciplinary
study that requires diverse expertise in biology, chemistry, physics,
and engineering, aiming to understand biological processes at
single-molecule level against ensemble averaging. Investigations
of single-molecule biophysics have enabled direct measurement of
single-molecule properties that were not even previously feasible
by any ensemble methods. These achievements include, but are not
limited to, direct measurement of the elastic property of an individual
strand of nucleic acids, direct manipulation of nucleic acids or protein
molecules, optical imaging of cellular processes in a nanometer
resolution, direct torque measurement of a supercoiled DNA, and
several others. The fast development of the field has also stimulated
the invention and evolution of a large variety of emerging single-
molecule tools, which have enabled new concepts and applications
of bioanalysis.
Though there is an urgent need to systematically summarize these
achievements, it is too much for any individual review article to
achieve a full coverage with sufficient details. Written by young
experts in the field of single-molecule research, this book aims to
provide a systematic and in-depth recap of representative topics
of single-molecule bioanalysis. The book contains six chapters that
cover topics on nanopores, optical tweezers, single-molecule FRET,
DNA origami sensors, magnetic tweezers, and ABEL trap. Each
chapter provides the general concept and a brief history of the
methods, technical fundamentals, diversified forms of the methods,
and the representative applications of the methods. This makes
the book ideal as a textbook for a graduate-level course. In fact, the
materials in this book were indeed summarized from the lecture
notes of a graduate course supervised by the book editor at Nanjing
University since 2015.