Fluorescent Analogs of Biomolecular Building Blocks

Marcus Wilhelmsson is an Associate Professor of Biophysical Chemistry at Chalmers University of Technology, Göteborg, Sweden.  He earned his Ph.D. in 2003 and thereafter entered the field of DNA-nanotechnology. In 2008 he joined Chalmers University of Technology as an Assistant Professor, where he is now working on the design, characterization and application of novel fluorescent nucleic acid base analogues. Yitzhak Tor is a Professor of Chemistry and Biochemistry and the George and Carol Lattimer Professor at the University of California, San Diego. He earned his Ph.D. in 1990 from the Weizmann Institute of Science and conducted postdoctoral work at the California Institute of Technology. His first faculty appointment was at the University of Chicago, followed by the University of California, San Diego since 1994.

List of Contributors xv

Preface xix

1 Fluorescence Spectroscopy 1
Renatus W. Sinkeldam, L. Marcus Wilhelmsson, and Yitzhak Tor

1.1 Fundamentals of Fluorescence Spectroscopy 1

1.2 Common Fluorescence Spectroscopy Techniques 3

1.2.1 Steady-State Fluorescence Spectroscopy 3

1.2.2 Time-Resolved Fluorescence Spectroscopy 5

1.2.3 Fluorescence Anisotropy 6

1.2.4 Resonance Energy Transfer and Quenching 7

1.2.5 Fluorescence Microscopy and Single Molecule Spectroscopy 8

1.2.6 Fluorescence-Based in vivo Imaging 9

1.3 Summary and Perspective 10

References 10

2 Naturally Occurring and Synthetic Fluorescent Biomolecular Building Blocks 15
Renatus W. Sinkeldam and Yitzhak Tor

2.1 Introduction 15

2.2 Naturally Occurring Emissive Biomolecular Building Blocks 16

2.3 Synthetic Fluorescent Analogs of Biomolecular Building Blocks 18

2.3.1 Synthetic Emissive Analogs of Membranes Constituents 19

2.3.2 Synthetic Emissive Analogs of Amino Acids 22

2.3.3 Synthetic Emissive Analogs of Nucleosides 24

2.4 Summary and Perspective 31

References 32

3 Polarized Spectroscopy with Fluorescent Biomolecular Building Blocks 40
Bo Albinsson and Bengt Nordén

3.1 Transition Moments 40

3.2 Linear Dichroism 41

3.3 Magnetic Circular Dichroism 45

3.4 F̈orster Resonance Energy Transfer (FRET) 46

3.5 Fluorescence Anisotropy 47

3.6 Fluorescent Nucleobases 47

3.7 Fluorescent Peptide Chromophores 48

3.8 Site-Specific Linear Dichroism (SSLD) 50

3.9 Single-Molecule Fluorescence Resonance Energy Transfer (smFRET) 50

3.10 Single-Molecule Fluorescence-Detected Linear Dichroism (smFLD) 51

References 53

4 Fluorescent Proteins: The Show Must go on! 55
Gregor Jung

4.1 Introduction 55

4.2 Historical Survey 55

4.3 Photophysical Properties 57

4.3.1 Absorption Properties and Color Hue Modification 57

4.3.2 Chromophore Formation 61

4.3.3 Fluorescence Color and Dynamics 64

4.3.4 Directional Properties along with Optical Transitions 68

4.3.5 Energy Transfer and Energy Migration 69

4.4 Photochemical Reactions 71

4.4.1 Excited-state Proton Transfer (ESPT) 71

4.4.2 Isomerization Reactions: Reversible Photoswitching 73

4.4.3 Photoconversion: Irreversible Bond Rupture 74

4.4.4 Other Photochemical Reactions 75

4.5 Ion Sensitivity 75

4.5.1 Ground-State Equilibria of Protonation States 75

4.5.2 Quenching by Small Ions 76

4.6 Relation Microscopy–Spectroscopy for Fluorescent Proteins 77

4.6.1 Brightness Alteration from Cuvette to Microscopic Experiments 77

4.6.2 Lessons from Microspectrometry 78

4.6.3 Tools for Advanced Microscopic Techniques 79

4.7 Prospects and Outlook 82

Acknowledgments 82

References 82

5 Design and Application of Autofluorescent Proteins by Biological Incorporation of Intrinsically Fluorescent Noncanonical Amino Acids 91
Patrick M. Durkin and Nediljko Budisa

5.1 Introduction 91

5.2 Design and Synthesis of Fluorescent Building Blocks in Proteins 97

5.2.1 Extrinsic Fluorescent Labels 97

5.2.2 Intrinsic Fluorescent Labels 98

5.2.3 Extrinsic Labels Chemically Ligated using Cycloaddition Chemistry 108

5.2.4 Modification of the Genetic Code to Incorporate ncAAs 109

5.3 Application of Fluorescent Building Blocks in Proteins 111

5.3.1 Azatryptophans 111

5.3.2 FlAsH-EDT2 Extrinsic Labeling System 112

5.3.3 Huisgen Dipolar Cycloaddition System 114

5.4 Conclusions 117

5.5 Prospects and Outlook 118

5.5.1 Heteroatom-Containing Trp Analogs 119

5.5.2 Expanded Genetic Code – Orthogonal Pairs 119

Acknowledgments 120

References 120

6 Fluoromodules: Fluorescent Dye–Protein Complexes for Genetically Encodable Labels 124
Bruce A. Armitage

6.1 Introduction 124

6.2 Fluoromodule Development and Characterization 126

6.2.1 Fluorogenic Dyes 128

6.2.2 Fluorogen-Activating Protein (FAP) Optimization 131

6.2.3 Fluoromodule Recycling 132

6.3 Implementation 132

6.3.1 Fusion Constructs for Protein Tagging 132

6.3.2 Protein Tagging and pH Sensing 133

6.3.3 Super-Resolution Imaging 133

6.3.4 Protease Biosensors 133

6.4 Conclusions 134

6.5 Prospects and Outlook 134

Acknowledgments 134

References 134

7 Design of Environmentally Sensitive Fluorescent Nucleosides and their Applications 137
Subhendu Sekhar Bag and Isao Saito

7.1 Introduction 137

7.1.1 Solvatochromic Fluorophores 138

7.1.2 Origin of Solvatochromism 139

7.2 Solvatochromic Fluorescent Nucleoside Analogs 140

7.2.1 Designing Criteria for Solvatochromic Fluorescent Nucleosides 140

7.3 Fluorescently Labeled Nucleosides and Oligonucleotide Probes: Covalent Attachment of Solvatochromic Fluorophores Onto the Natural Bases 141

7.3.1 Base-Discriminating Fluorescent Nucleosides (BDF) 142

7.4 Nucleosides with Dual Fluorescence for Monitoring DNA Hybridization 153

7.5 Approach for Developing Environmentally Sensitive Fluorescent (ESF) Nucleosides 154

7.5.1 Concept for Designing ESF Nucleosides 154

7.5.2 Examples and Photophysical Properties of ESF Nucleosides 156

7.6 Base-Selective Fluorescent ESF Probe 163

7.6.1 Cytosine Selective ESF Probe 163

7.6.2 Thymine Selective Fluorescent ESF Probe 163

7.6.3 Specific Detection of Adenine by Exciplex Formation with Donor-Substituted ESF Guanosine 165

7.7 Molecular Beacon (MB) and ESF Nucleosides 167

7.7.1 Ends-Free and Self-Quenched MB 167

7.7.2 Single-Stranded Molecular Beacon Using ESF Nucleoside in a Bulge Structure 168

7.8 Summary and Future Outlook 169

Acknowledgments 170

References 170

8 Expanding The Nucleic Acid Chemist’s Toolbox: Fluorescent Cytidine Analogs 174
Kirby Chicas and Robert H.E. Hudson

8.1 Introduction 174

8.2 Design and Characterization of Fluorescent C Analogs 176

8.2.1 1,3-Diaza-2-Oxophenothiazine (tC) 177

8.2.2 1,3-Diaza-2-Oxophenoxazine (tCO) 178

8.2.3 7-Nitro-1,3-Diaza-2-Oxophenothiazine (tCnitro) 179

8.2.4 G-Clamp and 8-oxoG-Clamp 179

8.2.5 Ç and Çf  181

8.2.6 Benzopyridopyrimidine (BPP) 182

8.2.7 Napthopyridopyrimidine (NPP) 183

8.2.8 dChpp 183

8.2.9 dChpd, dCmpp, dCtpp, dCppp 184

8.2.10 dCPPI 184

8.2.11 dxC 185

8.2.12 rxC 186

8.2.13 Methylpyrrolo-dC (MepdC) 186

8.2.14 5-(Fur-2-yl)-2′-Deoxycytidine (CFU) 187

8.2.15 Thiophen-2-yl pC 187

8.2.16 Thiophene Fused pC 188

8.2.17 Thieno[3,4-d]-Cytidine (thC) 189

8.2.18 Triazole Appended 190

8.3 Implementation 190

8.3.1 PNA 192

8.3.2 DNA 196

8.3.3 RNA 200

8.4 Conclusions 202

8.5 Prospects and Outlook 202

Acknowledgments 203

References 203

9 Synthesis and Fluorescence Properties of Nucleosides with Pyrimidopyrimidine-Type Base Moieties 208
Kohji Seio, Takashi Kanamori, Akihiro Ohkubo, and Mitsuo Sekine

9.1 Introduction 209

9.2 Discovery, Design, and Synthesis of Pyrimidopyrimidine Nucleosides 209

9.2.1 Synthesis and Fluorescence Properties of dChpp 209

9.2.2 Design, Synthesis, and Fluorescence Properties of dCPPP, dCPPI, and dCPPI Derivatives 212

9.2.3 Fluorescence Properties of the Oligonucleotides Containing dCPPI 213

9.3 Implementation 215

9.3.1 Application to a DNA Triplex System 215

9.3.2 Double Labeling of an Oligonucleotide with dCPPI and 2-Aminopurine 219

9.4 Conclusions 220

9.5 Prospects and Outlook 221

References 221

10 Förster Resonance Energy Transfer (FRET) Between Nucleobase Analogues – a Tool for Detailed Structure and Dynamics Investigations 224
L. Marcus Wilhelmsson

10.1 Introduction 224

10.2 The Tricyclic Cytosine Family 226

10.2.1 Structural Aspects, Dynamics, and Ability to Serve as Cytosine Analogs 228

10.2.2 Photophysical Properties 231

10.3 Development of the First Nucleic Acid Base Analog FRET Pair 234

10.3.1 The Donor–Acceptor Pair tCO –tCnitro 235

10.3.2 Applications of Tricyclic Cytosines in FRET Measurements 237

10.4 Conclusions 238

10.5 Prospects and Outlook 238

Acknowledgments 239

References 239

11 Fluorescent Purine Analogs that Shed Light on DNA Structure and Function 242
Anaëlle Dumas, Guillaume Mata, and Nathan W. Luedtke

11.1 Introduction 242

11.2 Design, Photophysical Properties, and Applications of Purine Mimics 244

11.2.1 Early Examples of Fluorescent Purine Mimics 245

11.2.2 Chromophore-Conjugated Purine Analogs 246

11.2.3 Pteridines 250

11.2.4 Isomorphic Purine Analogs 251

11.2.5 Fused-Ring Purine Analogs 252

11.2.6 Substituted Purine Derivatives 253

11.3 Implementation 258

11.3.1 Probing G-Quadruplex Structures with 2PyG 259

11.3.2 Energy Transfer Quantification 261

11.3.3 Metal-Ion Localization to N7 264

11.4 Conclusions 265

11.5 Prospects and Outlook 265

Appendix 268

References 268

12 Design and Photophysics of Environmentally Sensitive Isomorphic Fluorescent Nucleosides 276
Renatus W. Sinkeldam and Yitzhak Tor

12.1 Introduction 276

12.2 Designing Environmentally Sensitive Emissive Nucleosides 279

12.2.1 Structural and Electronic Elements that Impart Environmental Sensitivity 279

12.2.2 Sensitivity to Polarity 279

12.2.3 Sensitivity to Viscosity 281

12.2.4 Sensitivity to pH 282

12.3 Two Isomorphic Environmentally Sensitive Designs 282

12.4 Probing Environmental Sensitivity 283

12.4.1 Probing Sensitivity to Polarity 283

12.4.2 Probing Sensitivity to Viscosity 286

12.4.3 Probing Sensitivity to pH 288

12.5 Recent Advancements in Isomorphic Fluorescent Nucleoside Analogs 291

12.6 Summary 293

12.7 Prospects and Outlook 294

Acknowledgments 294

References 294

13 Site-Specific Fluorescent Labeling of Nucleic Acids by Genetic Alphabet Expansion Using Unnatural Base Pair Systems 297
Michiko Kimoto, Rie Yamashige, and Ichiro Hirao

13.1 Introduction 297

13.2 Development of Unnatural Base Pair Systems and Their Applications 299

13.2.1 Site-Specific Fluorescent Labeling of DNA by Unnatural Base Pair Replication Systems 301

13.2.2 Site-Specific Fluorescent Labeling of RNA by Unnatural Base Pair Transcription Systems 307

13.3 Implementation 310

13.3.1 Fluorescence Sensor System Using an RNA Aptamer by Fluorophore-Linked y Labeling 310

13.3.2 Local Structure Analyses of Functional RNA Molecules by s Labeling 313

13.4 Conclusions 315

13.5 Prospects and Outlook 316

Acknowledgments 317

References 317

14 Fluorescent C-Nucleosides and their Oligomeric Assemblies 320
Pete Crisalli and Eric T. Kool

14.1 Introduction 320

14.2 Design, Synthesis, Characterization, and Properties of Fluorescent C-Glycoside Monomers 322

14.2.1 Design of Fluorescent C-Glycoside Monomers 322

14.2.2 Synthesis of Fluorescent C-Glycoside Monomers 323

14.2.3 Characterization and Properties of Fluorescent C-glycoside Monomers 325

14.3 Implementation of Fluorescent C-Glycoside Monomers 327

14.3.1 Environmentally Sensitive Fluorophores 327

14.3.2 Pyrene Nucleoside in DNA Applications 330

14.4 Oligomers of Fluorescent C-Glycosides: Design, Synthesis, and Properties 335

14.4.1 Design of Fluorescent C-Glycoside Oligomers 335

14.4.2 Synthesis of Fluorescent C-Glycoside Oligomers 336

14.4.3 Characterization and Properties of Fluorescent C-Glycoside Oligomers 337

14.5 Implementation of Fluorescent C-Glycoside Oligomers 342

14.5.1 ODFs as Chemosensors in the Solution State 342

14.5.2 ODFs as Sensors in the Solid State 347

14.5.3 Alternative Designs of Oligomeric Fluorescent Glycosides 351

14.5.4 General Conclusions: Oligomers of Fluorescent C-glycosides 352

14.6 Conclusions 353

14.7 Prospects and Outlook 353

Acknowledgments 354

References 354

15 Membrane Fluorescent Probes: Insights and Perspectives 356
Amitabha Chattopadhyay, Sandeep Shrivastava, and Arunima Chaudhuri

Abbreviations 356

15.1 Introduction 357

15.2 NBD-Labeled Lipids: Monitoring Slow Solvent Relaxation in Membranes 358

15.3 n-AS Membrane Probes: Depth-Dependent Solvent Relaxation as Membrane Dipstick 359

15.4 Pyrene: a Multiparameter Membrane Probe 362

15.5 Conclusion and Future Perspectives 362

Acknowledgments 364

References 364

16 Lipophilic Fluorescent Probes: Guides to the Complexity of Lipid Membranes 367
Marek Cebecauer and Radek Šachl

16.1 Introduction 367

16.2 Lipids, Lipid Bilayers, and Biomembranes 368

16.3 Lipid Phases, Phase Separation, and Lipid Ordering 370

16.4 Fluorescent Probes for Membrane Studies 370

16.4.1 Fluorescently Labeled Lipids 371

16.4.2 Environment-Sensitive Membrane Probes 373

16.4.3 Specialized Techniques Using Fluorescent Probes to Investigate Membrane Properties 380

16.5 Conclusions 386

16.6 Prospects and Outlook 386

Acknowledgments 386

References 387

17 Fluorescent Neurotransmitter Analogs 393
James N. Wilson

17.1 Introduction 393

17.1.1 Structure of Neurotransmitters 393

17.1.2 Regulation of Neurotransmitters 394

17.1.3 Native Fluorescence of Neurotransmitters 395

17.1.4 Fluorescent Histochemical Techniques 396

17.2 Design and Optical Properties of Fluorescent Neurotransmitters 397

17.2.1 Early Examples 397

17.2.2 Recent Examples 398

17.3 Applications of Fluorescent Neurotransmitters 400

17.3.1 Probing Binding Pockets with Fluorescent Neurotransmitters 400

17.3.2 Imaging Transport and Release of Fluorescent Neurotransmitters 401

17.3.3 Enzyme Substrates 403

17.4 Conclusions 404

17.5 Prospects and Outlook 405

Acknowledgments 405

References 406

Index 409