Contents

1 Structure of Nucleosides 1

1.1 Introduction 1
1.2 Pyrimidine and Purine Bases 2

1.2.1 Pyrimidines 3
1.2.2 Purines 4
1.2.3 Nomenclature of Pyrimidines and Purines 4
1.2.4 Abbreviations 4

1.3 Carbohydrate Moieties of Nucleosides 5
1.4 Bonding Between Carbohydrate Moiety and Heterocyclic Base 7

1.4.1 Purine Nucleosides 7
1.4.2 Pyrimidine Nucleosides 9

1.5 Size of the Oxide Ring in the Sugar 10
1.6 Configuration of the Glycoside (Anomeric) Center 12
1.7 Nomenclature and Abridged Formulas of Nucleosides 15
1.8 Minor Nucleosides 18
1.9 Pseudouridine 23
1.10 Nucleoside Antibiotics 26

1.10.1 Purine Nucleosides 26
1.10.2 Pyrimidine Nucleosides 30

1.11 Other Nucleosides 31
References 32

2 Properties of Nucleosides 33

2.1 Heterocyclic Bases 33

2.1.1 General Concepts 33
2.1.2 Tautomerism 35
2.1.3 Reactions with Electrophilic Reagents 39
2.1.4 Reactions with Nucleophilic Reagents 44
2.1.5 Addition Reactions 47
2.1.6 Reactions Involving Exocyclic Amino Groups 50

2.2 Reactions at the Carbohydrate Moiety 52

2.2.1 Substitution for Hydrogen Atoms in Hydroxyl Groups 52
2.2.2 Oxidation 56

2.3 Reactions Involving Heterocyclic Bases and the Carbohydrate Moiety 58
2.4 Stability of N-Glycosidic Bonds 60

2.4.1 Effect of the Heterocyclic Base Species 60
2.4.2 Effect of Substituents in the Carbohydrate Moiety 62
2.4.3 Mechanism of Hydrolysis of N-Glycosidic Bonds 63

2.5 Properties of Pseudouridine 66
References 70

3 Structure of Nucleotides 71

3.1 Introduction 71
3.2 Nomenclature and Isomerism 73
3.3 Structure of Nucleotides 77

3.3.1 Nucleoside 5'-Phosphates 77
3.3.2 Nucleoside 3- and 2-Phosphates 83
3.3.3 Nucleoside Cyclic Phosphates 86
3.3.4 Nucleoside 3'(2'),5-Diphosphates 88

3.4 General Comments Regarding the Structure of Monomer Units in Nucleic Acids 89
References 92

4 Properties of Nucleotides 93

4.1 Acid-Base Behavior 93

4.1.1 Ionization of Bases 93
4.1.2 Ionization of Hydroxyl Groups in Pentose 96
4.1.3 Ionization of the Phosphate Group 96

4.2 Formation of Derivatives with Metals 98
4.3 Reactions at Heterocyclic Bases and Pentose 99
4.4 Some Properties of the Phosphate Group (General Concepts) 105

4.4.1 Structure of the Phosphate Group and the Mechanism of Nucleophilic Substitution at the Phosphorus Atom 106
4.4.2 Catalysis of Nucleophilic Substitution at the Phosphorus Atom 116
4.4.3 Hydrolysis of Alkyl Phosphates 119
4.4.4 Cyclic Phosphates 125
4.4.5 b-Elimination Reactions 131

4.5 Reactions of Nucleotides Involving the Phosphate Group 132

4.5.1 Chemical and Enzymatic Dephosphorylation 132
4.5.2 Migration of the Phosphate Group 134
4.5.3 Alkylation of the Phosphate Group 134
4.5.4 Activation of the Phosphate Group in Nucleotides. Synthesis of Some Derivatives with Respect to the Phosphate Group 135
4.5.5 Acylation of the Phosphate Group 144

4.6 Properties of Nucleotide Derivatives with Substituents in the Phosphate Group 145

4.6.1 Nucleoside Cyclic Phosphates 146
4.6.2 Alkyl Esters of Nucleotides 152
4.6.3 Mixed Anhydrides of Nueleotides 161
4.6.4 Amides of Nucleotides 169

4.7 Hydrolysis of N-Glycosidic Bonds 178
References 180

5 Primary Structure of Nucleic Acids 181

5.1 Introduction 181
5.2 Major Types of Nucleic Acids, Their Isolation and Characteristics 182

5.2.1 DNA 182
5.2.2 RNA 183

5.3 Nature of Internucleotide Linkages 184

5.3.1 Internucleotide Linkage in DNA 185
5.3.2 Internucleotide Linkage in RNA 187

5.4 Nomenclature, Abridged Formulas and Abbreviations 193
5.5 Nucleotide Composition 196

5.5.1 Composition of DNA 197
5.5.2 Composition of RNA 198

5.6 Sequence of Nucleotide Units 198

5.6.1 Internucleotide Bond-Breaking Enzymes (Nucleases) 200
5.6.1.1 Ribonucleases 201
5.6.1.2 Deoxyribonucleases 204
5.6.1.3 Nonspecific Nucleases 206
5.6.2 Methods for Determining the Nucleotide Sequence in Oligonucleotides 208

References 222

6 Determination of the Primary Structure of Nucleic Acids 223

6.1 Introduction 223
6.2 DNA Mapping 225
6.3 Restriction Endonucleases 227
6.4 Controlled Chemical DNA Cleavage Method (Chemical Sequencing) 230

6.4.1 Basic Principle of the Method 230
6.4.2 Chemical Methods for Specific Cleavage of Polydeoxyribonucleotide Chain 232
6.4.3 Obtaining Individual DNA Fragments and 32P-Labeling 240
6.4.4 Polyacrylamide Gel Electrophoresis: High-Resolution System for Separating Oligo(poly)nucleotides According to Chain Length 245
6.4.5 Direct Reading of Nucleotide Sequence from Autoradiogram 246
6.4.6 Double-stranded DNA Sequencing Strategy 248

6.5 DNA Sequencing by Polymerase Copying Method (Enzymatic Sequencing Method) 252

6.5.1 Basic Principle of the Method. Priming and Termination of Enzymatic Synthesis of DNA Copies 252
6.5.2 Strategy for Sequencing Large Single-stranded DNAs by the Enzymatic Method 257

6.6 Comparison of Chemical and Enzymatic DNA Sequencing Methods 266
6.7 Determination of Nucleotide Sequences in RNA 268

6.7.1 Direct Chemical Sequencing of RNAs Labeled at the 3' End 268
6.7.2 Direct Enzymatic Sequencing of RNA 274
6.7.3 Sequencing of High-Molecular Weight RNAs by Polymerase Copying with the Aid of Reverse Transcriptase 279

6.8 Solid-Phase Sequencing of DNA and RNA 281
6.9 Polymerase Chain Reactions in the Analysis of the Primary Structure of Nucleic Acids 283
6.10 Automation of the Nucleic Acid Sequencing Process 287
6.11 Computers in Nucleic Acid Sequencing 292
References 301

7 Conformation of Nucleic Acid Components. Macromolecular Structure of Polynucleotides 303

7.1 Conformation of Nucleosides and Nucleotides 304

7.1.1 Heterocyclic Bases 304
7.1.2 Ribose and Deoxyribose 305
7.1.3 Orientation of the Heterocyclic Bases Relative to the Sugar 307

7.2 Intermolecular Interactions Between the Heterocyclic Bases 312

7.2.1 Complementary Interactions 314
7.2.2 Stacking Interactions 317

7.3 Conformation of Single-Stranded Oligonucleotides and Polynucleotides 319
7.4 Structure of Double- and Multiple-Stranded Polynucleotide Complexes 325
7.5 Complexes of Polynucleotides with Mono- and Oligonucleotides 331
References 334

8 Macromolecular Structure of DNA and RNA. 335

8.1 DNA 335

8.1.1 The Watson and Crick Model 335
8.1.2 Polymorphism of the Double Helix of DNA 337
8.1.3 Single-Crystal X-Ray Structures of DNA 341
8.1.4 Denaturation and Renaturation of DNA 343
8.1.5 Some Aspects of DNA Behavior in Solution 350
8.1.6 Supercoiling of DNA 351
8.1.7 Unusual DNA Structures 354
8.1.8 Interaction of Ligands with Double Helices of DNA 357

8.2 RNA. 363

8.2.1 Secondary Structure of RNA. Fresco-Alberts-Doty Model 363
8.2.2 Elements of Secondary Structure 366
8.2.3 Macromolecular Structure of Transfer RNAs 371
8.2.4 Three-Dimensional Structure of High-Molecular Weight RNAs 381

References 388

 

9 Chemical Properties of Polynucleotides. Modification of Nucleic Acids 389

9.1 Introduction 389

9.2 Reactions of Heterocyclic Bases of Polynucleotides 391

9.2.1 General Concepts 391
9.2.2 Modification of Heterocycle Bases 398
9.2.2.1 Reactions at Carbon Atoms 398
9.2.2.2 Reactions at Pyridine.Nitrogens 412
9.2.2.3 Reactions at Pyrrole Nitrogens and Exocyclic Amino Groups 417

9.3 Hydrolysis of N-Glycosidic Bonds 426

9.3.1 Direct Hydrolysis Methods 426
9.3.2 Indirect Hydrolysis Methods 427

9.4 Reactions of Carbohydrate Moieties, 428

9.4.1. Substitution for Hydrogen Atoms in Hydroxyl Groups 428
9.4.2 Oxidation of 3-Terminal cis-Glycol Group in RNA 433

9.5 Reactions of Nucleotide Phosphate Groups 434

9.5.1 Cleavage of Internucleotide Linkages 435
9.5.2 Cleavage of the Sugar Phosphate Backbone with the Aid of "Chemical Nucleases" 445
9.5.3 Reactions with Internucleotide Linkages Remaining Intact 448

9.6 Reactions of Terminal Phosphate Groups 449
9.7 Chemical Probing of Nucleic Acid Structure 455

9.7.1 Location of Modified Nucleotides 455
9.7.2 Systematic Chemical Probing of RNA Secondary and Tertiary Structure 458
9.7.2.1 Transfer RNA 458
9.7.2.2 Ribosomal RNA 462
9.7.2.3 Searching for Unusual RNA Structures 463
9.7.3 Studying Conformational Changes in DNA 465
9.7.4 Evaluation of Four-stranded G4-DNA 468

References 469

10 Catalytic Activity of Nucleic Acids 471

10.1 RNA subunit of Ribonuclease P 471
10.2 Tetrahymena Ribozyme 474
10.3 Ribozymes Based on Self-Cleaving RNAs 483
References 489

11 Synthesis of Nucleic Acids 491

11.1 Introduction 491
11.2 Historical Background 493
11.3 General Principles of Chemoenzymatic Synthesis of Duplex DNAs 495

11.3.1 Implementation of the Principle of Complementarity of Heterocyclic Bases in Nucleic Acids 495
11.3.2 Ways to Produce Duplex DNAs 498
11.3.3 Enzymatic Methods for Construction of Duplex DNAs 500
11.3.4 Chemical Methods for Assembling Duplex DNAs 501

11.4    Methods for Chemical Synthesis of Oligodeoxyribonucleotides 505

11.4.1 Nucleoside and Nucleotide Components for Synthesis by the Phosphodiester and Phosphotriester Methods 506
11.4.1.1 Blocking of the Hydroxyl Groups of Pentose and Amino Groups in Heterocyclic Bases 506
11.4.1.2 Protection of Phosphate Groups 509
11.4.1.3 Preparation of the Nucleoside and Nucleotide Components 509
11.4.1.4 Mechanisms of Internucleotide Linkage Formation 512
11.4.2 Chemical Synthesis of Oligodeoxyribonucleotides in Solution 515
11.4.2.1 Phosphodiester Method 515
11.4.2.2 Phosphotriester Method 518
11.4.2.3 Phosphite triester Method 527

11.5 Solid-Phase Synthesis of Oligodeoxyribonucleotides and Its Automation 529

11.5.1 Basic Principle of Solid-Phase Synthesis 531
11.5.2 Polymer Supports and Immobilization of the First Monomer Thereon 532
11.5.3 Phosphoramidite-Triester Method 534
11.5.4 Hydrophosphoryl Method 537
11.5.5 Phosphotriester Method 540
11.5.6 Automation of Synthesis 541
11.5.7 Multiple Simultaneous Synthesis on Polymer Segments 542
11.5.8 Separation and Purification of Synthetic Oligonucleotides 545

11.6 Synthetic Genes and Their Cloning 546

11.6.1 Methods of Duplex DNA Construction 546
11.6.2 Synthesis of Genes and Their Functionally Important Segments 546

References 573

Index 575