Current Protocols in Nucleic Acid Chemistry

Current Protocols in Nucleic Acid Chemistry

Online ISBN: 9780471142706

DOI: 10.1002/0471142700

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  1. Foreword
  2. Preface
  3. Selected Suppliers of Reagents and Equipment
  4. Chapter 1 Synthesis of Modified Nucleosides
    1. Introduction
    2. UNIT 1.1 Palladium-Mediated C5 Substitution of Pyrimidine Nucleosides
    3. UNIT 1.2 Enzymatic Synthesis of M1G-Deoxyribose
    4. UNIT 1.3 Synthesis of N2-Substituted Deoxyguanosine Nucleosides from 2-Fluoro-6-O-(Trimethylsilylethyl)-2′-Deoxyinosine
    5. UNIT 1.4 Unnatural Nucleosides with Unusual Base Pairing Properties
    6. UNIT 1.5 Development of a Universal Nucleobase and Modified Nucleobases for Expanding the Genetic Code
    7. UNIT 1.6 Syntheses of Specifically 15N-Labeled Adenosine and Guanosine
    8. UNIT 1.7 Synthesis of Protected 2′-Deoxy-2′-fluoro-β-D-arabinonucleosides
    9. UNIT 1.8 Synthesis, Characterization, and Application of Substituted Pyrazolopyrimidine Nucleosides
    10. UNIT 1.9 Synthesis of 1,5-Anhydrohexitol Building Blocks for Oligonucleotide Synthesis
    11. UNIT 1.10 Synthesis and Properties of 7-Substituted 7-Deazapurine (Pyrrolo[2,3-d]pyrimidine) 2′-Deoxyribonucleosides
    12. UNIT 1.11 Reduction of Ribonucleosides to 2′-Deoxyribonucleosides
    13. UNIT 1.12 Synthesis of Fluorinated Nucleosides
    14. UNIT 1.13 Synthesis of Ribonucleosides by Condensation Using Trimethylsilyl Triflate
    15. UNIT 1.14 Synthesis of 2′-O-β-d-Ribofuranosylnucleosides
    16. UNIT 1.15 Preparation of 2′-Deoxy-2′-Methylseleno-Modified Phosphoramidites and RNA
    17. UNIT 1.16 Palladium-Catalyzed Cross-Coupling Reactions in C6 Modifications of Purine Nucleosides
    18. UNIT 1.17 Nucleobase-Caged Phosphoramidites for Oligonucleotide Synthesis
    19. UNIT 1.18 Synthesis of Altritol Nucleoside Phosphoramidites for Oligonucleotide Synthesis
    20. UNIT 1.19 Synthesis of a 4-Selenothymidine Phosphoramidite and Incorporation into Oligonucleotides
    21. UNIT 1.20 Synthesis of 2′-Cyclohexenylnucleosides and Corresponding CeNA Building Blocks
    22. UNIT 1.21 Synthesis of 5-Formyl-2′-Deoxyuridine and Its Incorporation into Oligodeoxynucleotides
    23. UNIT 1.22 O6-(Benzotriazol-1-yl)inosine Derivatives for C6 Modification of Purine Nucleosides
    24. UNIT 1.23 Synthesis of a 2-Selenothymidine Phosphoramidite and Its Incorporation into Oligodeoxyribonucleotides
    25. UNIT 1.24 Synthesis of 2′,4′-Bridged Nucleosides Using a New Orthogonally Protected Sugar Synthon: 5-O-(tert-Butyldiphenylsilyl)-4-C-Hydroxymethyl-1,2-O-Isopropylidene-3-O-Napthyl-α-d-Allofuranose
    26. UNIT 1.25 Synthesis of the Tellurium-Derivatized Phosphoramidites and Their Incorporation into DNA Oligonucleotides
    27. UNIT 1.26 Two-Step, One-Pot Synthesis of Inosine, Guanosine, and 2′-Deoxyguanosine O6-Ethers via Intermediate O6-(Benzotriazol-1-yl) Derivatives
    28. UNIT 1.27 Aqueous-Phase Sonogashira Alkynylation to Synthesize 5-Substituted Pyrimidine and 8-Substituted Purine Nucleosides
    29. UNIT 1.28 Solid-Phase Chemical Synthesis of 5′-Triphosphate DNA, RNA, and Chemically Modified Oligonucleotides
    30. UNIT 1.29 Synthesis of a North-Methanocarba-Thymidine (N-MCT) Analog
    31. UNIT 1.30 Use of a Novel 5′-Regioselective Phosphitylating Reagent for One-Pot Synthesis of Nucleoside 5′-Triphosphates from Unprotected Nucleosides
    32. UNIT 1.31 Synthesis and Characterization of Benzylidene Acetal–Type Bridged Nucleic Acids (BA-BNA)
    33. UNIT 1.32 Design and Synthesis of Triazolyl-Donor/Acceptor Unnatural Nucleosides and Oligonucleotide Probes Containing Triazolyl-Phenanthrene Nucleoside
  5. Chapter 2 Protection of Nucleosides for Oligonucleotide Synthesis
    1. Introduction
    2. UNIT 2.1 Nucleobase Protection of Deoxyribo- and Ribonucleosides
    3. UNIT 2.2 Protection of 2′-Hydroxy Functions of Ribonucleosides
    4. UNIT 2.3 Protection of 5′-Hydroxy Functions of Nucleosides
    5. UNIT 2.4 A Base-Labile Protecting Group (Fluorenylmethoxycarbonyl) for the 5′-Hydroxy Function of Nucleosides
    6. UNIT 2.5 2′-Hydroxyl-Protecting Groups that are Either Photochemically Labile or Sensitive to Fluoride Ions
    7. UNIT 2.6 Deoxyribo- and Ribonucleoside H-Phosphonates
    8. UNIT 2.7 Deoxyribonucleoside Phosphoramidites
    9. UNIT 2.8 Regioselective 2′-Silylation of Purine Ribonucleosides for Phosphoramidite RNA Synthesis
    10. UNIT 2.9 Preparation of 2′-O-[(Triisopropylsilyl)oxy]methyl-protected Ribonucleosides
    11. UNIT 2.10 Preparation of 5′-Silyl-2′-Orthoester Ribonucleosides for Use in Oligoribonucleotide Synthesis
    12. UNIT 2.11 Enzymatic Regioselective Levulinylation of 2′-Deoxyribonucleosides and 2′-O-Methylribonucleosides
    13. UNIT 2.12 Nucleobase Protection with Allyloxycarbonyl
    14. UNIT 2.13 Universal 2-(4-Nitrophenyl)ethyl and 2-(4-Nitrophenyl)ethoxycarbonyl Protecting Groups for Nucleosides and Nucleotides
    15. UNIT 2.14 Chromophoric 5′-O-Silyl Protection of N-Protected 2′-ACE Ribonucleosides for Solid-Phase RNA Synthesis
    16. UNIT 2.15 Chemical Synthesis of Oligoribonucleotides with 2′-O-(2-Cyanoethoxymethyl)-Protected Phosphoramidites
    17. UNIT 2.16 Recent Advances in the Chemical Synthesis of RNA
    18. UNIT 2.18 Synthesis of Branched DNA Using Oxidatively Cleavable Tritylsulfenyl as a Hydroxy Protecting Group
    19. UNIT 2.17 Synthesis of 5′-O-DMT-2′-O-TBS Mononucleosides Using an Organic Catalyst
  6. Chapter 3 Synthesis of Unmodified Oligonucleotides
    1. Introduction
    2. UNIT 3.1 Solid-Phase Supports for Oligonucleotide Synthesis
    3. UNIT 3.2 Attachment of Nucleosides and Other Linkers to Solid-Phase Supports for Oligonucleotide Synthesis
    4. UNIT 3.3 Synthetic Strategies and Parameters Involved in the Synthesis of Oligodeoxyribonucleotides According to the Phosphoramidite Method
    5. UNIT 3.4 Synthesis of Oligodeoxyribo- and Oligoribonucleotides According to the H-Phosphonate Method
    6. UNIT 3.5 Strategies for Oligoribonucleotide Synthesis According to the Phosphoramidite Method
    7. UNIT 3.6 Oligoribonucleotides with 2′-O-(tert-Butyldimethylsilyl) Groups
    8. UNIT 3.7 Synthesis of Oligoribonucleotides Using the 2-Nitrobenzyloxymethyl Group for 2′-Hydroxyl Protection
    9. UNIT 3.8 Chemical Synthesis of RNA Sequences with 2′-O-[(Triisopropylsilyl)oxy]methyl-protected Ribonucleoside Phosphoramidites
    10. UNIT 3.9 3-(N-tert-Butylcarboxamido)-1-propyl and 4-Oxopentyl Groups for Phosphate/Thiophosphate Protection in Oligodeoxyribonucleotide Synthesis
    11. UNIT 3.10 DNA Synthesis Without Base Protection
    12. UNIT 3.11 The 4-Methylthio-1-Butyl Group for Phosphate/Thiophosphate Protection in Oligodeoxyribonucleotide Synthesis
    13. UNIT 3.12 Nucleoside Phosphoramidites Containing Cleavable Linkers
    14. UNIT 3.13 Microwave-Assisted Functionalization of Solid Supports for Rapid Loading of Nucleosides
    15. UNIT 3.14 Solution-Phase Synthesis of Di- and Trinucleotides Using Polymer-Supported Reagents
    16. UNIT 3.15 DNA Synthesis Without Base Protection Using the Phosphoramidite Approach
    17. UNIT 3.16 A Universal and Recyclable Solid Support for Oligonucleotide Synthesis
    18. UNIT 3.17 Release of DNA Oligonucleotides and Their Conjugates from Controlled-Pore Glass Under Thermolytic Conditions
    19. UNIT 3.18 Nonenzymatic Oligomerization of Activated Nucleotides on Hairpin Templates
    20. UNIT 3.19 Chemical Synthesis of RNA with Base-Labile 2′-O-(Pivaloyloxymethyl)-Protected Ribonucleoside Phosphoramidites
    21. UNIT 3.20 RNA Synthesis by Reverse Direction Process: Phosphoramidites and High Purity RNAs and Introduction of Ligands, Chromophores, and Modifications at 3′-End
    22. UNIT 3.21 Safe Deprotection Strategy for the Tert-Butyldimethylsilyl (TBS) Group During RNA Synthesis
    23. UNIT 3.22 2′-Hydroxy Protection of Ribonucleosides as 2-Cyano-2,2-Dimethylethanimine-N-Oxymethyl Ethers in Solid-Phase Synthesis of RNA Sequences
  7. Chapter 4 Synthesis of Modified Oligonucleotides and Conjugates
    1. Introduction
    2. UNIT 4.1 A Status Update of Modified Oligonucleotides for Chemotherapeutics Applications
    3. UNIT 4.2 Modification of the 5′ Terminus of Oligonucleotides for Attachment of Reporter and Conjugate Groups
    4. UNIT 4.3 Direct Attachment of Conjugate Groups to the 5′ Terminus of Oligodeoxyribonucleotides
    5. UNIT 4.4 Synthesis and Characterization of Chimeric 2-5A-DNA Oligonucleotides
    6. UNIT 4.5 Attachment of Reporter and Conjugate Groups to the 3′ Termini of Oligonucleotides
    7. UNIT 4.6 3′-Modified Oligonucleotides and their Conjugates
    8. UNIT 4.7 Synthesis and Purification of Oligonucleotide N3′[RIGHTWARDS ARROW]P5′ Phosphoramidates and their Phosphodiester and Phosphorothioate Chimeras
    9. UNIT 4.8 Incorporation of Halogenoalkyl, 2-Pyridyldithioalkyl, or Isothiocyanate Linkers into Ligands
    10. UNIT 4.9 Modification of the 5′ Terminus of Oligodeoxyribonucleotides for Conjugation with Ligands
    11. UNIT 4.10 Conjugation of 5′-Functionalized Oligodeoxyribonucleotides with Properly Functionalized Ligands
    12. UNIT 4.11 Synthesis and Purification of Peptide Nucleic Acids
    13. UNIT 4.12 Locked Nucleic Acids: Synthesis and Characterization of LNA-T Diol
    14. UNIT 4.13 Cellular Delivery of Locked Nucleic Acids (LNAs)
    15. UNIT 4.14 Solid-Phase Synthesis of Branched Oligonucleotides
    16. UNIT 4.15 Solid-Phase Synthesis of 2′-Deoxy-2′-fluoro- β-D-Oligoarabinonucleotides (2′F-ANA) and Their Phosphorothioate Derivatives
    17. UNIT 4.16 Chemistry of CpG DNA
    18. UNIT 4.17 Synthesis of Phosphorothioate Oligonucleotides with Stereodefined Phosphorothioate Linkages
    19. UNIT 4.18 Synthesis of Oligonucleotide Conjugates via Aqueous Diels-Alder Cycloaddition
    20. UNIT 4.19 5′-Iodination of Solid-Phase-Linked Oligodeoxyribonucleotides
    21. UNIT 4.20 Reversible Biotinylation of the 5′-Terminus of Oligodeoxyribonucleotides and its Application in Affinity Purification
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      UNIT 4.21 Uridine 2′-Carbamates: Facile Tools for Oligonucleotide 2′-Functionalization
    23. UNIT 4.22 Stepwise Solid-Phase Synthesis of Nucleopeptides
    24. UNIT 4.23 Synthesis of Oligoribonucleotides Containing N6-Alkyladenosine and 2-Methylthio-N6-Alkyladenosine
    25. UNIT 4.24 Oligodeoxyribonucleotide Analogs Functionalized with Phosphonoacetate and Thiophosphonoacetate Diesters
    26. UNIT 4.25 Base-Modified Oligodeoxyribonucleotides: Using Pyrrolo[2,3-d]pyrimidines to Replace Purines
    27. UNIT 4.26 An Aminooxy-Functionalized Non-Nucleosidic Phosphoramidite for the Construction of Multiantennary Oligonucleotide Glycoconjugates on a Solid Support
    28. UNIT 4.27 Large-Scale Preparation of Conjugated Oligonucleoside Phosphorothioates by the High-Efficiency Liquid-Phase (HELP) Method
    29. UNIT 4.28 Disulfide Conjugation of Peptides to Oligonucleotides and Their Analogs
    30. UNIT 4.29 Methoxyoxalamido Chemistry in the Synthesis of Tethered Phosphoramidites and Functionalized Oligonucleotides
    31. UNIT 4.30 Using Morpholinos to Control Gene Expression
    32. UNIT 4.31 Solid-Phase Oligonucleotide Labeling with DOTA
    33. UNIT 4.32 Synthesis of Peptide-Oligonucleotide Conjugates by Diels-Alder Cycloaddition in Water
    34. UNIT 4.33 Synthesis of Alkyne- and Azide-Modified Oligonucleotides and Their Cyclization by the CuAAC (Click) Reaction
    35. UNIT 4.34 DNA Oligonucleotides Containing Stereodefined Phosphorothioate Linkages in Selected Positions
    36. UNIT 4.35 Heat-Activatable Primers for Hot-Start PCR: Oligonucleotide Synthesis and Basic PCR Setup
    37. UNIT 4.36 Oligodeoxynucleotides Containing N1-Methyl-2′-Deoxyadenosine and N6-Methyl-2′-Deoxyadenosine
    38. UNIT 4.37 Nucleoside Modification with Boron Clusters and Their Metal Complexes
    39. UNIT 4.38 Carbohydrate-Oligonucleotide Conjugates
    40. UNIT 4.39 Synthesis of 2′-Deoxyoxanosine from 2′-Deoxyguanosine, Conversion to Its Phosphoramidite, and Incorporation into Oxanine-Containing Oligodeoxynucleotides
    41. UNIT 4.40 Synthesis of Glycerol Nucleic Acid (GNA) Phosphoramidite Monomers and Oligonucleotide Polymers
    42. UNIT 4.41 Synthesis of Peptide-Oligonucleotide Conjugates Using a Heterobifunctional Crosslinker
    43. UNIT 4.42 Time-Dependent Thermocontrol of the Hydrophilic and Lipophilic Properties of DNA Oligonucleotide Prodrugs
    44. UNIT 4.43 Preparation of C5-Functionalized Locked Nucleic Acids (LNAs)
    45. UNIT 4.44 Synthesis of Stable Aminoacyl-tRNA Analogs
    46. UNIT 4.45 Preparation of Photoresponsive DNA Tethering Ortho-Methylated Azobenzene as a Supra-Photoswitch
    47. UNIT 4.46 RNA Aptamers and Spiegelmers: Synthesis, Purification, and Post-Synthetic PEG Conjugation
    48. UNIT 4.47 Preparation of DNA Containing 5-Hydroxymethyl-2′-Deoxycytidine Modification Through Phosphoramidites with TBDMS as 5-Hydroxymethyl Protecting Group
    49. UNIT 4.48 Synthesis and Application of Highly Reactive Amino Linkers for Functional Oligonucleotides
    50. UNIT 4.49 Oligodeoxynucleotide Containing S-Functionalized 2′-Deoxy-6-Thioguanosine: Facile Tools for Base-Selective and Site-Specific Internal Modification of RNA
    51. UNIT 4.50 Preparation of Azido Containing Oligonucleotides Through Diazo Transfer Reaction
    52. UNIT 4.51 Synthesis of Threose Nucleic Acid (TNA) Phosphoramidite Monomers and Oligonucleotide Polymers
    53. UNIT 4.52 Convenient and Efficient Approach to the Permanent or Reversible Conjugation of RNA and DNA Sequences with Functional Groups
    54. UNIT 4.53 Synthesis of Oligodeoxynucleotides with 5′-Caps Binding RNA Targets
    55. UNIT 4.54 Synthesis of Threose Nucleic Acid (TNA) Triphosphates and Oligonucleotides by Polymerase-Mediated Primer Extension
    56. UNIT 4.55 Non-Nucleoside Phosphoramidites of Xanthene Dyes (FAM, JOE, and TAMRA) for Oligonucleotide Labeling
    57. UNIT 4.56 Preparation of Oligodeoxyribonucleotides Containing the Pyrimidine(6–4)pyrimidone Photoproduct by Using a Dinucleotide Building Block
    58. UNIT 4.57 Synthesis of Triazole-Nucleoside Phosphoramidites and Their Use in Solid-Phase Oligonucleotide Synthesis
    59. UNIT 4.58 Synthesis of 8-Oxoguanosine Phosphoramidite and Its Incorporation into Oligoribonucleotides
    60. UNIT 4.59 Synthesis of Acetylene-Substituted Probes with Benzene-Phosphate Backbones for RNA Labeling
    61. UNIT 4.60 Using Triple-Helix-Forming Peptide Nucleic Acids for Sequence-Selective Recognition of Double-Stranded RNA
  8. Chapter 5 Methods for Cross-Linking Nucleic Acids
    1. Introduction
    2. UNIT 5.1 Engineering Disulfide Cross-Links in RNA Using Thiol-Disulfide Interchange Chemistry
    3. UNIT 5.2 Chemical and Enzymatic Methods for Preparing Circular Single-Stranded DNAs
    4. UNIT 5.3 Engineering Specific Cross-Links in Nucleic Acids Using Glycol Linkers
    5. UNIT 5.4 Engineering Disulfide Cross-Links in RNA Via Air Oxidation
    6. UNIT 5.5 Use of Electrophilic Substitution to Form Site-Specific Cross-Links in DNA
    7. UNIT 5.6 Synthesis of Endcap Dimethoxytrityl Phosphoramidites for Endcapped Oligonucleotides
    8. UNIT 5.7 Engineering Terminal Disulfide Bonds Into DNA
    9. UNIT 5.8 Preparation of DNA and RNA Fragments Containing Guanine N2-Thioalkyl Tethers
    10. UNIT 5.9 Synthesis of Building Blocks and Oligonucleotides with {G}O6-Alkyl-O6{G} Cross-Links
    11. UNIT 5.10 Syntheses of DNA Duplexes That Contain a N4C-Alkyl-N4C Interstrand Cross-Link
    12. UNIT 5.11 Synthesis of Building Blocks and Oligonucleotides with {T}N3-Alkylene-N3{T} Cross-Links
    13. UNIT 5.12 DNA Interstrand Cross-Link Formation Using Furan as a Masked Reactive Aldehyde
    14. UNIT 5.13 Synthesis of Building Blocks and Oligonucleotides Containing {T}O4-Alkylene-O4{T} Interstrand Cross-Links
    15. UNIT 5.14 Synthesis of G-N2-(CH2)3-N2-G Trimethylene DNA Interstrand Cross-Links
    16. UNIT 5.15 Synthesis of Oligonucleotides Containing 4,5′,8-Trimethylpsoralen at the 2′-O Position and Their Cross-Linking Properties with RNAs
  9. Chapter 6 Chemical and Enzymatic Probes for Nucleic Acid Structure
    1. Introduction
    2. UNIT 6.1 Probing RNA Structure with Chemical Reagents and Enzymes
    3. UNIT 6.2 Probing Nucleic Acid Structure with Shape-Selective Rhodium and Ruthenium Complexes
    4. UNIT 6.3 Probing RNA Structure by Lead Cleavage
    5. UNIT 6.4 Probing Nucleic Acid Structure with Nickel- and Cobalt-Based Reagents
    6. UNIT 6.5 Probing RNA Structures with Hydroxyl Radicals
    7. UNIT 6.6 Chemical Reagents for Investigating the Major Groove of DNA
    8. UNIT 6.7 Probing DNA Structure with Hydroxyl Radicals
    9. UNIT 6.8 Probing RNA Structure and Metal-Binding Sites Using Terbium(III) Footprinting
    10. UNIT 6.9 Probing RNA Structure and Function by Nucleotide Analog Interference Mapping
    11. UNIT 6.10 Bisulfite Modification for Analysis of DNA Methylation
    12. UNIT 6.11 Wavelength-Selective Uncaging of Oligonucleotides
  10. Chapter 7 Biophysical Analysis of Nucleic Acids
    1. Introduction
    2. UNIT 7.1 Biophysical Analysis of Nucleic Acids
    3. UNIT 7.2 NMR Determination of Oligonucleotide Structure
    4. UNIT 7.3 Optical Methods
    5. UNIT 7.4 Calorimetry of Nucleic Acids
    6. UNIT 7.5 Molecular Modeling of Nucleic Acid Structure
    7. UNIT 7.6 Methods to Crystallize RNA
    8. UNIT 7.7 Recent Advances in RNA Structure Determination by NMR
    9. UNIT 7.8 Molecular Modeling of Nucleic Acid Structure: Energy and Sampling
    10. UNIT 7.9 Molecular Modeling of Nucleic Acid Structure: Electrostatics and Solvation
    11. UNIT 7.10 Molecular Modeling of Nucleic Acid Structure: Setup and Analysis
    12. UNIT 7.11 Characterization of DNA Structures by Circular Dichroism
    13. UNIT 7.12 Biophysical Analysis of Triple-Helix Formation
    14. UNIT 7.13 Diffraction Techniques in Structural Biology
    15. UNIT 7.14 Determination of Nucleic Acid Hydration Using Osmotic Stress
    16. UNIT 7.15 Use of Chromophoric Ligands to Visually Screen Co-Crystals of Putative Protein-Nucleic Acid Complexes
    17. UNIT 7.16 Liquid Chromatography-Mass Spectrometry Analysis of DNA Polymerase Reaction Products
    18. UNIT 7.17 Attachment of Nitroxide Spin Labels to Nucleic Acids for EPR
    19. UNIT 7.18 Nucleic Acid Structure Characterization by Small Angle X-Ray Scattering (SAXS)
    20. UNIT 7.19 Quantitative Analyses of Nucleic Acid Stability Under the Molecular Crowding Condition Induced by Cosolutes
    21. UNIT 7.20 NMR Analysis of Base-Pair Opening Kinetics in DNA
    22. UNIT 7.21 Steady-State Kinetic Analysis of DNA Polymerase Single-Nucleotide Incorporation Products
    23. UNIT 7.22 Detection of Hydrogen Bonds in Dynamic Regions of RNA by NMR Spectroscopy
  11. Chapter 8 Nucleic Acid Binding Molecules
    1. Introduction
    2. UNIT 8.1 Determination of Binding Mode: Intercalation
    3. UNIT 8.2 Determination of Binding Thermodynamics
    4. UNIT 8.3 A Competition Dialysis Assay for the Study of Structure-Selective Ligand Binding to Nucleic Acids
    5. UNIT 8.4 Chemistry of Minor Groove Binder–Oligonucleotide Conjugates
    6. UNIT 8.5 A Fluorescent Intercalator Displacement Assay for Establishing DNA Binding Selectivity and Affinity
    7. UNIT 8.6 Synthesis of Dimeric 2-Amino-1,8-Naphthyridine and Related DNA-Binding Molecules
    8. UNIT 8.7 ICON Probes: Synthesis and DNA Methylation Typing
    9. UNIT 8.8 Binding to the DNA Minor Groove by Heterocyclic Dications: From AT-Specific Monomers to GC Recognition with Dimers
  12. Chapter 9 Combinatorial Methods in Nucleic Acid Chemistry
    1. Introduction
    2. UNIT 9.1 Theoretical Principles of In Vitro Selection Using Combinatorial Nucleic Acid Libraries
    3. UNIT 9.2 Design, Synthesis, and Amplification of DNA Pools for In Vitro Selection
    4. UNIT 9.3 In Vitro Selection of RNA Aptamers to a Protein Target by Filter Immobilization
    5. UNIT 9.4 Selection for Catalytic Function with Nucleic Acids
    6. UNIT 9.5 In Vitro Selection of RNA Aptamers to a Small Molecule Target
    7. UNIT 9.6 In Vitro Selection Using Modified or Unnatural Nucleotides
    8. UNIT 9.7 The Continuous Evolution In Vitro Technique
    9. UNIT 9.8 An In Vitro Selection Protocol for Threose Nucleic Acid (TNA) Using DNA Display
    10. UNIT 9.9 Compartmentalized Self-Tagging for In Vitro-Directed Evolution of XNA Polymerases
  13. Chapter 10 Purification and Analysis of Synthetic Nucleic Acids and Components
    1. Introduction
    2. UNIT 10.1 Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry of Oligonucleotides
    3. UNIT 10.2 Electrospray Ionization Mass Spectrometry of Oligonucleotides
    4. UNIT 10.3 Overview of Purification and Analysis of Synthetic Nucleic Acids
    5. UNIT 10.4 Polyacrylamide Gel Electrophoresis (PAGE) of Synthetic Nucleic Acids
    6. UNIT 10.5 Analysis and Purification of Synthetic Nucleic Acids Using HPLC
    7. UNIT 10.6 Base Composition Analysis of Nucleosides Using HPLC
    8. UNIT 10.7 Cartridge Methods for Oligonucleotide Purification
    9. UNIT 10.8 Analysis of Oxidized DNA Fragments by Gel Electrophoresis
    10. UNIT 10.9 Capillary Electrophoresis of DNA
    11. UNIT 10.10 Sequencing Oligonucleotides by Enrichment of Coupling Failures Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
    12. UNIT 10.11 Mass Determination of Phosphoramidites
    13. UNIT 10.12 3,4-Diaminobenzophenone Matrix for Analysis of Oligonucleotides by MALDI-TOF Mass Spectrometry
    14. UNIT 10.13 Detection of Aberrant 2′-5′ Linkages in RNA by Anion Exchange
    15. UNIT 10.14 Purification of Synthetic Oligonucleotides via Catching by Polymerization
  14. Chapter 11 RNA Folding Pathways
    1. Introduction
    2. UNIT 11.1 RNA Folding Pathways
    3. UNIT 11.2 RNA Secondary Structure Prediction
    4. UNIT 11.3 Thermal Methods for the Analysis of RNA Folding Pathways
    5. UNIT 11.4 Probing RNA Folding Pathways by RNA Fingerprinting
    6. UNIT 11.5 Characterization of Tertiary Folding of RNA by Circular Dichroism and Urea
    7. UNIT 11.6 Time-Resolved Hydroxyl Radical Footprinting of RNA with X-Rays
    8. UNIT 11.7 Rapid Magnesium Chelation as a Method to Study Real-Time Tertiary Unfolding of RNA
    9. UNIT 11.8 Use of Fluorescence Spectroscopy to Elucidate RNA Folding Pathways
    10. UNIT 11.9 Use of Chemical Modification To Elucidate RNA Folding Pathways
    11. UNIT 11.10 Probing RNA Structural Dynamics and Function by Fluorescence Resonance Energy Transfer (FRET)
    12. UNIT 11.11 Site-Specific Fluorescent Labeling of Large RNAs with Pyrene
    13. UNIT 11.12 RNA Intramolecular Dynamics by Single-Molecule FRET
    14. UNIT 11.13 Characterization of Conformational Dynamics of Bistable RNA by Equilibrium and Non-Equilibrium NMR
  15. Chapter 12 Nucleic Acid-Based Microarrays and Nanostructures
    1. Introduction
    2. UNIT 12.1 Key Experimental Approaches in DNA Nanotechnology
    3. UNIT 12.2 Preparation of Gold Nanoparticle–DNA Conjugates
    4. UNIT 12.3 Synthesis of 5′-O-Phosphoramidites with a Photolabile 3′-O-Protecting Group
    5. UNIT 12.4 Derivatization of Glass and Polypropylene Surfaces
    6. UNIT 12.5 DNA Microarray Preparation by Light-Controlled In Situ Synthesis
    7. UNIT 12.6 Preparation of α-Oxo Semicarbazone Oligonucleotide Microarrays
    8. UNIT 12.7 Synthesis of Covalent Oligonucleotide-Streptavidin Conjugates and Their Application in DNA-Directed Immobilization (DDI) of Proteins
    9. UNIT 12.8 Recent Progress in DNA Origami Technology
    10. UNIT 12.9 DNA Origami: Synthesis and Self-Assembly
  16. Chapter 13 Nucleoside Phosphorylation and Related Modifications
    1. Introduction
    2. UNIT 13.1 Overview of the Synthesis of Nucleoside Phosphates and Polyphosphates
    3. UNIT 13.2 Chemoenzymatic Preparation of Nucleoside Triphosphates
    4. UNIT 13.3 Synthesis and Polymerase Incorporation of 5′-Amino-2′,5′-Dideoxy-5′-N-Triphosphate Nucleotides
    5. UNIT 13.4 Nucleoside-5′-Phosphoimidazolides: Reagents for Facile Synthesis of Dinucleoside Pyrophosphates
    6. UNIT 13.5 Synthesis of Methylenebis(phosphonate) Analogs of Dinucleotide Pyrophosphates
    7. UNIT 13.6 Chemical Phosphorylation of Deoxyribonucleosides and Thermolytic DNA Oligonucleotides
    8. UNIT 13.7 Stereoselective Synthesis of Sugar Nucleotides Using Neighboring Group Participation
    9. UNIT 13.8 Solid-Supported Diphosphitylating and Triphosphitylating Reagents for Nucleoside Modification
    10. UNIT 13.9 Solid-Supported Reagents for Synthesis of Nucleoside Monothiophosphates, Dithiodiphosphates, and Trithiotriphosphates
    11. UNIT 13.10 Gram-Scale Chemical Synthesis of 2′-Deoxynucleoside-5′-O-Triphosphates
    12. UNIT 13.11 Rapid and Efficient Synthesis of Nucleoside Polyphosphates and Their Conjugates Using Sulfonyl Imidazolium Salts
    13. UNIT 13.12 A Simple Chemical Synthesis of Sugar Nucleoside Diphosphates in Water
    14. UNIT 13.13 Chemical Synthesis of Dinucleotide Cap Analogs
  17. Chapter 14 Biologically Active Nucleosides
    1. Introduction
    2. UNIT 14.1 Synthesis of Acyclic Analogs of Adenosine
    3. UNIT 14.2 Synthesis of Acyclic Nucleoside Phosphonates
    4. UNIT 14.3 Synthesis of β-L-2′-Deoxythymidine (L-dT)
    5. UNIT 14.4 Synthesis of Carbovir and Abacavir from a Carbocyclic Precursor
    6. UNIT 14.5 Synthesis of 2′- and 3′-C-Methylribonucleosides
    7. UNIT 14.6 Synthesis of Fluoroneplanocin A
    8. UNIT 14.7 Synthesis of Entecavir and Its Novel Class of Analogs
    9. UNIT 14.8 One-Flask Synthesis of Cyclic Diguanosine Monophosphate (c-di-GMP)
    10. UNIT 14.9 Biotinylation of a Propargylated Cyclic (3′-5′) Diguanylic Acid and of Its Mono-6-Thioated Analog Under “Click” Conditions
    11. UNIT 14.10 Anti-HIV Nucleoside Phosphonate GS-9148 and Its Prodrug GS-9131: Scale Up of a 2′-F Modified Cyclic Nucleoside Phosphonate and Synthesis of Selected Amidate Prodrugs
    12. UNIT 14.11 Olefin Cross-Metathesis for the Synthesis of Alkenyl Acyclonucleoside Phosphonates
    13. UNIT 14.12 Practical Synthesis of 4′-Thioribonucleosides Starting from D-Ribose
  18. Chapter 15 Nucleoside Prodrugs and Delivery Strategies
    1. Introduction
    2. UNIT 15.1 Synthesis of Amino Acid Phosphoramidate Monoesters via H-Phosphonate Intermediates
    3. UNIT 15.2 Synthesis of Cidofovir and (S)-HPMPA Ether Lipid Prodrugs
    4. UNIT 15.3 Chemistry of bisSATE Mononucleotide Prodrugs
    5. UNIT 15.4 Synthesis of Peptidomimetic Conjugates of Cyclic Nucleoside Phosphonates
    6. UNIT 15.5 Synthesis of Phosphoramidate Prodrugs: ProTide Approach
  19. Chapter 16 RNA Silencing
    1. Introduction
    2. UNIT 16.1 Overview of Gene Silencing by RNA Interference
    3. UNIT 16.2 Preparation of Short Interfering RNA Containing the Modified Nucleosides 2-Thiouridine, Pseudouridine, or Dihydrouridine
    4. UNIT 16.3 Chemical Modification of siRNA
    5. UNIT 16.4 Synthesis of Dumbbell-Shaped Cyclic RNAs for RNA Interference
  20. Chapter 17 Quadruplex Formation
    1. Introduction
    2. UNIT 17.1 UV Melting of G-Quadruplexes
    3. UNIT 17.2 Overview of Formation of G-Quadruplex Structures
    4. UNIT 17.3 Resolution of Quadruplex Polymorphism by Size-Exclusion Chromatography
    5. UNIT 17.4 Analysis of Multidimensional G-Quadruplex Melting Curves
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      UNIT 17.5 Sequence, Stability, and Structure of G-Quadruplexes and Their Interactions with Drugs
    7. UNIT 17.6 Crystallography of DNA and RNA G-Quadruplex Nucleic Acids and Their Ligand Complexes
  21. Appendix 1 Standard Nomenclature, Data, and Abbreviations
    1. APPENDIX 1A Selected Abbreviations Used in This Manual
    2. APPENDIX 1B Characteristics of Nucleic Acids
    3. APPENDIX 1C IUPAC-IUB Joint Commission on Biochemical Nomenclature Abbreviations and Symbols for the Description of Conformations of Polynucleotide Chains
    4. APPENDIX 1D Nucleoside and Nucleotide Nomenclature
    5. APPENDIX 1E A Convenient Stereochemical Notation for P-Chiral Nucleotide Analogs
  22. Appendix 2 Laboratory Stock Solutions and Equipment
    1. APPENDIX 2A Common Buffers and Stock Solutions
  23. Appendix 3 Commonly Used Techniques
    1. APPENDIX 3A References to Commonly Used Techniques
    2. APPENDIX 3B Denaturing Polyacrylamide Gel Electrophoresis
    3. APPENDIX 3C Introduction to the Synthesis and Purification of Oligonucleotides
    4. APPENDIX 3D Thin-Layer Chromatography
    5. APPENDIX 3E Column Chromatography
  24. Appendix 4 Resources
    1. APPENDIX 4A Useful Nucleic Acid Chemistry Web Sites