Current Protocols in Nucleic Acid Chemistry
Table of Contents

Foreword
Preface
Contributors

1 Synthesis of Modified Nucleosides

1.1 Palladium-Mediated C5 Substitution of Pyrimidine Nucleosides
1.2 Enzymatic Synthesis of M1G-Deoxyribose
1.3 Synthesis of N2 Substituted Deoxyguanosine Nucleosides from 2-Fluoro-6-O-(Trimethylsilyethyl)-2'-Deoxyinosine
1.4 Unnatural Nucleosides with Unusual Base Pairing Properties
1.5 Development of a Universal Nucleobase and Modified Nucleobases for Expanding the Genetic Code
1.6 Syntheses of Specifically 15N-Labeled Adenosine and Guanosine
1.7 Synthesis of Protected 2’-Deoxy-2’-fluoro- b-D-Arabinonucleosides
1.8 Synthesis, Characterization, and Application of Substituted Pyrazolopyrimidine Nucleosides
1.9 Synthesis of 1,5-Anhydrohexitol Building Blocks for Oligonucleotide Synthesis

2 Protection of Nucleosides for Oligonucleotide Synthesis

2.1 Nucleobase Protection of Deoxyribo- and Ribonucleosides
2.2 Protection of the 2'-Hydroxy Functions of Ribonucleosides
2.3 Protection of the 5'-Hydroxy Functions of Nucleosides
2.4 A Base-Labile Protecting Group for the 5'-Hydroxy Function of Nucleosides
2.5 2'-Hydroxyl-Protecting Groups That Are Either Photochemically Labile or Sensitive to Fluoride Ions
2.6 Deoxyribo- and Ribonucleoside H-Phosphonates
2.7 Deoxyribonucleoside Phosphoramidites
2.8 Regioselective 2'-Silylation of Purine Ribonucleosides for Phosphoramidite RNA Synthesis
2.9 Preparation of 2'-O-[(Triisopropylsilyl)oxy]methyl-protected Ribonucleosides
2.10 Preparation of 5-Silyl-2-Orthoester Ribonucleosides for Use in Oligoribonucleotide Synthesis

3 Synthesis of Unmodified Oligonucleotides

3.1 Solid-Phase Supports for Oligonucleotide Synthesis
3.2 Attachment of Nucleosides to Solid-Phase Supports
3.3 Synthetic Strategies and Parameters Involved in the Synthesis of Oligodeoxyribonucleotides According to the Phosphoramidite Method
3.4 Synthetic Strategies and Parameters Involved in the Synthesis of Oligodeoxyribo- and Oligoribonucleotides According to the H-Phosphonate Method
3.5 Strategies for Oligoribonucleotide Synthesis According to the Phosphoramidite Method
3.6 Oligoribonucleotides with 2'-O-(Tert-Butyldimethylsilyl) Groups
3.7 Synthesis of Oligoribonucleotides Using the 2-Nitrobenzyloxymethyl Group for 2'-Hydroxyl Protection
3.8 Chemical Synthesis of RNA Sequences with 2'-O-[(Triisopropylsilyl)oxy]methyl-protected Ribonucleoside Phosphoramidites
3.9 3-(N-tert-Butylcarboxamido)-1-propyl and 4-Oxopentyl Groups for Phosphate/Thiophosphate Protection in Oligodeoxyribonucleotide Synthesis

4 Synthesis of Modified Oligonucleotides and Conjugates

4.1 A Brief History, Status, and Perspective of Modified Oligonuculeotides for Chemotherapeutic Applications
4.2 Modification of the 5' Terminus of Oligonucleotides for the Attachment of Reporter and Conjugate Groups
4.3 Direct Attachment of Conjugate Groups to the 5' Terminus of Oligodeoxyribonucleotides
4.4 Chimeric 2-5A-DNA Oligonucleotides
4.5 Attachment of Reporter and Conjugate Groups to the 3' Termini of Oligonucleotides
4.6 3'-Modified Oligonucleotides and their Conjugates
4.7 Synthesis and Purification of Oligonucleotide N3'®P5' Phosphoramidates and their Phosphodiester and Phosphorothioate Chimeras
4.8 Incorporation of Halogenoalkyl, 2-Pyridyldithioalkyl, or Isothiocyanate Linkers into Ligands
4.9 Modification of the 5' Terminus of Oligodeoxyribonucleotides for Conjugation with Ligands
4.10 Conjugation of 5'-Functionalized Oligodeoxyribonucleotides with Properly Functionalized Ligands
4.11 Synthesis and Purification of Peptide Nucleic Acids
4.12 Synthesis and Characterization of Locked Nucleic Acids
4.13 Cellular Delivery of Locked Nucleic Acids (LNAs)
4.14 Solid-Phase Synthesis of Branched Oligonucleotides
4.15 Solid-Phase Synthesis of 2’-Deoxy-2’-fluoro- b-D-Oligoarabinonucleotides (2’F-ANA) and Their Phosphorothioate Derivatives
4.16 Chemistry of CpG DNA
4.17 Synthesis of Phosphorothioate Oligonucleotides with Stereodefined Phosphorothioate Linkages (New, September 2003)
4.18 Synthesis of Oligonucleotide Conjugates via Aqueous Diels-Alder Cycloaddition (New, September 2003)
3.10 DNA Synthesis Without Base Protection
3.11 The 4-Methylthio-1-Butyl Group for Phosphate/Thiophosphate Protection in Oligodeoxyribonucleotide Synthesis 4.19 5'-Iodination of Solid-Phase-Linked Oligodeoxyribonucleotides
4.20 Reversible Biotinylation of the 5'-Terminus of Oligodeoxyribonucleotides and Its Application in Affinity Purification
4.21 Uridine 2-Carbamates: Facile Tools for Oligonucleotide 2-Functionalization
4.22 Stepwise Solid-Phase Synthesis of Nucleopeptides
4.23 Synthesis of Oligoribonucleotides Containing N6-Alkyladenosine and 2-Methylthio-N6-Alkyladenosine
4.24 Oligodeoxyribonucleotide Analogs Functionalized with Phosphonoacetate and Thiophosphonoacetate Diesters

5 Methods for Cross-Linking Nucleic Acids

5.1 Engineering Disulfide Cross-Links in RNA Using Thiol-Disulfide Interchange Chemistry
5.2 Chemical and Enzymatic Methods for Preparing Circular Single-Stranded DNAs
5.3 Engineering Specific Cross-Links in Nucleic Acids Using Glycol Linkers
5.4 Engineering Disulfide Cross-Links in RNA Via Air Oxidation
5.5 Use of Electrophilic Substitution to Form Site-Specific Cross-Links in DNA
5.6 Synthesis of Endcap Dimethoxytrityl Phosphoramidites for Endcapped Oligonucleotides
5.7 Engineering Terminal Disulfide Bonds Into DNA (New, June 2003)

6 Chemical and Enzymatic Probes for Nucleic Acid Structure

6.1 Probing RNA Structure with Chemical Reagents and Enzymes
6.2 Probing Nucleic Acid Structure with Shape-Selective Rhodium and Ruthenium Complexes
6.3 Probing RNA Structure by Lead Cleavage
6.4 Probing Nucleic Acid Structure with Nickel and Cobalt-Based Reagents
6.5 Probing RNA Structures with Hydroxyl Radicals
6.6 Chemical Reagents for Investigating the Major Groove of DNA
6.7 Probing DNA Structure with Hydroxyl Radicals
6.8 Probing RNA Structure and Metal-Binding Sites Using Terbium(III) Footprinting
6.9 Probing RNA Structure and Function by Nucleotide Analog Interference Mapping

7 Biophysical Analysis of Nucleic Acids

7.1 Biophysical Analysis of Nucleic Acids
7.2 NMR Determination of Oligonucleotide Structure
7.3 Optical Methods
7.4 Calorimetry of Nucleic Acids
7.5 Molecular Modeling of Nucleic Acid Structure
7.6 Methods to Crystallize RNA
7.7 Recent Advances in RNA Structure Determination by NMR
7.8 Molecular Modeling of Nucleic Acid Structure: Energy and Sampling
7.9 Molecular Modeling of Nucleic Acid Structure: Electrostatics and Solvation
7.10 Molecular Modeling of Nucleic Acid Structure: Setup and Analysis
7.11 Characterization of DNA Structures by Circular Dichroism

8 Nucleic Acid Binding Molecules

8.1 Determination of Binding Mode: Intercalation
8.2 Determination of Binding Thermodynamics
8.3 A Competition Dialysis Assay for the Study of Structure-Selective Ligand Binding to Nucleic Acids
8.4 Chemistry of Minor Groove Binder-Oligonucleotide Conjugates (New, June 2003)

9 Combinatorial Methods in Nucleic Acid Chemistry

9.1 Theoretical Principles of In Vitro Selection Using Combinatorial Nucleic Acid Libraries
9.2 Design, Synthesis, and Amplification of DNA Pools for In Vitro Selection
9.3 In Vitro Selection of RNA Aptamers to a Protein Target by Filter Immobilization
9.4 Selection for Catalytic Function with Nucleic Acids
9.5 In Vitro Selection of RNA Aptamers to a Small Molecule Target
9.6 In Vitro Selection Using Modified or Unnatural Nucleotides

10 Purification and Analysis of Synthetic Nucleic Acids and Components

10.1 Analysis of Oligonucleotides by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
10.2 Analysis of Oligonucleotides by Electrospray Ionization Mass Spectrometry
10.3 Purification and Analysis of Synthetic Nucleic Acids
10.4 PAGE of Synthetic Nucleic Acids
10.5 HPLC Analysis and Purification of Synthetic Nucleic Acids
10.6 Base Composition Analysis of Nucleosides Using HPLC
10.7 Cartridge Methods for Oligonucleotide Purification
10.9 Capillary Electrophoresis of DNA(New, June 2003)

11 RNA Folding Pathways

11.1 RNA Folding Pathways
11.2 RNA Secondary Structure Prediction
11.3 Thermal Methods for the Analysis of RNA Folding Pathways
11.4 Probing RNA Folding Pathways by RNA Fingerprinting
11.5 Characterization of Tertiary Folding of RNA by Circular Dichroism and Urea
11.6 Time-Resolved Hydroxyl Radical Footprinting of RNA with X-rays
11.7 Rapid Magnesium Chelation as a Method to Study Real-Time Tertiary Unfolding of RNA
11.8 Use of Fluorescence Spectroscopy to Elucidate RNA Folding Pathways
11.9 Use of Chemical Modification to Elucidate RNA Folding Pathways
11.10 Probing RNA Structural Dynamics and Function by Fluorescence Resonance Energy Transfer (FRET)
11.11 Site-Specific Fluorescent Labeling of Large RNAs with Pyrene

12 DNA Nanotechnology

12.1 Key Experimental Approaches in DNA Nanotechnology
12.2 Preparation of Gold Nanoparticle-DNA Conjugates
12.3 Synthesis of 5-O-Phosphoramidites with a Photolabile 3-O-Protecting Group )
12.4 Derivatization of Glass and Polypropylene Surfaces HTML PDF (Size 109KB)
12.6 Preparation of -Oxo Semicarbazone Oligonucleotide Microarrays

Appendices

1 Standard Nomenclature, Data, and Abbreviations
A Abbreviations
B Characteristics of Nucleic Acids
C IUPAC-IUB Joint Commission on Biochemical Nomenclature Abbreviations and Symbols for Conformation of Polynucleotide Chains
D Nucleoside and Nucleotide Nomenclature

2 Laboratory Stock Solutions and Equipment
A Common Buffers and Stock Solutions

3 Commonly Used Techniques
A References to Molecular Biology Techniques
B Denaturing Polyacrylamide Gel Electrophoresis
C Introduction to the Synthesis and Purification of Oligonucleotides
D Thin-Layer Chromatography
E Column Chromatography
4 Resources
A Useful Nucleic Acid Chemistry Web Sites

Suppliers Appendix
Index

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