Nonenzymatic Oligomerization of Activated Nucleotides on Hairpin Templates

Eun‐Kyong Kim1, Christopher Switzer1

1 Department of Chemistry, University of California, Riverside, California
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.18
DOI:  10.1002/0471142700.nc0318s39
Online Posting Date:  December, 2009
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This unit describes a protocol for nonenzymatic oligomerization of activated ribonucleotides on DNA hairpins appended by templates containing threofuranosyl nucleic acid (TNA). TNA‐cytidylate templates effectively promote oligomerization of 2‐MeImpG, and give 3′,5′‐linked oligomerization products predominantly, with good base‐pairing fidelity. Although the rates of oligomerization depend on TNA content, after 3 days of incubation, oligomerization products are apparent, and full‐length products are present after 10 days. Characterization of product phosphodiester bond regiochemistry is accomplished by digestion with RNase T1. Additionally, exposure of oligomerization products to calf intestinal alkaline phosphatase enables detection of any endcapping due to pyrophosphate formation. Base‐pairing fidelity is assessed by challenging the template to oligomerize 2‐MeImpA. The protocols described for nonenzymatic, template‐directed synthesis in this unit are applicable to oligomerization of activated monomers on templates of different compositions, with respect to both base identity and polymer backbone. Curr. Protoc. Nucleic Acid Chem. 39:3.18.1‐3.18.13. © 2009 by John Wiley & Sons, Inc.

Keywords: nonenzymatic; template‐directed; oligomerization; RNA; DNA; TNA; prebiotic

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Activated Nucleotide Monomers
  • Basic Protocol 2: Nonenzymatic Oligomerization of RNA by TNA Templates
  • Support Protocol 1: Synthesis of TNA‐Bearing Hairpin Oligonucleotides
  • Alternate Protocol 1: Base‐Pairing Fidelity and Effects of Different Divalent Ions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation of Activated Nucleotide Monomers

  • Adenosine 5′‐monophosphate (AMP; Sigma)
  • Guanosine 5′‐monophosphate (GMP; Sigma)
  • 2‐methylimidazole (2‐MeIm), 99% (Aldrich)
  • Dimethyl sulfoxide (DMSO), distilled from CaH 2
  • Nitrogen (or argon) gas, dry
  • N,N‐Dimethylformamide (DMF), water < 50 ppm (Acros Organics)
  • Triphenylphosphine (Ph 3P), 99% (Aldrich)
  • 2,2′‐Dipyridyl disulfide (PySSPy, AldrithiolT‐2), 98% (Aldrich)
  • Triethylamine (Et 3N, TEA), distilled from KOH and stored over KOH
  • 11:2:7 (v/v/v) n‐propanol/ammonium hydroxide/water
  • Acetone (Fisher Scientific)
  • Diethyl ether, anhydrous (Fisher Scientific)
  • Saturated anhydrous sodium perchlorate (NaClO 4; Aldrich) in acetone
  • Phosphorus pentoxide (P 2O 5), anhydrous, 99+% (Acros Organics)
  • Sodium hydroxide (NaOH), certified ACS (Fisher scientific)
  • 50‐mL round‐bottom flasks
  • Magnetic stir bars and plate
  • TLC silica gel plates with UV indicator, Merck silica gel 60 F254
  • Benchtop centrifuge
  • Desiccator
  • High vacuum oil pump
  • Additional reagents and equipment for performing TLC ( appendix 3D)

Basic Protocol 2: Nonenzymatic Oligomerization of RNA by TNA Templates

  • Templates: Hairpin oligonucleotides bearing TNA‐C residues ( protocol 3)
  • T4 polynucleotide kinase and dilution buffer (USB)
  • 10× kinase buffer; 5:1:1:3 (v/v/v/v) 1.4 M Tris⋅Cl, pH 7.6/1.0 M MgCl 2/500 mM dithiothreitol (DTT)/H 2O
  • [γ‐32P]ATP, 6000 Ci/mmol, 10 mCi/mL (Perkin‐Elmer & Analytical Science)
  • 660 µM ATP
  • Loading buffer (7 M urea in 1× TBE buffer containing 0.1 % xylene cyanol and 0.1 % bromphenol blue)
  • 7 M urea/20% denaturing polyacrylamide gel
  • 50 mM TEAA buffer, pH 7.0 (see recipe)
  • 2 M NaCl
  • 1 M MgCl 2
  • 1 M 2,6‐lutidine⋅HCl, pH 8.0 (see recipe)
  • 2‐MeImpG ( protocol 1)
  • 2‐MeImpA ( protocol 1)
  • 0.5 M EDTA, pH 8.0 ( appendix 2A)
  • Ribonuclease T1 (RNase T1; Boehringer Mannheim)
  • Tris‐EDTA buffer (10 mM Tris⋅Cl, pH 7.4/1 mM EDTA, pH 8.0)
  • Calf intestinal alkaline phosphatase (CIAP)
  • 10× phosphatase buffer (20 mM Tris⋅Cl, pH 8.0/10 mM MgCl 2)
  • 1.5‐mL microcentrifuge tubes
  • Vortex mixer
  • Benchtop centrifuge
  • 37°C incubator
  • Heating block
  • Plastic wrap
  • Autoradiographic film
  • Developer (Kodak GBX)
  • Fixer (Kodak GBX)
  • Thin knife
  • 14‐mL culture tubes
  • Rotomix 50800 shaker (Thermolyne)
  • Lyophilizer
  • Sephadex G‐25 NAP columns, preswollen in H 2O (GE Healthcare)
  • Geiger counter
  • Phosphorimager screen
  • Typhoon 9410 imaging scanner (Amersham Biosciences)
  • ImageQuant software (Molecular Dynamics)
  • Additional reagents and equipment for DNA quantification by UV spectrophotometry (unit 5.2) and polyacrylamide gel electrophoresis (PAGE; appendix 3B)
NOTE: Appropriate precautions must be taken when working with radioactive chemicals to avoid contamination of personnel conducting the study and surroundings. The radioactive waste should be collected and disposed of appropriately following the Nuclear Regulatory Commission (NRC) and institutional guidelines that are provided by the institutional radiation safety officer.NOTE: Use autoclaved deionized water for all reagents.
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Literature Cited

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