Selection for Catalytic Function with Nucleic Acids

Ronald R. Breaker1

1 Yale University, New Haven, Connecticut
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 9.4
DOI:  10.1002/0471142700.nc0904s00
Online Posting Date:  May, 2001
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Abstract

For in vitro selection of catalytic polynucleotides, each new protocol must be designed to harness the desired catalytic activity to help propel the selection process itself. This unit gives guidelines for design of in vitro selection experiments for catalytic function. It outlines several representative protocols as examples of successful selection experiments, providing a conceptual basis for the design and implementation of new selective‚Äźamplification protocols for nucleic acids.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: In Vitro Selection of Group I Ribozyme Variants
  • Support Protocol 1: Mutagenic PCR
  • Basic Protocol 2: In Vitro Selection of RNase P Ribozyme Variants
  • Basic Protocol 3: In Vitro Selection of New Kinase Ribozymes from Random Sequence
  • Basic Protocol 4: Continuous Evolution of Ligase Ribozymes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: In Vitro Selection of Group I Ribozyme Variants

  Materials
  • 2× selection buffer: 60 mM EPPS[4‐(2‐hydroxethyl)‐1‐piperazinepropanesulfonic acid; pH 7.5 at 23°C)/20 mM MgCl 2
  • Substrate oligonucleotide solution (200 pmol/µL)
  • 100% ethanol, −20°C
  • TE buffer, pH 7.5 at 23°C ( appendix 2A)
  • 10× 3SR buffer: 500 mM Tris⋅Cl ( appendix 2A; pH 7.5 at 23°C)/100 mM MgCl 2/50 mM dithiothreitol (DTT)
  • 10× 3SR NTP/dNTP mix: 20 mM each of the four ribonucleoside‐5′‐triphosphates and 2 mM each of the four deoxyribonucleoside‐5′ ‐triphosphates (see appendix 2A for details of NTP and dNTP preparation and storage)
  • DNA primer 1: complementary to the 3′ tail of the modified ribozymes (20 pmol/µL)
  • DNA primer 2: homologous to the 5′ end of each ribozyme (20 pmol/µL)
  • 50 U/µL T7 RNAP (New England Biolabs)
  • 10 U/µL avian myoblastosis virus reverse transcriptase (AMV RT; Amersham)
  • 0.3 N NaOH
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 10× PCR buffer: 100 mM Tris⋅Cl (pH 8.3 at 23°C)/15 mM MgCl 2/500 mM KCl/0.1% (w/v) gelatin
  • 10× PCR dNTP mix: 2 mM each of the four deoxyribonucleoside‐5′‐triphosphates (see appendix 2A for details of dNTP preparation and storage)
  • DNA primer 3: complementary to the original 3′ terminus of the unmodified ribozymes (20 pmol/µL)
  • 5 U/µL Taq DNA polymerase (Boehringer Mannheim) diluted 20:1 in distilled H 2O to 0.25 U/µL final concentration
  • Additional reagents and equipment for agarose gel electrophoresis (e.g., CPMB UNIT ) and DNA sequencing ( appendix 3B)

Support Protocol 1: Mutagenic PCR

  Materials
  • DNA Primer 2: homologous to the 5′ end of each ribozyme (20 pmol/µL)
  • DNA Primer 3: complementary to the original 3′ terminus of the unmodified ribozymes (20 pmol/µL)
  • DNA template: 5 pmole/µL
  • 10× mutagenic PCR buffer: 100 mM Tris⋅Cl (pH 8.3 at 23°C)/70 mM MgCl 2/500 mM KCl/0.1% (w/v) gelatin
  • 10× mutagenic PCR dNTP mix: 2 mM each of dGTP and dATP/10 mM each of dCTP and dTTP
  • 5 mM MnCl 2
  • 5 U/µL Taq DNA polymerase (Boehringer Mannheim) diluted 20:1 in dH 2O to 0.25 U/µL final concentration

Basic Protocol 2: In Vitro Selection of RNase P Ribozyme Variants

  Materials
  • 10× transcription buffer (see recipe)
  • 10× transcription NTP mix: 10 mM each of the four ribonucleoside‐5′‐triphosphates
  • 50% (v/v) aqueous glycerol
  • Double‐stranded template DNA (at 10 pmol/µL)
  • 100 mM guanosine‐5′‐phosphorothioate (GMPS; Amersham Life Sciences–NucleixPlus)
  • 10 mCi/mL [α‐32P]GTP (3000 Ci/mmol)
  • 50 U/µL T7 RNAP (New England Biolabs)
  • 0.5 M EDTA, pH 8.0 ( appendix 2A)
  • 100% ethanol, −20°C
  • 2× urea loading buffer ( appendix 2A)
  • Gel elution buffer (see recipe)
  • TE buffer, pH 7.5 ( appendix 2A)
  • Sulfolink gel (aqueous slurry of beaded agarose; Pierce)
  • Loading buffer: 40% methanol/20 mM sodium phosphate (pH 8.9)/0.1% SDS ( appendix 2A)/5 mM EDTA
  • Nonpool RNAs: e.g., Bacillus subtilis rRNA
  • Wash buffer: 3 M NaCl/50 mM Tris⋅Cl (pH 8; appendix 2A)/5 mM EDTA
  • Reaction buffer −Mg2+ (see recipe)
  • 1 M MgCl 2
  • Glycogen
  • Sterile razor blade
  • Liquid scintillation counter
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (PAGE; e.g., appendix 3B or CPMB UNIT ) and RT‐PCR (unit 9.3)

Basic Protocol 3: In Vitro Selection of New Kinase Ribozymes from Random Sequence

  Materials
  • 2× kinase selection buffer (see recipe)
  • 100 mM adenosine 5′‐O‐(3‐thiotriphosphate) (ATP‐γS; Sigma)
  • 100% ethanol, −20°C
  • Binding buffer: 1 mM EDTA/25 mM HEPES (pH 7.4 at 23°C)
  • Thiopyridine‐activated thiopropyl Sepharose 6B (Amersham Pharmacia Biotech)
  • Wash buffer: 1 M NaCl/5 mM EDTA/25 mM HEPES (pH 7.4 at 23°C)
  • Urea solution: 3 M urea/5 mM EDTA
  • 0.1 M 2‐mercaptoethanol
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • Chromatographic column

Basic Protocol 4: Continuous Evolution of Ligase Ribozymes

  Materials
  • 10× reaction buffer (see recipe)
  • 10× 3SR NTP/dNTP mix: 20 mM each of the four ribonucleoside‐5′‐triphosphates and 2 mM each of the four deoxyribonucleoside‐5′‐triphosphates (see appendix 2A for NTP and dNTP preparation details)
  • DNA primer 1: complementary to the 3′ tail of the pool RNAs (20 pmol/µL)
  • Substrate oligonucleotide: DNA/RNA chimera that encodes the T7 promoter sequence (20 pmol/µL)
  • 40 U/µL T7 RNAP (New England Biolabs)
  • 200 U/µL Moloney murine leukemia virus reverse transcriptase (MMLV RT; Amersham)
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Figures

Videos

Literature Cited

Literature Cited
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