In Vitro Selection Using Modified or Unnatural Nucleotides

Gwendolyn M. Stovall1, Robert S. Bedenbaugh1, Shruti Singh1, Adam J. Meyer1, Paul J. Hatala2, Andrew D. Ellington1, Bradley Hall2

1 The University of Texas at Austin, Austin, Texas, 2 Altermune Technologies LLC, Austin, Texas
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 9.6
DOI:  10.1002/0471142700.nc0906s56
Online Posting Date:  March, 2014
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Incorporation of modified nucleotides into in vitro RNA or DNA selections offers many potential advantages, such as the increased stability of selected nucleic acids against nuclease degradation, improved affinities, expanded chemical functionality, and increased library diversity. This unit provides useful information and protocols for in vitro selection using modified nucleotides. It includes a discussion of when to use modified nucleotides; protocols for evaluating and optimizing transcription reactions, as well as confirming the incorporation of the modified nucleotides; protocols for evaluating modified nucleotide transcripts as template in reverse transcription reactions; protocols for the evaluation of the fidelity of modified nucleotides in the replication and the regeneration of the pool; and a protocol to compare modified nucleotide pools and selection conditions. Curr. Protoc. Nucleic Acid Chem. 56:9.6.1‐9.6.33. © 2014 by John Wiley & Sons, Inc.

Keywords: in vitro selection; aptamer; ribozymes; deoxyribozymes; modified nucleotides; unnatural nucleotides

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Table of Contents

  • Introduction
  • Strategic Planning
  • Determination of Suitability of Modified Nucleotides for In Vitro Selection
  • Basic Protocol 1: Evaluation and Optimization of Transcription with a Modified Nucleotide
  • Basic Protocol 2: Confirmation of the Presence of Modified Nucleotides
  • Basic Protocol 3: Evaluation of Modified RNA as a Template for Reverse Transcriptase
  • Basic Protocol 4: Determination of the Fidelity of Replication
  • Determination of Modified Pool Most Suitable for In Vitro Aptamer Selection
  • Basic Protocol 5: Comparing Modified Nucleotide Pools and Selection Conditions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Evaluation and Optimization of Transcription with a Modified Nucleotide

  • AmpliScribe T7 high‐yield transcription kit (Epicentre) containing:
    • 10× transcription (TNX) buffer
    • T7 enzyme solution
    • 100 mM ATP, CTP, GTP, and UTP solutions
    • 10× reaction mix
    • 100 mM dithiothreitol (DTT)
    • T7 RNA polymerase
  • DuraScribe T7 transcription kit (Epicentre) for modified NTP incorporation, kit includes:
    • DuraScribe T7 enzyme solution
    • 50 mM ATP and GTP solutions
    • 50 mM 2′‐deoxy‐2′‐fluoro‐CTP and 2′‐deoxy‐2′‐fluoro‐UTP solutions
    • 10× reaction mix
    • 100 mM dithiothreitol (DTT)
  • 100 mM modified nucleotide
  • 500 ng DNA clone of known sequence composition (dsDNA containing a T7 promoter)
  • 3000 Ci/mmol α‐32P‐labeled ATP (e.g., Perkin Elmer)
  • Nuclease‐free water
  • 2× denaturing stop dye (see recipe)
  • 10% (w/v) denaturing polyacrylamide gel (see recipe in appendix 3B), 0.8‐mm thick
  • 500 ng purified DNA pool or randomized clone per reaction (dsDNA containing a T7 promoter) (unit 9.2)
  • Thermal cycler or 37°C to 42°C and 70°C water baths or heating blocks
  • Gel blotting paper (Bio‐Rad)
  • Plastic wrap
  • Gel dryer with vacuum (e.g., Bio‐Rad)
  • Phosphorimager screen
  • Phosphorimager and image analysis software (e.g., GE Healthcare Life Sciences, ImageQuant)
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (PAGE; appendix 3B)

Basic Protocol 2: Confirmation of the Presence of Modified Nucleotides

  • Transcribed RNA pools, modified and unmodified (see protocol 1 without radiolabeled ATP)
  • Nuclease P1 digestion mix (see recipe)
  • Alkaline phosphatase reaction mix (see recipe)
  • HPLC mobile phase solution: 5% (v/v) methanol in 0.1 M sodium phosphate, pH 6.0 ( appendix 2A)
  • Thermal cycler or 37°C and 50°C water baths
  • HPLC with reversed‐phase C18 column (5 µm, 250 × 4.5–mm; Waters.) and UV detector

Basic Protocol 3: Evaluation of Modified RNA as a Template for Reverse Transcriptase

  • Transcribed RNA pools, modified and unmodified (prepared as in protocol 1 but without radiolabeled ATP)
  • 100 µM reverse (3′‐end) primer
  • 3000 Ci/mmol α‐32P‐labeled dATP (e.g., Perkin Elmer)
  • 10 mM dNTP mix (containing 10 mM each of dATP, dCTP, dGTP, and dTTP)
  • ThermoScript reverse transcriptase kit (Invitrogen, Life Technologies) containing:
    • 5× buffer
    • 100 mM DTT
    • RNase OUT (ribonuclease inhibitor)
    • Reverse transcriptase (RT) enzyme
  • 2× denaturing stop dye (see recipe)
  • 10% (w/v) denaturing polyacrylamide gel (see recipe in appendix 3B), 0.8‐mm thick
  • Thermal cycler
  • Gel blotting paper (Bio‐Rad)
  • Plastic wrap
  • Gel dryer with heat and vacuum (e.g., Bio‐Rad)
  • Phosphorimager screen
  • Phosphorimager
  • Image analysis software (e.g., GE Healthcare Life Sciences ImageQuant)
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (PAGE; appendix 3B)

Basic Protocol 4: Determination of the Fidelity of Replication

  • 32P‐end‐labeled nucleic acid pools with specific modifications (modified pool in one tube, unmodified in another; refer to unit 9.5, Support Protocol 1, for the radiolabeling protocol)
  • Binding buffer: this buffer should promote binding and be specific to the aptamer application; common examples are phosphate‐buffered saline (PBS) and PCR buffer, e.g., 10 mM Tris·Cl, pH 8.4, 50 mM KCl, and 1.5 mM MgCl 2 (for more discussion, see unit 9.5)
  • Target molecule (range of concentrations from 1 × 10−7 M to 1 × 10−12 M)
  • Methanol
  • 0.5 M potassium hydroxide (KOH)
  • Thermal cycler or 37°C and 65°C water baths or heating blocks
  • Minifold I dot blot system, 96‐well plate vacuum manifold (e.g., Whatman)
  • Nylon membranes (e.g., Hybond N+ nylon sheets)
  • 0.45‐µm nitrocellulose transfer and immobilization membrane (e.g., Protran BA‐83, Whatman)
  • Vacuum pump or water aspirator
  • 80°C oven
  • Phosphorimager screen
  • Phosphorimager and image analysis software (e.g., GE Healthcare Life Sciences ImageQuant)
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