In Vitro Selection of RNA Aptamers to a Small Molecule Target

Vlad Codrea1, Michelle Hayner2, Bradley Hall2, Sulay Jhaveri3, Andrew Ellington1

1 Department of Chemistry and Biochemistry, University of Texas, Austin, Texas, 2 Freshman Research Initiative, University of Texas, Austin, Texas, 3 Finnegan, Washington, D.C.
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 9.5
DOI:  10.1002/0471142700.nc0905s40
Online Posting Date:  March, 2010
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Abstract

This unit describes the selection of aptamers from a single-stranded RNA pool that bind to small molecule targets. Aptamers generated by this type of selection experiment can potentially function as receptors for small molecules in numerous applications, including medical diagnostics, therapeutics, and environmental monitoring. This unit describes two modes of selection, one by column filtration and one by batch selection. Curr. Protoc. Nucleic Acid Chem. 40:9.5.1-9.5.23. © 2010 by John Wiley & Sons, Inc.

Keywords: aptamer; affinity matrix; target immobilization; small molecule

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol: Isolating a Functionally Enriched Pool by Column Selection
  • Alternate Protocol: Isolating a Functionally Enriched Pool by Batch Selection
  • Support Protocol 1: Assaying the Accumulation of Binding Species
  • Support Protocol 2: Measuring Binding Constants (Kd)
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Isolating a Functionally Enriched Pool by Column Selection

 Materials
  • Resin (Table 9.5.1)
  • Affinity resin with immobilized target molecule (see Strategic Planning and Table 9.5.1)
  • Binding buffer (see Critical Parameters)
  • RNA pool (units 9.2 & 9.3)
  • Affinity elution buffer (see Critical Parameters)
  • 3 M sodium acetate (appendix 2A)
  • 100% and 70% (v/v) ethanol, room temperature and ice cold, respectively
  • 2-mL, 0.8 × 4-cm columns with cap and lid (e.g., Poly-Prep or Econo-Prep columns, Bio-Rad)
  • 65 to 75°C water bath
  • 13-mL collection tubes (e.g., Sarstedt)

Alternate Protocol: Isolating a Functionally Enriched Pool by Batch Selection

 Additional Materials (also see Basic Protocol)
  • Magnetic beads (BioMag, Bangs Laboratories), with and without immobilized small molecule target (see Strategic Planning)
  • Magnet or magnetic concentrator (GenScript; http://www.genscript.com/)

Support Protocol 1: Assaying the Accumulation of Binding Species

 Additional Materials (also see Basic Protocol and Alternate Protocol)
  • Radiolabeled RNA pool (unit 9.3)
  • 0.45-µm, 13-mm HAWP nitrocellulose filter disks (Millipore)
  • Glass plate
  • Phosphor imager (e.g., GE Healthcare Life Sciences) and screen or X-ray film and densitometer

Support Protocol 2: Measuring Binding Constants (Kd)

 Materials
  • Radiolabeled RNA (unit 9.3)
  • Affinity resin (Table 9.5.1)
  • Binding buffer (see Critical Parameters)
  • Affinity elution buffer (see Critical Parameters)
  • Minifold 1 Dot-Blot apparatus (Whatman)
  • Nylon transfer membrane (Hybond N+, GE Healthcare Life Sciences)
  • 80°C oven2 mL, 0.8 × 4–cm columns with cap and lid (e.g., Poly-Prep or Econo-Prep columns, Bio-Rad)
  • Glass plate
  • Phosphor imager (GE Healthcare Life Sciences) and screen or X-ray film and densitometer
  • Additional reagents and equipment for generating radiolabeled RNA aptamer or pool (unit 9.3)
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Figures

  •  FigureFigure 9.5.1 Steps involved in the selection of immobilized targets. Selection begins by incubating the pool with resin alone. The nonbinding species are collected and incubated with resin-immobilized target. Unbound and weakly bound species are separated from tightly bound species through successive washes. The bound pool is affinity eluted from the immobilized target with soluble target. The sieved pool can be amplified by some combination of reverse transcription, PCR, and transcription.
    View Image
  •  FigureFigure 9.5.2 Resin-based selection supports. (A) A schematic drawing of beads conjugated to a target via a long linker. Each bead can potentially bind multiple targets. (B) Column selection utilizes a target immobilized onto a resin and packed into a gel bed. The pool is in the mobile phase and can interact with target as it passes by the bead-based immobile phase. (C) Batch selection relies on the collection and dispersion of derivatized beads (such as magnetic particles). The pool initially interacts with beads dispersed in solution. Beads are collected magnetically or through centrifugation, and bound pool is collected along with the beads in the solid phase.
    View Image
  •  FigureFigure 9.5.3 Cycle course PCR. After binding species are collected, they can be amplified. Washes 0, 2, and 3 and the eluate are collected and a cycle course PCR amplification is carried out to determine the relative quantities of nucleic acid pool that is bound. For each sample, aliquots are removed from the PCR after 6, 8, 10, and 12 cycles, and separated on a 3.8% agarose gel. W0 is a positive control for amplification. The successive washes (W2, W3) yield visible bands at later cycles, indicating that weakly bound nucleic acid is being removed. The eluate (E1) amplified earlier than the last wash, suggesting enrichment of tightly bound species relative to weakly bound species.
    View Image
  •  FigureFigure 9.5.4 Isocratic elution. (A) After loading radiolabeled RNA on an affinity column, fractions are eluted with either binding buffer or binding buffer containing solubilized target. The RNA in each fraction is quantitated after spotting on a nylon filter. (B) Radioactive signal is plotted across fractions for the three different experiments. The fraction volume containing the maximum signal is used in the calculation of Kd values.
    View Image

Videos

Literature Cited

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