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 1: Isolating a Functionally Enriched Pool by Column Selection
  • Alternate Protocol 1: 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 1: Isolating a Functionally Enriched Pool by Column Selection

  Materials
  • Resin (Table 9.5.1)
  • Affinity resin with immobilized target molecule (see and Table 9.5.1)
  • Binding buffer (see )
  • RNA pool (units 9.2& 9.3)
  • Affinity elution buffer (see )
  • 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 1: Isolating a Functionally Enriched Pool by Batch Selection

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

Support Protocol 1: Assaying the Accumulation of Binding Species

  • 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 )
  • Affinity elution buffer (see )
  • 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

Videos

Literature Cited

Literature Cited
   Axen, R., Porath, J., and Ernback, S. 1967. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature 214:1302‐1304.
   Berens, C., Thain, A., and Schroeder, R. 2001. A tetracycline‐binding RNA aptamer. Bioorg. Med. Chem. 9:2549‐2556.
   Cekan, P., Jonsson, E.O., and Sigurdsson, S.T. 2009. Folding of the cocaine aptamer studied by EPR and fluorescence spectroscopies using the bifunctional spectroscopic probe C. Nucleic Acids Res. 37:3990‐3995.
   Connell, G.J. and Yarus, M. 1994. RNAs with dual specificity and dual RNAs with similar specificity. Science 264:1137‐1141.
   Connell, G.J., Illangesekare, M., and Yarus, M. 1993. Three small ribooligonucleotides with specific arginine sites. Biochemistry 32:5497‐5502.
   Cruz‐Aguado, J.A. and Penner, G. 2008. Determination of ochratoxin a with a DNA aptamer. J. Agric. Food Chem. 56:10456‐10461.
   Ellington, A.D. 1994. Empirical explorations of sequence space‐host‐guest chemistry in the RNA world. Berichte Der Bunsen‐Gesellschaft‐Physical Chemistry Chemical Physics 98:1115‐1121.
   Ellington, A.D. and Szostak, J.W. 1990. In vitro selection of RNA molecules that bind specific ligands. Nature 346:818‐822.
   Famulok, M. 1994. Molecular recognition of amino acids by RNA aptamers: An L‐citrulline binding RNA motif and its evolution into an L‐arginine binder. J. Am. Chem. Soc. 116:1698‐1706.
   Famulok, M. 1999. Oligonucleotide aptamers that recognize small molecules. Curr. Opin. Struct. Biol. 9:324‐329.
   Geiger, A., Burgstaller, P., von der Eltz, H., Roeder, A., and Famulok, M. 1996. RNA aptamers that bind L‐arginine with sub‐micromolar dissociation constants and high enantioselectivity. Nucleic Acids Res. 24:1029‐1036.
   Goertz, P.W., Cox, J.C., and Ellington, A.D. 2004. Automated selection of aminoglycoside aptamers. J. Assoc. Lab. Automat. 9:150‐154.
   Holeman, L.A., Robinson, S.L., Szostak, J.W., and Wilson, C. 1998. Isolation and characterization of fluorophore‐binding RNA aptamers. Fold. Des. 3:423‐431.
   Huizenga, D.E. and Szostak, J.W. 1995. A DNA aptamer that binds adenosine and ATP. Biochemistry 34:656‐665.
   Jenison, R.D., Gill, S.C., Pardi, A., and Polisky, B. 1994. High resolution molecular discrimination by RNA. Science 263:1425‐1429.
   Jhaveri, S.D., Rajendaran, M., and Ellington, A.D. 2000. In vitro selection of signaling aptamers. Nat. Biotechnol. 18:1293‐1297.
   Joeng, C.B., Niazi, J.H., Lee, S.J., and Gu, M.B. 2009. ssDNA aptamers that recognize diclofenac and 2‐anilinophenylacetic acid. Bioorg. Med. Chem. 17:5380‐5387.
   Kato, T., Takemura, T., Yano, K., Ikebukuro, K., and Karube, I. 2000. In vitro selection of DNA aptamers which bind to cholic acid. Biochim. Biophys. Acta. 1493:12‐18.
   Kawakami, J., Imanaka, H., Yokota, Y., and Sugimoto, N. 2000. In vitro selection of aptamers that act with Zn2+. J. Inorg. Biochem. 82:197‐206.
   Kawazoe, N., Teramoto, N., Ichinari, H., Imanishi, Y., and Ito, Y. 2001. In vitro selection of nonnatural ribozyme‐catalyzing porphyrin metalation. Biomacromolecules 2:681‐686.
   Kramer, F.R., Mills, D.R., Cole, P.E., Nishihara, T., and Speigelman, S. 1974. Evolution of in vitro sequence and phenotype of a mutant RNA resistant to ethidium bromide. J. Mol. Biol. 89:719‐736.
   Lato, S.M., Boles, A.R., and Ellington, A.D. 1995. In vitro selection of RNA lectins: Using combinatorial chemistry to interpret ribozyme evolution. Chem. Biol. 2:291‐303.
   Lauhon, C.T. and Szostak, J.W. 1995. RNA aptamers that bind flavin and nicotinamide redox cofactors. J. Am. Chem. Soc. 117:1246‐1257.
   Lee, J.F., Hesselberth, J.R., Meyers, L.A., and Ellington, A.D. 2004. Aptamer database. Nucleic Acids Res. 32:D95‐D100.
   Levisohn, R. and Speigleman, S. 1969. Further extracellular Darwinian experiments with replicating RNA molecules: Diverse variants isolated under different selective conditions. Proc. Natl. Acad. Sci. U.S.A. 63:805‐811.
   Li, Y.F., Geyer, C.R., and Sen, D. 1996. Recognition of anionic porphyrins by DNA aptamers. Biochemistry 35:6911‐6922.
   Majerfeld, I. and Yarus, M. 1998. Isoleucine: RNA sites with associated coding sequences. RNA 4:471‐478.
   Mann, D., Reinemann, C., Stoltenburg, R., and Strehlitz, B. 2005. In vitro selection of DNA aptamers binding ethanolamine. Biochem. Biophys. Res. Commun. 338:1928‐1934.
   Mannironi, C., Scerch, C., Fruscoloni, P., and Tocchini‐Valentini, G.P. 2000. Molecular recognition of amino acids by RNA aptamers: The evolution into an L‐tyrosine binder of a dopamine‐binding RNA motif. RNA 6:520‐527.
   Masud, M.M., Kuwahara, M., Ozaki, H., and Sawai, H. 2004. Sialyllactose‐binding modified DNA aptamer bearing additional functionality by SELEX. Bioorg. Med. Chem. 12:1111‐1120.
   Mills, D.R., Peterson, R.L., and Speigelman, S. 1967. An extracellular Darwinian experiment with a self‐duplicating nucleic acid molecule. Proc. Natl. Acad. Sci. U.S.A. 58:217‐224.
   Miyachi, Y., Shimizu, N., Ogino, C., Fukuda, H., and Kondo, A. 2009. Selection of a DNA aptamer that binds 8‐OHdG using GMP‐agarose. Bioorg. Med. Chem. Lett. 19:3619‐3622.
   Okazawa, A., Maeda, H., Fukusaki, E., Katakura, Y., and Kobayashi, A. 2000. In vitro selection of hematoporphyrin binding DNA aptamers. Bioorg. Med. Chem. Lett. 10:2653‐2656.
   Patel, D.J. and Suri, A.K. 2000. Structure, recognition, and discrimination in RNA aptamer complexes with cofactors, amino acids, drugs, and aminoglycoside antibiotics. J. Biotechnol. 74:39‐60.
   Sassanfar, M. and Szostak, J.W. 1993. An RNA motif that binds ATP. Nature 364:550‐553.
   Shoji, A., Kuwahara, M., Ozaki, H., and Sawai, H. 2007. Modified DNA aptamer that binds the (R)‐isomer of a thalidomide derivative with high enantioselectivity. J. Am. Chem. Soc. 129:1456‐1464.
   Stoltenburg, R., Reinemann, C., and Strehlitz, B. 2007. SELEX: A (r)evolutionary method to generate high‐affinity nucleic acid ligands. Biomol. Eng. 24:381‐403.
   Wallace, S.T. and Schroeder, R. 1998. In vitro selection and characterization of streptomycin‐binding RNAs: Recognition discrimination between antibiotics. RNA 4:112‐123.
   Wallis, M.G., Vonahsen, U., Schroeder, R., and Famulok, M. 1995. A novel RNA motif for neomycin recognition. Chem. Biol. 2:543‐552.
   Walsh, R. and DeRosa, M.C. 2009. Retention of function in the DNA homolog of the RNA dopamine aptamer. Biochem. Biophys. Res. Commun. 388:732‐735.
   Wilson, C., Nix, J., and Szostak, J. 1998. Functional requirements for specific ligand recognition by a biotin‐binding RNA pseudoknot. Biochemistry 37:14410‐14419.
   Windbichler, N., and Schroeder, R. 2006. Isolation of specific RNA‐binding proteins using the streptomycin‐binding RNA aptamer. Nat. Protoc. 1:637‐640.
   Wochner, A., Menger, M., Orgel, D., Cech, B., Rimmele, M., Erdmann, V.A., and Glokler, J. 2007. A DNA aptamer with high affinity and specificity for therapeutic anthracyclines. Anal. Biochem. 373:34‐42.
   Wrzesinski, J. and Jozwiakowski, S.K. 2008. Structural basis for recognition of Co2+ by RNA aptamers. FEBS. J. 275:1651‐1662.
   Xu, W. and Ellington, A.D. 1996. Anti‐peptide aptamers recognize amino acid sequence and bind a protein epitope. Proc. Natl. Acad. Sci. U.S.A. 93:7475‐7480.
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