Abstract
This method describes a microarray-based platform to perform nucleic acid selections. Chemical ligands to which a nucleic acid binder is desired are immobilized onto an agarose microarray surface; the array is then incubated with an RNA library. Bound RNA library members are harvested directly from the array surface via gel excision at the position on the array where a ligand was immobilized. The RNA is then amplified via RT-PCR, cloned, and sequenced. This method has the following advantages over traditional resin-based Systematic Evolution of Ligands by Exponential Enrichment (SELEX): (1) multiple selections can be completed in parallel on a single microarray surface; (2) kinetic biases in the selections are mitigated since all RNA binders are harvested from an array via gel excision; (3) the amount of chemical ligand needed to perform a selection is minimized; (4) selections do not require expensive resins or equipment; and (5) the matrix used for selections is inexpensive and easy to prepare. Although this protocol was demonstrated for RNA selections, it should be applicable for any nucleic acid selection.
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References
Ellington, A. D., and Szostak, J. W. (1990) In vitro selection of RNA molecules that bind specific ligands, Nature 346, 818–822.
Ellington, A. D., and Szostak, J. W. (1992) Selection in vitro of single-stranded DNA molecules that fold into specific ligand-binding structures, Nature 355, 850–852.
Osborne, S. E., and Ellington, A. D. (1997) Nucleic acid selection and the challenge of combinatorial chemistry, Chem. Rev. 97, 349–370.
Tuerk, C., and Gold, L. (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase, Science 249, 505–510.
Cox, J. C., and Ellington, A. D. (2001) Automated selection of anti-protein aptamers, Bioorg. Med. Chem. 9, 2525–2531.
Giver, L., Bartel, D. P., Zapp, M. L., Green, M. R., and Ellington, A. D. (1993) Selection and design of high-affinity RNA ligands for HIV-1 REV, Gene 137, 19–24.
Hesselberth, J. R., Miller, D., Robertus, J., and Ellington, A. D. (2000) In vitro selection of RNA molecules that inhibit the activity of ricin A-chain, J. Biol. Chem. 275, 4937–4942.
Daniels, D. A., Chen, H., Hicke, B. J., Swiderek, K. M., and Gold, L. (2003) A tenascin-C aptamer identified by tumor cell SELEX: Systematic evolution of ligands by exponential enrichment, Proc. Natl. Acad. Sci. U.S.A. 100, 15416–15421.
Minunni, M., Scarano, S., and Mascini, M. (2008) Affinity-based biosensors as promising tools for gene doping detection, Trends Biotechnol. 26, 236–243.
Famulok, M., Hartig, J. S., and Mayer, G. (2007) Functional aptamers and aptazymes in biotechnology, diagnostics, and therapy, Chem. Rev. 107, 3715–3743.
Wu, C. C., Sabet, M., Hayashi, T., Tawatao, R., Fierer, J., Carson, D. A., Guiney, D. G., and Corr, M. (2008) In vivo efficacy of a phosphodiester TLR-9 aptamer and its beneficial effect in a pulmonary anthrax infection model, Cell Immunol. 251, 78–85.
Keefe, A. D., and Schaub, R. G. (2008) Aptamers as candidate therapeutics for cardiovascular indications, Curr. Opin. Pharmacol. 8, 147–152.
Lorsch, J. R., and Szostak, J. W. (1994) In vitro evolution of new ribozymes with polynucleotide kinase activity, Nature 371, 31–36.
Bartel, D. P., and Szostak, J. W. (1993) Isolation of new ribozymes from a large pool of random sequences [see comment], Science 261, 1411–1418.
Mendonsa, S. D., and Bowser, M. T. (2004) In vitro evolution of functional DNA using capillary electrophoresis, J. Am. Chem. Soc. 126, 20–21.
Klug, S. J., and Famulok, M. (1994) All you wanted to know about SELEX, Mol. Biol. Rep. 20, 97–107.
Schena, M., Shalon, D., Davis, R. W., and Brown, P. O. (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray, Science 270, 467–470.
MacBeath, G., and Schreiber, S. L. (2000) Printing proteins as microarrays for high-throughput function determination, Science 289, 1760–1763.
Houseman, B. T., and Mrksich, M. (2002) Carbohydrate arrays for the evaluation of protein binding and enzymatic modification, Chem. Biol. 9, 400–401.
Barrett, O. J., Childs, J. L., and Disney, M. D. (2006) Chemical microarrays to identify ligands that bind pathogenic cells, Chembiochem 7, 1882–1885.
Disney, M. D., Magnet, S., Blanchard, J. S., and Seeberger, P. H. (2004) Aminoglycoside microarrays to study antibiotic resistance, Angew. Chem. Int. Ed. 43, 1591–1594.
Bradner, J. E., McPherson, O. M., and Koehler, A. N. (2006) A method for the covalent capture and screening of diverse small molecules in a microarray format, Nat. Prot. 1, 2344–2352.
Duffner, J. L., Clemons, P. A., and Koehler, A. N. (2007) A pipeline for ligand discovery using small-molecule microarrays, Curr. Opin. Chem. Biol. 11, 74–82.
MacBeath, G., Koehler, A. N., and Schreiber, S. L. (1999) Printing small molecules as microarrays and detecting protein-ligand interactions en masse, J. Am. Chem. Soc. 121, 7967–7968.
Afanassiev, V., Hannemann, V., and Wolfl, S. (2000) Preparation of DNA and protein micro arrays on glass slides coated with an agarose film, Nucleic Acids Res. 28, E66.
Dufva, M., Petronis, S., Jensen, L. B., Krag, C., and Christensen, C. B. (2004) Characterization of an inexpensive, nontoxic, and highly sensitive microarray substrate. Biotechniques 37, 286–292, 294, 296.
Childs-Disney, J. L., Wu, M., Pushechnikov, A., Aminova, O., and Disney, M. D. (2007) A small molecule microarray platform to select RNA internal loop-ligand interactions, ACS Chem. Biol. 2, 745–754.
Disney, M. D., Labuda, L. P., Paul, D. J., Poplawski, S. G., Pushechnikov, A., Tran, T., Velagapudi, S. P., Wu, M., and Childs-Disney, J. L. (2008) Two-dimensional combinatorial screening identifies specific aminoglycoside-RNA internal loop partners, J. Am. Chem. Soc. 130, 11185–11194.
Chan, T. R., Hilgraf, R., Sharpless, B., and Fokin, V. V. (2004) Polytriazoles as copper(I)-stabilizing ligand in catalysis, Org. Lett. 6, 2853–2855.
Kolb, H. C., Finn, M. G., and Sharpless, K. B. (2001) Click chemistry: diverse chemical function from a few good reactions, Angew. Chem. Int. Ed. 40, 2004–2021.
Milligan, J. F., and Uhlenbeck, O. C. (1989) Synthesis of small RNAs using T7 RNA polymerase, Methods Enzymol. 180, 51–62.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, Vol. 1, Cold Spring Harbor, NY.
Peyret, N., Seneviratne, P. A., Allawi, H. T., and SantaLucia Jr, J. (1999) Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A-A, C-C, G-G, and T-T mismatches, Biochemistry 38, 3468–3477.
SantaLucia Jr, J. (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics, Proc. Natl. Acad. Sci. U.S.A. 95, 1460–1465.
Acknowledgments
We thank Professor Jessica Disney for careful proofreading of the manuscript. This work was supported by funding from the University at Buffalo, the NYS Center of Excellence and Bioinformatics and Life Sciences, a New Investigator Award from the Camille and Henry Dreyfus Foundation, a Cottrell Scholar Award from the Research Corporation, a NYSTAR J. D. Watson Young Investigator Award, and the National Institutes of Health (GM079235).
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Aminova, O., Disney, M.D. (2010). A Microarray-Based Method to Perform Nucleic Acid Selections. In: Uttamchandani, M., Yao, S. (eds) Small Molecule Microarrays. Methods in Molecular Biology, vol 669. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-845-4_17
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DOI: https://doi.org/10.1007/978-1-60761-845-4_17
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