Name: primer-free aptamer selection using a random dna library
Uploaded: 2010-07-26T17:00:00
Duration: 11 min 14 s
Description: Watch this Scientific Journal Video about Primer-Free Aptamer Selection Using A Random DNA Library at JoVE.com

We thank Craig Paul at the Microarray Core Facility for his help. This work was supported by NIH/NCI grant #CA118591 from the IMAT Program. SLD also acknowledges a Pennsylvania Space Grant Consortium Fellowship for financial support. This publication was also supported by the Pennsylvania State University Materials Research Institute Nano Fabrication Network and the National Science Foundation Cooperative Agreement No. 0335765, National Nanotechnology Infrastructure Network, with Cornell University.

<ol>
	<li>Kulbachinskiy, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+selection+of+aptamers+to+protein+targets.">Methods for selection of aptamers to protein targets.</a> Biochemistry (Moscow). 72, 1505-1518 (2006).</li><li>Mairal, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamers:+molecular+tools+for+analytical+applications.">Aptamers: molecular tools for analytical applications.</a> Anal Bioanal Chem. 390, 989-1007 (2008).</li><li>Stoltenburg, R., Reinemann, C., Strehlitz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SELEX+--+A+(r)evolutionary+method+to+generate+high-affinity+nucleic+acid+ligands.">SELEX -- A (r)evolutionary method to generate high-affinity nucleic acid ligands.</a> Biomolec Engineering. 24, 381-403 (2007).</li><li>Que-Gewirth, N. S., Sullenger, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+progress+and+prospects:+RNA+aptamers.">Gene therapy progress and prospects: RNA aptamers.</a> Gene Ther. 14, 283-2891 (2007).</li><li>Cowperthwaite, M. C., Ellington, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinformatic+analysis+of+the+contribution+of+primer+sequences+to+aptamer+structures.">Bioinformatic analysis of the contribution of primer sequences to aptamer structures.</a> J Mol Evol. 67, 95-102 (2008).</li><li>Shtatland, T., Gill, S. C., Javornik, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+Escherichia+coli+RNA+with+bacteriophage+MS2+coat+protein:+genomic+SELEX.">Interactions of Escherichia coli RNA with bacteriophage MS2 coat protein: genomic SELEX.</a> Nucleic Acids Res. 28, E93-E93 (2000).</li><li>Jarosch, F., Buchner, K., Klussmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+selection+using+a+dual+RNA+library+that+allows+primerless+selection.">In vitro selection using a dual RNA library that allows primerless selection.</a> Nucleic Acids Res. 34, E86-E86 (2006).</li><li>Vater, A., Jarosch, F., Buchner, K., Klussmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+bioactive+Spiegelmers+to+migraine-associated+calcitonin+gene-related+peptide+rapidly+identified+by+a+novel+approach:+tailored-SELEX.">Short bioactive Spiegelmers to migraine-associated calcitonin gene-related peptide rapidly identified by a novel approach: tailored-SELEX.</a> Nucleic Acids Res. 31, E130-E130 (2003).</li><li>Wen, J. D., Gray, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+genomic+sequences+that+bind+tightly+to+Ff+gene+5+protein:+primer-free+genomic+SELEX.">Selection of genomic sequences that bind tightly to Ff gene 5 protein: primer-free genomic SELEX.</a> Nucleic Acids Res. 32, E182-E182 (2004).</li><li>Musheev, M. U., Krylov, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+aptamers+by+systematic+evolution+of+ligands+by+exponential+enrichment:+addressing+the+polymerase+chain+reaction+issue.">Selection of aptamers by systematic evolution of ligands by exponential enrichment: addressing the polymerase chain reaction issue.</a> Anal Chim Acta. 564, 91-916 (2006).</li><li>Pan, W. H., Clawson, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer-free+aptamer+selection+using+a+random+DNA+library.">Primer-free aptamer selection using a random DNA library.</a> Methods Mol Biol. , (2009).</li><li>Pan, W., Xin, P., Clawson, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal-Primer+and+Primer-Free+SELEX+Protocols+for+Selection+of+Aptamers+from+Random+DNA+Libraries.">Minimal-Primer and Primer-Free SELEX Protocols for Selection of Aptamers from Random DNA Libraries.</a> BioTechniques. 44, 351-360 (2008).</li><li>Nicewarner-Pena, S., Freeman, G., Reiss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submicrometer+metallic+barcodes.">Submicrometer metallic barcodes.</a> Science. 294, 137-141 (2001).</li><li>JC, H. u. l. t. e. e. n., CR, M. a. r. t. i. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+template-based+method+for+the+preparation+of+nanomaterials.">A general template-based method for the preparation of nanomaterials.</a> J Materials Chem. 7, 1075-1087 (1997).</li></ol>

No conflicts of interest declared.

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primer-free aptamer selection using a random dna library

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues.
Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10.
We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format.

Watch this Scientific Journal Video about Primer-Free Aptamer Selection Using A Random DNA Library at JoVE.com

Primer-Free Aptamer Selection Using A Random DNA Library

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified ...

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