Non-SELEX: selection of aptamers without intermediate amplification of candidate oligonucleotides

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Abstract

Aptamers are typically selected from libraries of random DNA (or RNA) sequences through systematic evolution of ligands by exponential enrichment (SELEX), which involves several rounds of alternating steps of partitioning of candidate oligonucleotides and their PCR amplification. Here we describe a protocol for non-SELEX selection of aptamers — a process that involves repetitive steps of partitioning with no amplification between them. Non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), which is a highly efficient affinity method, is used for partitioning. NECEEM also facilitates monitoring of bulk affinity of enriched libraries at every step of partitioning and screening of individual clones for their affinity to the target. NECEEM allows all clones to be screened prior to sequencing, so that only clones with suitable binding parameters are sequenced. The entire protocol can be completed in 1 wk, whereas conventional SELEX protocols take several weeks even in a specialized industrial facility.

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Figure 1
Figure 2
Figure 3: Schematic representation of NECEEM-based determination of DNA affinity to the target (T).
Figure 4: Choosing an aptamer-collection window.
Figure 5
Figure 6: Gel-free CE analysis of PCR products.
Figure 7: NECEEM-based screening of clones for binding affinity after asymmetric PCR.
Figure 8: NECEEM-based affinity analysis of a chemically synthesized aptamer for h-Ras protein.

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Acknowledgements

This work was supported by the Natural Sciences and Engineering Research Council of Canada.

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Correspondence to Sergey N Krylov.

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Berezovski, M., Musheev, M., Drabovich, A. et al. Non-SELEX: selection of aptamers without intermediate amplification of candidate oligonucleotides. Nat Protoc 1, 1359–1369 (2006) doi:10.1038/nprot.2006.200

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