Abstract
The identification of nucleic acid aptamers would be advanced if they could be obtained after fewer rounds of selection and amplification. In this paper the identification of bivalent aptamers for thrombin by SELEX and single-step selection are compared using next generation sequencing and motif finding informatics. Results show that similar aptamers are identified by both methods. This is significant because it shows that next generation sequencing and motif finding informatics have the potential to simplify the selection of aptamers by avoiding multiple rounds of enzymatic transcription and amplification.
Highlights
Nucleic acid aptamers are high affinity binding molecules that have applications in diagnostics, therapy and separation science. They are normally identified by screening combinatorial libraries of typically 1012–1016 oligonucleotides for nucleic acid sequences that bind to a chosen target molecule by a process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment) that consists of multiple cycles of selection and PCR amplification.[1,2,3]
Individual oligonucleotides consist of a 30 base combinatorial sequence bracketed by APT-15 and Apt-29, and primer sequences for PCR
Counter-selection was carried out with a mixture consisting of equal amounts of uncoated streptavidin beads, beads coated with biotin-PEG and beads coated with human serum albumin (HSA)
Summary
Nucleic acid aptamers are high affinity binding molecules that have applications in diagnostics, therapy and separation science. They are normally identified by screening combinatorial (randomized) libraries of typically 1012–1016 oligonucleotides for nucleic acid sequences that bind to a chosen target molecule by a process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment) that consists of multiple cycles of selection and PCR amplification.[1,2,3] In the selection step oligonucleotides compete for binding sites on the target molecule and in the amplification step the remaining pool of oligonucleotides is enriched with sequences that bind. The stabilities and/or affinities of aptamers based on natural nucleotide bases can be improved by incorporation of chemically modified bases, but these are more difficult to amplify. Gold and colleagues have used SELEX to identify high affinity aptamers based on chemically modified DNA bases, but these bases must be transcribed onto natural bases for amplification and reversetranscribed back onto modified bases for selection.[5,6,7] These complications would be avoided if it was possible to identify aptamers without multiple cycles of selection and amplification
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