Aptamers oligonucleotide sequences

Biological Oligonucleotide sequence in aptamers for specific and sensitive target binding 119... 

An EMA consists of selection via an inhibition assay, crossover, and mutation of the oligonucleotide sequence in silica. Eirst, a set of 10 DNA sequences, each of which contains a portion of the original aptamer sequence as well as a randomized region, is generated by a computer and synthesized chemically. The inhibitory activity of each aptamer for the target enzyme is measured and... 

Aptamers represent an attractive alternative to antibodies as bioelements. They are short ssDNA or RNA oligonucleotide sequences (20-40 bases) that fold into a 3D-structure that has a high selectivity and affinity for a defined target [10]. The 3D-stracture is stabilized after binding to the target. Aptamers are smaller in size compared to antibodies and they can be synthesized chemically so that no... 

By similar logic, protein affinity Hbraries have been constmcted to identify protein—protein combining sites, as in antibody—antigen interaction (19) and recombinant Hbraries have been made which produce a repertoire of antibodies in E. coli (20). In another case, a potential DNA-based therapeutic strategy has been studied (21). DNAs from a partially randomized Hbrary were selected to bind thrombin in vitro. Oligonucleotides, termed aptamers that bound thrombin shared a conserved sequence 14—17 nucleotides long. 

Combinatorial Hbraries are limited by the number of sequences that can be synthesized. For example, a Hbrary consisting of one molecule each of a 60-nucleotide sequence randomized at each position, would have a mass of >10 g, weU beyond the capacity for synthesis and manipulation. Thus, even if nucleotide addition is random at all the steps during synthesis of the oligonucleotide only a minority of the sequences can be present in the output from a laboratory-scale chemical DNA synthesis reaction. In analyzing these random but incomplete Hbraries, the protocol is efficient enough to allow selection of aptamers of lowest dissociation constants (K ) from the mixture after a small number of repetitive selection and amplification cycles. Once a smaller population of oligonucleotides is amplified, the aptamer sequences can be used as the basis for constmcting a less complex Hbrary for further selection. 

Enrichment of high affinity candidates is usually achieved in 8 to 15 rounds of SELEX. Each rotmd takes approximately 2 days to perform. The process has been automated using robotic liquid handlers both for DNA (SomaLogic) and RNA aptamers (Cox, 2002). Next, the sequenced aptamer is prepared in bulk by conventional DNA synthesis chemistry and purified, then the aptamer arrayed onto a solid support. Thus, an aptamer is ready for application within 2 to 3 mo. Because the sequence is known, preparation of additional aptamer is easily accomplished using conventional oligonucleotide chemical synthesis. 

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