Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment.
Bottom Line: Although high-throughput sequencing (HTS) promises to significantly facilitate systematic evolution of ligands by exponential enrichment (SELEX) analysis, the enormous datasets generated in the process pose new challenges for identifying those rare, high-affinity aptamers present in a given pool.We also demonstrate the importance of using reference targets to eliminate binding candidates with reduced specificity.Finally, we demonstrate the power of this selection process for identifying cross-species aptamers that can bind human receptors and cross-react with their murine orthologs.
Affiliation: Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA.Show MeSH
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Mentions: To isolate aptamers recognizing human IL-10RA with high affinity and specificity, we performed in vitro selection for five rounds, gradually decreasing the amount of target protein (Figure 1A and Supplementary Table S1). RNA recovered from rounds 2 to 6 was sequenced as described previously (14). On average, 3 × 106 (2 × 106 to 5 × 106) reads were recovered from each pool. After demultiplexing, as described in the Materials and Methods section, we used a hash-based algorithm to determine the prevalence of individual sequences. We define ‘bystander’ sequences as those encountered only once in the dataset in a single copy and ‘true aptamer’ sequences as present in multiple rounds and/or in multiple copies (Table 1). As described previously, we used k-mer distance to cluster related sequences (12). Highly similar sequences with as many as three mismatches/deletions/insertions were associated with the same cluster. The results, including aptamer sequences, their prevalence in each pool, and their cluster association, are hosted online on NCBI servers in a MySQL database format as described previously (15).
Affiliation: Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA.