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Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment.

Levay A, Brenneman R, Hoinka J, Sant D, Cardone M, Trinchieri G, Przytycka TM, Berezhnoy A - Nucleic Acids Res. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA.

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Convergence of the in vitro selection begins at round 5. (A) Schematic representation of in vitro selection process and its key improvements: at amplification step we employed emulsion PCR to preserve variability of the selected library by physical separation of amplification of individual DNA molecules; at fractionation step we introduced parallel binding there identical aliquotes of the same library had been reacted with different targets (depicted in black), for example (H)uman protein, (M)urine ortholog of it and (C)ontrol, to deduce aptamers’ binding patterns in highly parallel format. Bound RNA portions from hIL-10 SELEX rounds 2–5 were isolated, mass sequenced and analyzed for cluster formation. (B) Fractions of single copy aptamers (unenriched, open bars), aptamers with multiple copies but did not belonging to any cluster (unclustered, gray bars) and aptamers belonging to a cluster plotted (clustered, black bars). (C) Total number of individual clusters (blue bars, left Y axis) and average number of members per cluster (red bars, right Y axis) in each round.
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Figure 1: Convergence of the in vitro selection begins at round 5. (A) Schematic representation of in vitro selection process and its key improvements: at amplification step we employed emulsion PCR to preserve variability of the selected library by physical separation of amplification of individual DNA molecules; at fractionation step we introduced parallel binding there identical aliquotes of the same library had been reacted with different targets (depicted in black), for example (H)uman protein, (M)urine ortholog of it and (C)ontrol, to deduce aptamers’ binding patterns in highly parallel format. Bound RNA portions from hIL-10 SELEX rounds 2–5 were isolated, mass sequenced and analyzed for cluster formation. (B) Fractions of single copy aptamers (unenriched, open bars), aptamers with multiple copies but did not belonging to any cluster (unclustered, gray bars) and aptamers belonging to a cluster plotted (clustered, black bars). (C) Total number of individual clusters (blue bars, left Y axis) and average number of members per cluster (red bars, right Y axis) in each round.

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).


Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment.

Levay A, Brenneman R, Hoinka J, Sant D, Cardone M, Trinchieri G, Przytycka TM, Berezhnoy A - Nucleic Acids Res. (2015)

Convergence of the in vitro selection begins at round 5. (A) Schematic representation of in vitro selection process and its key improvements: at amplification step we employed emulsion PCR to preserve variability of the selected library by physical separation of amplification of individual DNA molecules; at fractionation step we introduced parallel binding there identical aliquotes of the same library had been reacted with different targets (depicted in black), for example (H)uman protein, (M)urine ortholog of it and (C)ontrol, to deduce aptamers’ binding patterns in highly parallel format. Bound RNA portions from hIL-10 SELEX rounds 2–5 were isolated, mass sequenced and analyzed for cluster formation. (B) Fractions of single copy aptamers (unenriched, open bars), aptamers with multiple copies but did not belonging to any cluster (unclustered, gray bars) and aptamers belonging to a cluster plotted (clustered, black bars). (C) Total number of individual clusters (blue bars, left Y axis) and average number of members per cluster (red bars, right Y axis) in each round.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4499151&req=5

Figure 1: Convergence of the in vitro selection begins at round 5. (A) Schematic representation of in vitro selection process and its key improvements: at amplification step we employed emulsion PCR to preserve variability of the selected library by physical separation of amplification of individual DNA molecules; at fractionation step we introduced parallel binding there identical aliquotes of the same library had been reacted with different targets (depicted in black), for example (H)uman protein, (M)urine ortholog of it and (C)ontrol, to deduce aptamers’ binding patterns in highly parallel format. Bound RNA portions from hIL-10 SELEX rounds 2–5 were isolated, mass sequenced and analyzed for cluster formation. (B) Fractions of single copy aptamers (unenriched, open bars), aptamers with multiple copies but did not belonging to any cluster (unclustered, gray bars) and aptamers belonging to a cluster plotted (clustered, black bars). (C) Total number of individual clusters (blue bars, left Y axis) and average number of members per cluster (red bars, right Y axis) in each round.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA.

Show MeSH
Related in: MedlinePlus