Abstract
Aptamers are high-affinity ligands selected from DNA or RNA libraries via SELEX, a repetitive in vitro process of sequential selection and amplification steps. RNA SELEX is more complicated than DNA SELEX because of the additional transcription and reverse transcription steps. Here, we report a new selection scheme, RAPID-SELEX (RNA Aptamer Isolation via Dual-cycles SELEX), that simplifies this process by systematically skipping unnecessary amplification steps. Using affinity microcolumns, we were able to complete a multiplex selection for protein targets, CHK2 and UBLCP1, in a third of the time required for analogous selections using a conventional SELEX approach. High-throughput sequencing of the enriched pools from both RAPID and SELEX revealed many identical candidate aptamers from the starting pool of 5×1015 sequences. For CHK2, the same sequence was preferentially enriched in both selections as the top candidate and was found to bind to its respective target. These results demonstrate the efficiency and, most importantly, the robustness of our selection scheme. RAPID provides a generalized approach that can be used with any selection technology to accelerate the rate of aptamer discovery, without compromising selection performance.
Highlights
Aptamers are high-affinity ligands selected from large libraries of random oligonucleotides that can contain up to 1016 unique sequences
RAPID SELEX is capable of isolating aptamers in less time than conventional SELEX
We found that the two Cycle 6 pools bound with comparable affinity, the RAPID pool bound slightly better (,1.5-fold higher)
Summary
Aptamers are high-affinity ligands selected from large libraries of random oligonucleotides that can contain up to 1016 unique sequences. SELEX (Systematic Evolution of Ligands by EXponential enrichment) [1,2,3], an in vitro selection method, can isolate aptamers with high-affinity and specificity for a wide range of target molecules from DNA or RNA libraries [4,5,6]. This is achieved by iteratively selecting and amplifying target-bound sequences to preferentially enrich those sequences with the highest affinity to the target. A disproportionate amount of time and effort is dedicated to amplifying RNA pools compared to the actual selection steps where aptamer enrichment takes place
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