High Throughput Screening of Aptamers For Human Thrombin and Factor IXa

Abstract

The traditional method for discovery of DNA/RNA aptamers is in vitro evolution (SELEX), where multiple cycles of partitioning and amplification enrich aptamer candidates from a pool containing randomized segments of length, m. Previous to our work the consensus sequence for alpha-thrombin aptamers (ThbA) was found via five rounds of SELEX in a DNA pool with m=60, while factor IXa aptamers (FIXaA) were discovered in an RNA library of m=40 after 8 rounds. We isolated the same ThbA, FIXaA and a novel carbohydrate aptamer (CA) to validate our new method, High Throughput Screening of Aptamers. HTSA uses a single partitioning step, PCR, and counts survivors by massively parallel sequencing. We found the minimal ThbA in a library of DNA hairpins loops (m=15) containing 56,000 copies of each of the 1.1 billion possible sequences. We distinguished two sequence motifs well above the background. The ThbA motif contains the consensus (counted 46,000 times) and dozens of related sequences. The leading candidate in the CA family (29,000 counts) is a novel aptamer that binds glucose (Kd=1,400 nM) and alpha-methyl mannoside (Kd=500 nM). A known FIXaA was counted 52,000 times from a library of RNA hairpins (m=16 with 14,000 copies of each sequence). HTSA simplifies and shortens the discovery process, exhaustively searches the space of sequences within a library, simplifies characterization of the core binding domain, reduces the quantity of the target required, eliminates cycling artifacts, allows multiplexing of targets, and requires no complex automation.