Stepping Library-Based Post-SELEX Strategy Approaching to the Minimized Aptamer in SPR

When evolved from SELEX (systematic evolution of ligands by exponential enrichment), aptamers are generally about 70-130 nucleotides in length and needed to be effectively truncated for further diagnosis or therapeutic uses. Post-SELEX optimization is then aroused to simplify the aptamer sequence and improve the affinity property. In this work, we report a new post-SELEX strategy based on a stepping library for the first time. With a hypothesis that one nucleobase can influence the whole binding affinity through its adjacent base stacking and potential steric hydrogen bonding interaction, we designed a stepping library composed of all probable nucleotide truncation directions. We employed an aptamer 807-39nt toward EPO-alpha as a model, and surface plasmon resonance (SPR) as an efficient screening and evaluation method to optimize all label-free sequences in the library. We have successfully picked out In27 as the minimized aptamer from a mini library of only 35 sequences. Aptamer In27 has a sole loop, without the original stem portion of the initial aptamer, but retains the whole binding affinity. We have also defined the key nucleotide contribution by site mutagenesis with natural bases, and finally produced a degenerated sequence with higher or the same good affinities. Furthermore, we explored different binding behaviors between aptamer In27 and other recognition molecule such as agglutinin, monoclonal antibody, or receptor by competition or binding assays. Our work provides a new and efficient post-SELEX optimization strategy, as well as a minimized aptamer In27 with an explicit degenerated sequence and a defined binding behavior. That would enhance their great potential in future diagnosis and therapy.