Over-Represented DNA Libraries Containing Two Hydrophobic (Het)aryl-Linked Nucleotides for Expedient Single-Round Selection of a Specific Aptamer Targeting Human Insulin Receptor

Discovery of functional nucleic acid ligands and catalytic motifs from a vast sequence space of randomized libraries is mostly based on time-consuming and demanding iterative rounds of in vitro selection. Here, we present a streamlined method for rapid identification of nucleobase-modified aptamers in a single selection round by combining chemically modified over-represented libraries, consecutive partition steps, and sequence abundance distribution analysis coupled with clustering. This approach generates distinct clusters of sequences, avoiding the need for massive screening of aptamer candidates. We demonstrated the proof-of-principle and effectiveness of the single-round selection approach resulting in a human insulin receptor aptamer, moreover, this method offers the expedient ability to screen multiple targets, various selection conditions, or differently modified libraries in a parallelized manner. The modified aptamer composed of two hydrophobic indole- and phenyl-linked nucleotides and two natural nucleotides exhibited picomolar binding affinity and exquisite specificity, capable of distinguishing subtle amino acid variations. Additionally, we determined the structure of the aptamer-receptor complex using cryo-electron microscopy, confirming the crucial role of hydrophobic aromatic modifications for the interactions of the aptamer with the target protein. These results highlight the synergistic effect of the components involved and demonstrate the capability of this methodology to generate highly potent and specific modified aptamers without tedious iterative selection rounds and extensive sequence space, thus accelerating aptamer discovery for therapeutic and diagnostic applications.