Combinatorial and rational synthesis of complex, base-modified aptamer libraries on microarrays

Chemically modifying the backbone, sugar, or nucleobase moieties of nucleic acids greatly expands their functional repertoire. Nucleobase modifications have received particular attention for their proven ability to generate a greater diversity of intra- and intermolecular interactions. This broader interaction landscape has facilitated advances in the identification of aptamers with high affinity to different target molecules, including proteins. As common practice, SELEX (Systematic Evolution of Ligands by EXponential enrichment) is used to select the best binders from a large pool of random oligonucleotides. However, the combinatorial space of SELEX is usually limited to n = 4 different nucleotides and it does not account for position-specific effects of target binding. In contrast, photolithographic synthesis of oligonucleotide microarrays allows for (a) full control of the position of a modified nucleotide in a sequence, (b) the implementation of multiple nucleic acid building blocks in addition to the four canonical nucleotides, and (c) the chemical synthesis of hundreds of thousands of unique sequence variants in parallel and on the same surface. In this work, we explored photolithographic microarray synthesis with two uracil analogs carrying the side chain of the amino acids serine and tyrosine on position C5 (dU-Ser, dU-Tyr). Hybridization experiments showed an enhanced duplex stability of homopolymer strands of these modified nucleotides compared both to dU and dT. Additionally, we synthesized permutation libraries of two known streptavidin aptamers by substituting the dT by dU, dT, dU-Ser and dU-Tyr in all possible combinations. In binding assays, we observed positions at which the presence of modified dUs increases protein binding, and locate other sites that do not tolerate chemical modifications. In summary, this project highlights the power of oligonucleotide microarrays in studying affinity patterns in aptamers and demonstrates the value of incorporating base-modified nucleotides in photolithographic synthesis.