Synthesis of highly complex phosphorothioate-modified oligonucleotides on microarrays

The phosphorothioate bond (PS) is one of the most common chemical modifications of DNA and RNA and replaces one of the non-bridging oxygen atoms of the phosphodiester (PO) bond with a sulfur atom. Ubiquitous in oligonucleotide drug design, as it equips most FDA-approved therapeutics, it imparts resistance against nuclease degradation. Racemic PS bonds tend to lower duplex stability, but how and where PS bonds most affect it is difficult to predict. A high-throughput approach such as in situ nucleic acid microarray synthesis can potentially help to map out these effects. In this paper, we adapt the sulfurization process to microarray photolithography and carry out the combinatorial synthesis of PO and PS oligos that includes every combination of PO and PS across a set of three different sequences. We were able to map sensitivity to modification and to correlate hybridization signal to melting temperature. Phosphorothioate microarrays should be an ideal platform to accelerate the process of identifying ideal patterns of modifications in a therapeutically relevant context.