FANArrays: a high-throughput nucleic acid synthesis platform

The therapeutic potential of nucleic acid drugs has started to be explored and tapped into many decades ago, but actual drugs reaching the pharmaceutical market appeared less than ten years ago. This discrepancy between the theorized usefulness of nucleic acids to combat genetic and viral diseases and the reality of FDA-approved therapies, made all the more apparent given the conjoined efforts of both the academic and industrial worlds, can be explained with the multiple hurdles that field must overcome in order to design and obtain potent DNA or RNA-based oligonucleotide drugs. One of them is scale and throughput. To design effective nucleic acid therapeutics, the sequence must specifically target the intended gene and avoid off-target effects. In addition, chemical modifications of oligonucleotides, which are fundamental in DNA/RNA drug design, are carefully selected and introduced in order to increase stability, deliverability and potency but the amount and location of modifications must be fine-tuned so as to maintain an optimal balance between these three properties. This optimization process is very iterative and extremely time-consuming due to the lack of synthetic approaches that can performed combinatorial nucleic acid synthesis. Microarrays have this extraordinary property that they can simultaneously synthesize thousands upon thousands of molecules with exact control over structure and location. In particular, the photolithographic approach to microarray fabrication with nucleic acids allows for >780000 unique sequences to be synthesized on the same surface at the same time, whether DNA, RNA, or oligonucleotide analogs. One nucleoside analog that we recently introduced in microarrays is FANA, and it is a particularly relevant chemical modification in the field of nucleic acid therapeutics. Like every modification, the introduction of FANA into DNA or RNA appears to be sensitive to location and number. We therefore aim to synthesize microarrays that can include all possible DNA/FANA combinations in a given sequence (for example, a DNA sequence with 18 bases gives 2 18 possible combinations, or 262144 oligonucleotides) and perform a simple hybridization assay to a complementary RNA sequence to immediately identify positions that are sensitive to the introduction of FANA. We also intend to use these complex FANA-containing oligonucleotide antisense libraries to perform enzymatic assays on the RNA, expecting to link FANA position to antisense efficacy. Libraries of oligonucleotides will also be prepared to study the effect of mismatches and off-target activity. Additional chemical modifications, like the indispensable phosphorothioate (PS), will also be tested in combinatorial libraries of phosphate group modifications. We can also foresee the preparation of very complex systems where FANA and PS modifications can simultaneously be incorporated into antisense microarray libraries. Nucleic acid candidates with ideal properties on microarrays will be tested in more conventional settings thanks to our collaborators at McGill University (Prof. Masad Damha). In so doing, we aim to produce and showcase a novel platform that can potentially accelerate the process of oligonucleotide drug discovery.