Search for: All records

Splice-modulating antisense oligonucleotides (ASOs) are precision RNA-based drugs that are becoming an established modality to treat human disease. Previously, we reported the discovery of ASOs that target a novel, putative intronic RNA structure to rescue splicing of multiple pathogenic variants of F8 exon 16 that cause hemophilia A. However, the conventional approach to discovering splice-modulating ASOs is both laborious and expensive. Here, we describe a novel approach that integrates data-driven RNA structure prediction and community science to discover splice-modulating ASOs. Using a splicing-deficient pathogenic variant of F8 exon 16 as a model, we show that 25% of the top-scoring molecules designed in the Eterna OpenASO challenge have a statistically significant impact on enhancing exon 16 splicing. Additionally, we show that a distinct combination of ASOs designed by Eterna players can additively enhance the inclusion of the splicing-deficient exon 16 variant. Together, our data suggests that crowdsourcing designs from a community of citizen scientists may accelerate and complement traditional avenues for the discovery of splice-modulating ASOs with potential to treat human disease. 

Abstract The ribosome is a ribonucleoprotein complex found in all domains of life. Its role is to catalyze protein synthesis, the messenger RNA (mRNA)-templated formation of amide bonds between α-amino acid monomers. Amide bond formation occurs within a highly conserved region of the large ribosomal subunit known as the peptidyl transferase center (PTC). Here we describe the step-wise design and characterization of mini-PTC 1.1, a 284-nucleotide RNA that recapitulates many essential features of the Escherichia coli PTC. Mini-PTC 1.1 folds into a PTC-like structure under physiological conditions, even in the absence of r-proteins, and engages small molecule analogs of A- and P-site tRNAs. The sequence of mini-PTC 1.1 differs from the wild type E. coli ribosome at 12 nucleotides that were installed by a cohort of citizen scientists using the on-line video game Eterna. These base changes improve both the secondary structure and tertiary folding of mini-PTC 1.1 as well as its ability to bind small molecule substrate analogs. Here, the combined input from Eterna citizen-scientists and RNA structural analysis provides a robust workflow for the design of a minimal PTC that recapitulates many features of an intact ribosome.