Sato, S.-i. | bioRxiv Science

bioRxiv2025

Expanded gene targeting in RNA hacking with G-tract-supply Staple oligomer

RNA hacking (RNAh) is a gene regulation technology that employs a short oligonucleotide, termed a Staple oligomer, to induce the formation of RNA G-quadruplex structures on target mRNAs. While RNAh has the potential to target approximately 65% of human mRNAs, its applicability to the remaining genes is restricted by the sequence constraints. Herein, we present the G-tract-supply Staple oligomer (Gs-Staple oligomer), designed to expand the range of targetable mRNAs within the RNAh framework. Incorporating G-tracts into Staple oligomers alleviates the sequence constraints, enabling access to a broader range of mRNA targets. Gs-Staple oligomers effectively suppressed the translation of target proteins in mammalian cells and in vivo. Furthermore, the gene suppression could be precisely modulated by adjusting the linker length between the G-tracts. These findings have significantly expanded the versatility of RNAh, suggesting its potential for further development while highlighting its potential to be utilized as a nucleic acid-based tool for research and clinical medicine.

bioengineering↗

bioRxiv2023

In Vivo mRNA Hacking with Staple Oligomers Prevents Myocardial Hypertrophy

Summary paragraphThe elucidation of gene-silencing mechanisms by RNA interference (RNAi) and antisense oligomers has drawn increasing attention to nucleic acid medicine. However, several challenges remain to be overcome, such as in vivo stability1, target selectivity 2,3, drug delivery4,5, and induced innate immunity6. Here, we report a new, versatile, and highly-selective method to hack RNA by controlling RNA structure using short oligonucleotides (RNA hacking: RNAh) in living cells. The oligonucleotide, named Staple oligomer, hybridizes specifically to a target mRNA and artificially induces an RNA higher-order structure, RNA G-quadruplex (RGq)7, on the mRNA. As a result, the RGq allows effective suppression of the target protein translation. This technology does not require cooperation with bioprocesses including enzymatic reactions as in RNAi or antisense technologies, permitting the introduction of artificial nucleic acids into Staple oligomers to increase their in vivo stability without compromising their effectiveness. The method was validated by translational regulation of the mRNAs of TPM3, MYD88, and TRPC6, in a cell-free system and in living mammalian cells. In vivo application of the technology to TRPC6 mRNA allowed us to prevent cardiac hypertrophy in transverse aortic constriction (TAC)-treated mice with no detectable off-target effects. This technology provides new insights into gene therapy after RNAi and antisense technologies.

molecular biology↗