Elucidating the effect of a rationally designed nanostructured form-switching ASO (NaFASO) for targeting long non-coding RNA to alleviate Japanese encephalitis virus infection

RNA therapeutic modalities such as antisense oligonucleotides (ASOs) have emerged as promising tools to target previously "undruggable" targets. Despite their great promise as precision therapeutic agents, their clinical adoption remains limited due to production costs, sequence-length restrictions, limited structural heterogeneity, and the generation of environmentally hazardous waste during synthesis. Biocatalytic synthesis strategies provide a sustainable alternative; however, their reliance on specialized enzymes and precursors often limits sequence diversity and scalability. To address these limitations, we report the design and biocatalytic synthesis of a novel circular ASO: Nanostructured Form-switching Antisense Oligonucleotide (NaFASO) for targeting Japanese Encephalitis Virus (JEV) infection-associated host long non-coding RNA (lncRNA) JINR1 (JEV-induced non-coding RNA1) in SH-SY5Y cells. The novel modular architecture in NaFASO has been designed to have a metastable stem that separates the functional antisense domain from the splint-padlock circularizing region, ensuring both structural integrity and efficient target engagement. The serum- and nuclease-stable NaFASOs achieved knockdown of the lncRNA JINR1 during JEV infection, resulting in a reduction in JEV replication and neuronal cell death. NaFASO-mediated JINR1 depletion also resulted in downregulation of the JEV replication-associated gene GRP78. Together, these findings establish NaFASO as a first-of-its-kind structure-switching circular ASO platform for combating JEV infection, combining stability, efficacy, and environmental sustainability. Beyond the JEV, the generalizability of this design suggests broad applicability for targeting diverse RNA species implicated in genetic disorders, viral infections, and cancer, thus highlighting a promising paradigm for developing next-generation transformational nucleic acid therapeutics.