Targeting lncRNA JINR1 with programmable Circular Active Nano DNAzyme (CANDe) suppresses Japanese Encephalitis Virus infection

Oligonucleotide therapeutics such as antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) enable sequence-specific gene silencing but rely on endogenous cellular machinery and often require extensive chemical modification for stability and efficacy. DNAzymes offer a mechanistically distinct alternative through intrinsic catalytic RNA cleavage; however, their therapeutic translation has been limited by nuclease susceptibility, structural constraints, and synthetic challenges. Here, we report the development of Circular Active Nano DNAzyme (CANDe), an enzymatically synthesized circular DNAzyme platform designed to enhance stability without backbone modification. The therapeutic potential of CANDe constructs was investigated against Japanese Encephalitis Virus (JEV) infection associated host long-noncoding RNA JINR1 (LINC01518). CANDe constructs were generated via splint-assisted ligation and incorporate modular elements, including catalytic cores (8-17 or 10-23), target-binding arms, and structural stems. Circularization conferred marked resistance to exonuclease-mediated degradation compared to linear DNA, maintaining structural integrity under nuclease-rich conditions.,CANDe targeting the lncRNA JINR1 achieved effective JINR1 knockdown in SHSY-5Y with and without JEV infection. This was accompanied by reduced expression JEV RNA and titers. In line with this, CANDe constructs attenuated of virus-induced cytotoxicity and apoptosis. Among the constructs, 10-23-based CANDe targeting the JINR1-1 site exhibited the strongest overall activity. These findings establish CANDe as a modular, modification-free DNAzyme platform that combines catalytic efficiency with enhanced stability, enabling effective host-directed antiviral intervention. This approach highlights topological engineering as a viable alternative to chemical modification for advancing DNAzyme-based therapeutics.