Therapeutic DNAzyme targeting conserved N gene sequences of pan-coronaviruses: Dual antiviral mechanisms of RNA cleavage and immune activation.

Coronaviruses frequently mutate and cross species, rendering most strain-specific drugs and antibodies less effective and leaving familial-level threats unresolved. The nucleocapsid (N) protein, with highly conserved N- and C-terminal domains, plays a central role in viral replication and immune evasion, making it an ideal target for broad-spectrum antiviral development. We designed DNAzyme targeting conserved N gene sequences from SARS-CoV and multiple SARS-CoV-2 variants, identifying N4 with strong in vitro RNA cleavage activity (Kobs = 0.053 min-1, 54.56% cleavage rate) and 80% N mRNA knockdown in transfected cells. To enhance its pharmacological properties, N4 was chemically modified with FANA, generating FANA4. Compared with N4, FANA4 exhibited a sixfold increase in serum half-life (8 h to 48 h), a twofold improvement in cleavage efficiency, and > 95% cellular uptake. In N-overexpressing and virus-infected cell models, FANA4 suppressed N protein expression by 99.02% and reduced viral replication by 85%. In vivo, intranasal administration decreased lung viral load by 3-fold without observable toxicity. Transcriptomic profiling revealed ERK/MAPK pathway activation, supporting a dual antiviral mechanism of sequence-specific RNA cleavage and host immunity enhancement. These findings highlight FANA4 as a promising, broad-spectrum nucleic acid therapeutic candidate for combating current and emerging coronaviruses.