PROTEIN-FREE MOLECULAR PLATFORM FOR H5N1 VIRUS DIAGNOSIS: RIBOZYME CLEAVAGE-DIRECTED SIGNAL AMPLIFICATION VIA BRANCHED HCR

The global spread of highly pathogenic avian influenza (HPAI) subtype H5N1 represents a severe health crisis, marked by recurrent spillover events into mammals and substantial losses in the commercial poultry industry. In Brazil, the world's second-largest producer of chicken meat, confirmed cases in wild and commercial birds have led to the extension of the national animal health emergency. Although early diagnosis is critical to mitigate pandemic risks, conventional methods such as RT-PCR and ELISA are limited by their dependence on heat-sensitive protein enzymes, complex infrastructure, and strict cold-chain requirements. To address these logistical constraints, this project proposes an innovative, low-cost, and rapid molecular diagnostic platform independent of protein-based enzymatic activity. The system combines two complementary molecular mechanisms: sequence-specific target viral RNA recognition and cleavage by hammerhead ribozymes, followed by signal amplification using a non-linear hybridization chain reaction (bHCR) coupled with DNAzyme activity. Conserved and specific regions within the HA and NA genes of the H5N1 virus were identified to define suitable target sites. Rational design of the ribozymes and hairpin-shaped DNA reporter molecules (H1, H2, H3, and H4) was conducted using RiboSoft and a computational program tool developed by our research group. Synthetic RNA targets and ribozymes were generated by in vitro transcription, and catalytic activity was evaluated through in vitro cleavage assays. We hypothesized that the initiator released upon ribozyme cleavage promotes sequential opening of the DNA hairpins. This process separates the fluorophore/quencher pair (FAM/BHQ1) on the H2 probe, producing a fluorescence signal detected by a spectrofluorometer. Out of three ribozymes tested, two exhibited target-specific catalytic activity: ribozyme 8 against HA and ribozyme 14 against NA. In fluorescence assays, signal generation was observed in the presence of synthetic 18-nt target DNAs and hairpin reporters, achieving a limit of detection of 12.5 nM compared to the negative control. Consequently, these findings highlight the platform's potential for robust, decentralized epidemiological surveillance, including in remote settings.