Synthesis and characterization of non-infectious hop latent viroid circular RNA using group I self-splicing ribozyme system
DOI:
https://doi.org/10.7124/bc.000B12Keywords:
Hop latent viroid, cannabis, Group I Permutated Intron, in vitro transcription, circRNAAbstract
Aim. Hop latent viroid (HLVd) poses a major threat to Cannabis sativa cultivation, with up to 90% infection rates and 70% THC loss. Reliable non-infectious positive controls are needed for diagnostics. Methods. We synthesized non-infectious HLVd circular RNA using a group I self-splicing ribozyme system. The product was characterized by RNase R digestion, RT-PCR, and sequencing to confirm circularization and the ribozyme-derived joining sequence. Results. Structural analysis showed the synthetic RNA retained high stability (ΔG = –90.4 kcal/mol) compared to native HLVd (ΔG = –93.8 kcal/mol), with introduced mutations preventing replication. The molecule remained stable from –20°C to 37°C for 10 days and performed consistently as a PCR positive control. The infection assays in cannabis confirmed its non-infectious nature. Conclusion. The synthetic HLVd circRNA provides a safe, stable positive control for diagnostics and a model for viroid research, addressing a key gap in current detection methods.References
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