Arli Aditya PARIKESIT, Arif Nur Muhammad ANSORI, Moh. Royhan AFNANI
The European Chemistry and Biotechnology Journal - 2026;3(6):35-49
Methanogenic archaea are responsible for roughly 14% of global methane emissions, so disrupting their methane production could substantially lower these emissions. The methyl-coenzyme M reductase (MCR) complex, encoded by mcrA , mcrB , and mcrG , catalyzes the final step of methane formation. Here we used computational methods to design a small interfering RNA (siRNA), conceived as an RNA interference (RNAi)-like antisense oligonucleotide, that targets a region of the mcrA gene conserved across eight methanogen species spanning five taxonomic orders. A 14-step computational pipeline was used to retrieve mcrA sequences, align them, identify a 40-nucleotide window with 96.9% mean conservation, and select the best 19-nucleotide guide strand by energy scoring. The guide strand (5'-UGCCUGCUUUGAUGCCUGC-3') targets the mRNA sequence 5'-GCAGGCAUCAAAGCAGGCA-3' and has a GC content of 57.9%. ViennaRNA analysis gave a target mRNA minimum free energy (MFE) of -8.20 kcal/mol and a guide-strand MFE of -2.10 kcal/mol; co-folding of the siRNA-mRNA duplex yielded an MFE of -41.20 kcal/mol, indicating a stable pairing. Structural-diversity analysis identified 22 siRNA and 94 mRNA conformations within 5 kcal/mol of the MFE, and the target site was highly accessible (mean unpaired probability of 0.581). RNA-RNA pairing in an antiparallel A-form duplex showed 49 Watson-Crick hydrogen bonds, 36 pi-stacking interactions, and 5 metal-coordinating contacts across the 19 base pairs. All-atom molecular dynamics at 300 K using OpenMM (AMBER14 force field, GBn2 implicit solvent) showed a mean potential energy of about -3,754 kcal/mol, a backbone RMSD that converged to a plateau of 8.2 Å, and a stable radius of gyration (17.4 Å), consistent with a flexible single-stranded guide. This conserved, multispecies target makes the designed guide a strong candidate for experimental validation as a biotechnological route to reduce livestock methane emissions.
