Research Articles
DOI DOI: 10.62063/ecb-88

Computational design of a siRNA targeting the methyl-coenzyme M reductase A (mcrA) gene for methane cessation in methanogenic archaea

Arli Aditya Parikesit1, Arif Nur Muhammad Ansori2, Moh. Royhan Afnani3
  1. 1 i3L University
  2. 2 Universitas Airlangga
  3. 3 Universitas Gadjah Mada

Abstract

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.

How to Cite

Parikesit, A. A., Ansori, A. N. M., & Afnani, M. R. (2026). Computational design of a siRNA targeting the methyl-coenzyme M reductase A (mcrA) gene for methane cessation in methanogenic archaea. The European Chemistry and Biotechnology Journal, (6). https://doi.org/10.62063/ecb-88
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