Advancing siRNA Therapeutics targeting MCT-4: A Multifaceted approach integrating Arithmetical Designing, Screening, and molecular dynamics validation.

Monocarboxylate transporter 4 (MCT-4) is involved in various metabolic processes which are crucial in maintaining cellular pH and energy metabolism, and thus influence the tumor microenvironment. The study is aimed to rationally design effective Small interfering RNA (siRNA) that can silence MCT-4. We utilized a comprehensive workflow integrating multiple tools such as siDirect version 2.0, Oligowalk and i-score designer, to evaluate sequence features and predict target site accessibility, Guanine-Cytosine (GC) content and thermodynamic stability. Five (M1, M2, M3, M4 and M5) siRNAs sequences were retrived and subjected to further scrutiny on the account of off-target elimation, sequence conservation, secondary structure formation, and thermodynamic properties. The M1 demonstrated off targets and the M2 sequence showed secondry conformation and therefore M3, M4 and M5 were considered for further evaluation. Additionally, molecular docking and simulations (50 ns) were conducted with human Argonaute 2 protein (h-Arg-2). The post- molecular dynamics (MD) analysis revealed M4 (5'UUGAAGAAGACACUGACGG3') as a most appropriate siRNA candidate agsint MCT-4 on the basis of Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), and H-Bond results. The Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis was also performed to further validate the selected siRNA candidates, which further affirmed M4 (5'UUGAAGAAGACACUGACGG3') as an potential candidate for future in-vitro and in-vivo evaluation.