Fig 2.

In silico λ-dynamics workflow for screening potential binders to the inosine and m⁶A antibodies. A three-step process was used to filter candidates from a library of 48 ribonucleosides for in vitro antibody binding validation. (1) For each mutant library candidate, a λ-dynamics simulation was conducted to calculate a relative binding free energy between the mutant and its respective native ribonucleoside ligand (inosine or m⁶A). (2) All ribonucleosides that unbound during these simulations were deemed unfavorable and excluded from further processing. (3) Mutant bases with relative binding free energies deemed favorable (ΔΔG_bind ≤ −0.7 kcal/mol) were selected for in vitro validation with binding assays based on commercial availability.