The well-studied interaction between the MS2 coat protein and its cognate hairpin was used to test the utility of the methylphosphonate linkage as a phosphate analog. A nitrocellulose filter binding assay was used to measure the change in binding affinity upon introduction of a single methylphosphonate stereoisomer at 13 different positions in the RNA hairpin. Comparing these data to the available crystal structure of the complex shows that all phosphates that are in proximity to the protein show a weaker binding affinity when substituted with a phosphorothioate and control positions show no change. However, in two cases, a methylphosphonate isomer either increased or decreased the binding affinity where no interaction can be detected in the crystal structure. It is possible that methylphosphonate substitutions at these positions affect the structure or flexibility of the hairpin. The utility of the methylphosphonate substitution is compared to phosphate ethylation and phosphorothioate substitution experiments previously performed on the same system.