Evaluation of methylphosphonates as analogs for detecting phosphate contacts in RNA-protein complexes

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