Data Analytics Pipeline for RNA Structure Analysis via SHAPE

Coxsackievirus B3 (CVB3) is a cardiovirulent enterovirus from the family Picornaviridae. The RNA genome houses an internal ribosome entry site (IRES) in the 5’ untranslated region (5’UTR) that enables cap-independent translation. Ample evidence suggests that the structure of the 5’UTR is a critical element for virulence. We probe RNA structure in solution using base-specific modifying agents such as dimethyl sulfate as well as backbone targeting agents such as N-methylisatoic anhydride used in Selective 2’-Hydroxyl Acylation Analyzed by Primer Extension (SHAPE). We have developed a pipeline that merges and evaluates base-specific and SHAPE data together with statistical analyses that provides confidence intervals for reactivity values. Combining the “2%-8% rule” for normalization with base-specific mean and standard deviation calculations, ANOVA and multiple comparison procedures, we generate confidence intervals for each position, thereby verifying resulting secondary and tertiary structure models. Our datasets demonstrate that reactivity of each nucleotide base primarily parallels modification of the backbone, but not at every position. Using reactivity values validated by our statistical analyses, we are now in position to provide base-by-base analysis of RNA structural transitions. Understanding these transitions extends our previous comparative analysis of genomes from virulent and avirulent serotypes and sequential structural states during RNA-protein interaction