Methods for high-throughput analysis of RNA structure and dynamics Public Deposited

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  • March 20, 2019
  • Mortimer, Stefanie Ann Ward
    • Affiliation: College of Arts and Sciences, Department of Chemistry
  • RNA sequences fold back on themselves to form secondary and tertiary structures that are difficult to predict. Knowledge of these structures and the time-resolved mechanism by which RNA molecules fold into these structures are necessary for a full understanding of structure-function relationships in RNA biology. A newly developed technology, called SHAPE, provides accurate and quantitative RNA structure information. SHAPE chemistry was developed using N-methylisatoic anhydride (NMIA), which is only moderately electrophilic and requires tens of minutes to form ribose 2'-O-adducts. In this work I design and evaluate several classes of significantly more useful reagents for SHAPE chemistry and create a new way to assess RNA tertiary structure in an experimentally straightforward way. First, I design and synthesize a faster reacting reagent for SHAPE chemistry, 1-methyl-7-nitroisatoic anhydride (1M7), based on the NMIA scaffold. With 1M7, single nucleotide resolution interrogation of RNA structure is complete in 70 seconds and appears to be the ideal reagent for equilibrium analysis of RNA structure. Second, I apply SHAPE reagents of varying electrophilicity to identify a class of nucleotides with slow conformational dynamics. The observation is that select C2´-endo nucleotides undergo extremely slow conformational changes on the order of ~10-4 sec-1. Third, I extend SHAPE chemistry to a benzoyl cyanide scaffold to make possible facile time-resolved kinetic studies of RNA in ~1 s snapshots. I then use SHAPE chemistry to follow the time-dependent folding of an RNase P specificity domain RNA and identify a slow folding region in the RNA. I am able to attribute this slow folding step to the conformational dynamics of a single nucleotide. Finally, I show that show that N,N-(dimethylamino)dimethylchlorosilane (DMAS-Cl) reacts selectively at the guanosine N2 position. Critically, DMAS-Cl reactivity yields a near-perfect measure (r greater than or equal to 0.82) of solvent accessibility for this position in a folded RNA. This silane-based chemistry represents a significant improvement over classical approaches that employ carbon electrophiles for probing solvent accessibility at the base pairing face of guanosine in RNA.
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  • Weeks, Kevin
  • Open access

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