Genome Technology Center

The Application and Development of 2’-Hydroxyl Chemistry for RNA Structure Analysis

Dr. Bin Wang
Department of Chemistry, University of North Carolina at Chapel Hill

Understanding the global conformation of an RNA is an important first step in describing its function. However, classical approaches for obtaining model-independent constraints on RNA structure require significant experimental and hands-on effort. We have developed a high-throughput technology that allows RNA structure to be monitored at single nucleotide resolution. This technology, termed Selective 2’-Hydroxyl Acylation analyzed by Primer Extension (SHAPE), can be used to map the structure of any RNA under arbitrary solution and biological environments.

We are applying this technology to analyze the effects of Mg2+ and tobramycin binding on the folding of tRNAAsp at single nucleotide resolution. As expected, tRNAAsp folding is highly sensitive to Mg2+. Decreasing the Mg2+ concentration does not cause simple unfolding – it causes structural rearrangement where the D-loop and variable loop pair. Mg2+-induced refolding is not hierarchical as in usually assumed. Addition of the aminoglycoside antibiotic tobramycin disrupts the native fold in two distinct transitions, involving loss of tertiary interactions between the T- and D-loops followed by the complete unfolding of the D-stem. The ability to monitor ligand binding at single nucleotide resolution supports a surprisingly complex effect on RNA structure.

SHAPE primer extension products are separated and detected by slab gel electrophoresis and capillary gel electrophoresis. In the long run, polymer microchip-based capillary gel electrophoresis shows the most promise in SHAPE technology due to its decreased time of analysis, reduced consumption of reagents and analytes, and decreased cost in manufacture, use, and disposal.

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