Structural studies of a double-stranded RNA from trypanosome RNA editing by small-angle X-ray scattering

We used small-angle X-ray scattering (SAXS) to evaluate the solution structure of a double-stranded RNA with 32 base pairs. We wanted to compare the solution structure to the crystal structure to assess the impact of the crystal lattice on the overall conformation of the RNA. The RNA was designed to self-anneal and form a head-to-head fusion of two identical mRNA/oligo(U) tail domains (the U-helix) from a trypanosome RNA editing substrate formed by the annealing of a guide RNA to a pre-edited mRNA. This substrate is from the U insertion/deletion RNA editing system of trypanosomes. Each strand in the fusion RNA had 16 purines from the pre-mRNA followed by 16 uracils (Us) from the U-tail at the 3' end of the guide RNA. The strands were designed to form a double helix with blunt ends, but each strand had the potential to form hairpins and single-stranded RNA helices. Hairpins could form by the 3' oligouridylate tract folding back to hybridize with the 5' oligopurine tract and forming an intervening loop. Single-stranded helices could form by the stacking of bases in the polypurine tract. Some of the 16 Us 3' to the polypurine tract may have been unstacked and in random coils. Our SAXS studies showed that the RNA formed a mix of single-stranded structures in the absence of MgCl2. In the presence of MgCl2 at concentrations similar to those in the crystal, the solution structure was consistent with the double-stranded, blunt-ended structure, in agreement with the crystal structure. Here we describe the preparation of RNA samples, data collection with an in-house SAXS instrument designed for biological samples, and the processing and modeling of the scattering data.