The structure of a pre-mRNA molecule in solution determined with a site directed cross-linking reagent.

We describe the use of site specific psoralen (SSP) to determine the solution structure of a segment of the human beta globin pre-mRNA. In these experiments, SSP is first delivered as monoadducts to specific nucleotides in the pre-mRNA and subsequently used to form intramolecular RNA-RNA cross-links. The use of this reagent greatly decreases the number of the cross-linked products as compared to generalized psoralen cross-linking. The experiments confirm the locations of previously determined aminomethyltrimethylpsoralen (AMT) cross-links in the human precursor mRNA. In addition, new cross-links consistent with an alternative secondary structure and a small number of cross-links that represent higher order interactions have been determined. Altogether, 42 of 47 cross-links identified in this analysis can be accounted for in a small number of alternative secondary structures and higher order interactions. The site directed cross-linking technique will be useful for the precise determination of RNA secondary and tertiary structures under a variety of experimental conditions.