The objective of this proposal is to develop a new method (linked- oligonucleotide probes) for the analysis of RNA structure in solution. To do this, the strengths of chemical synthesis and enzymology have been combined in a technique which is expected to be more effective than either crosslinking or chemical probes. This approach requires the synthesis of molecules that contain two short oligonucleotides, covalently linked, by a flexible tether of defined length. One of the oligonucleotides will be constructed of normal deoxynucleotides, the other, of 2'-OMe ribonucleotides. Each will be complementary to a single-stranded region present in the RNA under investigation. Simultaneous hybridization of both ends of this molecule to RNA will occur only if the flexible tether that separates the two oligonucleotides can traverse the complementary sites. Because of the substrate specificity of RNase H, treatment of the hybrid with this enzyme will cleave the RNA exclusively at the site of the RNA:DNA hybrid. Determination of the lengths of the RNA fragments generated will identify the cleavage site to nucleotide resolution. Through the use of tethers of varying length, the distance in space between the two complementary sites on the macromolecule can be defined. Because the linked-oligonucleotide probes can be synthesized either entirely or predominantly using automated solid phase methods, this technique will be more versatile than conventional crosslinking. Because the data can be interpreted only in terms of tertiary structure, this technique will be more straightforward than chemical or enzymatic higher-order structural analysis. This application will focus on the use of this technique to investigate the structure of native RNA molecules in solution. However, the methods described can be applied to the study of ribonucleoprotein structure, as well as RNA:protein and RNA:DNA interactions in solution. Specific Aims 1 and 2 focus on the development of this technique using structurally well characterized yeast tRNAphe. In the first of these Specific Aims, the 2'-OMe pentanucleotide 5'-UmGmGmUmGm, complementary to the 3'-end of tRNAphe, will be linked by an abasic phosphodiester tether to tetraoligonucleotides complementary to either the anticodon, D- or T-loops. By targeting these three loops, we will define a relationship between the length of the tether and the distance in A units. In Specific Aim 2, the two oligonucleotides will be linked by a poly (diaminopentane) linker. Electrostatic effects may enhance the affinity of these probes for RNA, resulting in superior molecular rulers. In Specific Aim 3, the techniques developed in Specific Aim 1 and 2 will be applied towards the study of the three dimensional structure of a catalytic ribozyme. Our goal is to use this information to determine which residues constitute the enzyme active site..