The major goal of the proposed research plan is to determine whether the chemical probes used to modify TAR RNA and Tat peptides have induced significant structural perturbations and reduced the capacity to form a productive complex. In order to confirm the biological significance of their affinity-cleavage and photo-crosslinking results, the Principal Investigator and his Co-Principal Investigator must verify that the chemical alterations associated with the introduction of structural probes have not significantly affected the RNA or peptides. Incorporation of structural probes into RNA sequences may perturb major determinants of nucleic acid conformation, such as base pairing, base stacking, and phosphate backbone geometry which may change the three-dimensional structure of RNA. To minimize this problem, the researchers will make single site modifications at different positions in the RNA sequence and test for destabilization in RNA structure. They will confirm the folding of chemically-modified RNA into the correct functional structure by: (1) formation of a specific Tat-TAR complex; (b) CD and UV melting measurements; and (c) NMR spectroscopic techniques to determine any major structural changes in the chemically-modified RNA; they will compare these results to those from unmodified RNA. Similarly, chemical modification of Tat fragments could cause a change in the folded structure of the peptide. The researchers will conduct initial evaluations by examining the RNA-binding capabilities of the modified peptides and measuring the binding constants. They will conduct NMR studies on free peptides in solution and RNA-peptide complexes. If the modification of the peptides has led to a different mode of TAR recognition, these results could be of potential significant for drug discovery. The free TAR structure is very flexible; compounds that recognize TAR by locking the RNA structure into a conformation that is incompetent for binding would provide potentially very effective Tat inhibitors.
