Structure-based design of a dimeric RNA-peptide complex.

The arginine-rich RNA-binding domain of bovine immunodeficiency virus (BIV) Tat adopts a beta-hairpin conformation upon binding to the major groove of BIV TAR. Based on its NMR structure, we modeled dimeric arrangements in which two adjacent TAR sites might be recognized with high affinity by a dimeric peptide. Some dimeric RNAs efficiently bound two unlinked BIV Tat peptides in vitro, but could not bind even one monomeric peptide in vivo, as monitored by transcriptional activation of human immunodeficiency virus long terminal repeat reporters. Results with additional reporters suggest that extending the RNA helix in the dimeric arrangements inhibits peptide binding by decreasing major groove accessibility. In contrast, a dimeric peptide efficiently bound an optimally arranged dimeric TAR in vivo, and bound with an affinity at least 10-fold higher than the monomeric peptide in vitro. Mutating specific nucleotides in each RNA 'half-site' or specific amino acids in each beta-hairpin of the dimeric peptide substantially decreased binding affinity, providing evidence for the modeled dimer-dimer interaction. These studies provide a starting point for identifying dimeric RNA-protein interactions with even higher binding affinities and specificities.