Design of well-folded beta-hairpin peptides for molecular recognition of RNA and improved resistance to proteolysis.

摘要

Ribonucleic acid (RNA) plays a vital role in many biological processes of the cell, which makes it an attractive target for drug discovery. In this work, small beta-hairpin peptides were used to better understand RNA molecular recognition. Specifically, peptides were designed with the capability to bind via aromatic, electrostatic, and hydrogen bonding interactions to the single stranded RNA.;To investigate RNA recognition using a conjugated system of different binding motifs, a beta-hairpin peptide was connected to an RNA intercalator. It was found that the individual parts did not bind well to RNA, but the conjugated system binds RNA with a dissociation constant of 3.7 muM. RNase footprinting experiments showed that the intercalator threads the stem region while the peptide interacts with an internal bulge region, and that the beta-hairpin structure of the peptide is important for binding.;Combinatorial chemistry and de novo peptide design were used to improve binding of the BIV Tat (bTat) peptide for BIV TAR RNA (bTAR). A combinatorial library was designed which incorporated natural and unnatural residues in the N-terminal region of bTat with the purpose of inducing interactions with the bTAR hairpin loop region. Two peptides were isolated from fluorescent library screens, one which had one less charge than bTat and contained an unnatural homophenylalanine residue. It was determined by gel shift assays that the two peptides bound equally as well to bTAR as the native sequence. In a separate project, strong cation-pi side chain interactions were used to promote beta-hairpin structure of a bTAR-binding peptide without the need for cyclization. The side-chain interactions were enough to stabilize the hairpin structure; however some RNA binding affinity was lost.;To successfully develop peptide drugs that bind RNA, the receptors need to be stable to proteolytic degradation. For this purpose, it was investigated whether stable beta-hairpin peptides have increased resistance to proteolysis due to their structure. A series of peptides with ranging amount of thermodynamic stability were designed and digested using a variety of specific and non-specific proteases. It was determined that increased thermodynamic stability of the beta-hairpin peptides does correlate to an increase in proteolytic stability.