dc.contributor.author	Zhang, Wenyu	en
dc.contributor.committeechair	Santos, Webster L.	en
dc.contributor.committeemember	Gandour, Richard D.	en
dc.contributor.committeemember	Capelluto, Daniel G.	en
dc.contributor.committeemember	Kingston, David G. I.	en
dc.contributor.department	Chemistry	en
dc.date.accessioned	2015-10-07T06:00:21Z	en
dc.date.available	2015-10-07T06:00:21Z	en
dc.date.issued	2014-04-14	en
dc.description.abstract	RNAs have gained significant attention in recent years because they can fold into well-defined secondary or tertiary structures. These three dimensional architectures provide interfaces for specific RNA-RNA or RNA-protein interactions that are essential for biological processes in a living system. These discoveries greatly increased interest in RNA as a potential drug target for the treatment of diseases. Two of the most studied RNA based regulatory systems are HIV-1 trans-activating response element (TAR)/Tat replication pathway and Rev response element (RRE)/Rev export pathway. To efficiently target TAR and RRE RNA, we designed and synthesized three generations of branched peptide libraries that resulted in medium sized molecules. The first generation of BPs were discovered from screening a one-bead one-compound library (4,096 compounds) against HIV-1 TAR RNA. One peptide FL4 displayed a binding affinity of 600 nM to TAR RNA, which is tighter than its native protein counterpart, Tat. Biophysical characterization of these BP demonstrated that "branches" in BPs impart multivalency, and they are cell permeable and non-toxic. The second generation peptides were discovered from an on-bead high-throughput screening of a 3.3.4 branched peptide boronic acids (BPBAs) library that bind selectively to the tertiary structure of RRE IIB. The library comprised of 46,656 unique sequences. We demonstrate that our highest affinity BPBA (BPBA1) selectively binds RRE IIB in the presence of competitor tRNAs as well as against six RRE IIB structural variants. Further, we show that the boronic acid moieties afford a novel binding mode towards RNA that is tunable; their Lewis acidity has critical effects on binding affinity. In addition, biophysical characterizations provide evidence that "branching" in these peptides is a key structural motif for multivalent interactions with the target RNA. Finally, RNA footprinting studies revealed that the BPBA1 binding site encompasses a large surface area that spans both the upper stem as well as the internal loop regions of RRE IIB. BPBA1 is cell permeable and non-toxic. In the next generation of branched peptides, a 3.3.4 branched peptide library composed of 4,096 unique sequences that featured boronic acid and acridine moieties was designed. We chose acridine as the amino acid side chain due to its potential for π-stacking interaction that provides high binding affinity to RNA target. The library was screened against HIV-1 RRE IIB RNA. Fifteen peptides were sequenced and four contained acridine alone and/or in conjunction with boronic acid moieties displayed dissociation constants lower than 100 nM. The ribonuclease protection assays of A7, a sequence that contains both boronic acid and acridine residues, showed a similar protection pattern compared to previous peptide BPBA1, suggesting that the 3.3.4 branched peptides shared similar structural elements and contacted comparable regions of the RRE IIB RNA. The results from this research indicated that "branching" in peptides imparts multivalent interactions to the RNA, and that functional groups such as boronic acid and acridine are key structural features for efficient binding and selectivity for the folded RNA target. We demonstrated that the branched peptides are cell permeable and non-toxic.	en
dc.description.degree	Ph. D.	en
dc.format.medium	ETD	en
dc.identifier.other	vt_gsexam:2640	en
dc.identifier.uri	http://hdl.handle.net/10919/56731	en
dc.publisher	Virginia Tech	en
dc.rights	In Copyright	en
dc.rights.uri	http://rightsstatements.org/vocab/InC/1.0/	en
dc.subject	HIV-1	en
dc.subject	TAR RNA	en
dc.subject	RRE RNA	en
dc.subject	Branched Peptide Library	en
dc.subject	High-throughput Screening	en
dc.subject	Branched Peptide Boronic Acids	en
dc.subject	Acridine Branched Peptides	en
dc.subject	Unnatural Amino Acids	en
dc.title	Targeting HIV-1 RNAs with Medium Sized Branched Peptides Featuring Boron and Acridine-Branched Peptide Library Design, Synthesis, High-Throughput Screening and Validation	en
dc.type	Dissertation	en
thesis.degree.discipline	Chemistry	en
thesis.degree.grantor	Virginia Polytechnic Institute and State University	en
thesis.degree.level	doctoral	en
thesis.degree.name	Ph. D.	en

Targeting HIV-1 RNAs with Medium Sized Branched Peptides Featuring Boron and Acridine-Branched Peptide Library Design, Synthesis, High-Throughput Screening and Validation

Files

Original bundle

Collections