| Abstract 1: An estimated 1.2 million people in the United States are living with the human immunodeficiency virus (HIV), as reported by the Center for Disease Control (CDC). Because mutations constantly occur throughout the HIV genome, a cure has yet to be found to eradicate the virus. Instead, treatment with Highly Active Antiretroviral Therapy (HAART) effectively postpones the progression of HIV to AIDS (Acquired Immune Deficiency Syndrome) by eliminating most circulating viruses through drugs that target reverse transcription, integration, or HIV-1 fusion and entry. While this combination therapy has been shown to be very effective, it is unable to target viral reservoirs in patients, allowing emerging viruses to rapidly repopulate the body if therapy is interrupted. Thus, an important gap in current HIV treatment is the absence of drugs that block the emergence of the virus from latently infected cells, principally residing amongst resting CD4+ cells.;The viral transactivator protein (Tat) is required for viral gene expression for both the exponential growth of the virus and the activation of integrated, but latent, proviral genomes. Since the emergence of the virus from latency relies on Tat-dependent transcription, inhibitors of Tat-function are expected to be potent blockers of viral escape from latency. My thesis explores three approaches to developing potent, proteolytically stable, selective inhibitors of HIV-1 viral replication.;My primary focus was determining the in vitro binding affinity, selectivity and specificity of a small library of improved peptides, followed by elucidation of the tertiary structure of the lead peptide-RNA complex. The lead peptide, JB-181, bound selectively to HIV-1 TAR RNA at 28 pM, was specific to HIV-1 TAR RNA in the presence of other competing, structurally similar RNAs, and its 1.6 A, NMR resolved tertiary structure revealed more intimate contacts between the peptide's hydrophobic residues and the HIV-1 TAR RNA relative to previously designed peptides of its class. My second focus was to evaluate the activity of a new class of peptide mimics -- gamma-AA peptides (based on the gamma-PNA backbone) designed to target and inhibit the TAR-Tat interaction. These gamma-AA peptides can project the same number of functional groups as peptides of equivalent length, suggesting that they could structurally mimic an RNA-binding protein. They can be modified with virtually limitless potential by introducing a wide variety of functional groups and are resistant to proteolytic degradation. My third focus was to implement the in silico free energy perturbation (FEP) method to design new peptide inhibitors of HIV-1 transcription, particularly those inclusive of nonstandard amino acids.;Abstract 2: Two decades of genome sequencing have uncovered tens of thousands of biologically important non-coding RNAs with largely unknown function. Like other non-coding RNAs (ncRNA), long intervening noncoding RNAs (lincRNAs) do not encode proteins, but are clearly identified by capping and polyadenylation, unlike other long noncoding RNAs. They regulate key biological processes such as transcription and chromosomal inactivation, but how this occurs is unclear. The 4.5kb lincRNA Cyrano was discovered in zebrafish and regulates nasal and eye development during embryogenesis. While there is little sequence conservation from one species to the next across much of the 4.5kb Cyrano transcript, a highly conserved 300nt region is found in mammalian and fish genomes, including human and mouse. Mutations in this conserved region cause developmental defects in zebrafish embryos in embryogenesis.;Using free energy minimization folding, comparative sequence analysis, and Selective 2'-Hydroxyl Acylation and Primer Extension (SHAPE), I determined the secondary structure of the 300 nucleotide conserved region of Cyrano. Secondary structure provides insight to how this RNA folds and functions during embryogenesis, albeit through RNA - RNA or protein -- RNA interactions, as well as form the start for three-dimensional structural elucidation. (Abstract shortened by UMI.). |
