Title

Aminoglycoside binding to the packaging region of HIV-1 RNA

Date of Award

2001

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Advisor(s)

James C. Dabrowiak

Keywords

Aminoglycoside, Packaging region, HIV-1, RNA, Immune deficiency

Subject Categories

Biochemistry | Biochemistry, Biophysics, and Structural Biology | Chemistry | Life Sciences

Abstract

The ψ region of HIV-1 spans ∼200 nucleotides (nt) near the 5 ' end of the RNA genome. This region has been shown to play a significant role in encapsidation and transciptional regulation of the viral RNA. As a result, the ψ region has the potential to be a target for drugs. In this study, a 176 nt model (LAI strain) of the ψ region, representing positions 213-388, was investigated with natural aminoglycosides (paromomycin and neomycin) and modified analogues of neomycin (neo-neo, neo-acridine and neo-guanidinium) using footprinting and Circular dichroism (CD) techniques. Footprinting analysis was conducted using the ribonuclease RNase I under optimized "single-hit" kinetic cutting conditions. Intensity data obtained from autogradiograms was used to generate a collection of footprinting plots for each drug. Analysis of the footprinting plots revealed a primary binding site at SL1 for paromomycin and neomycin, with a binding constant of 4.5 × 10 5 M -1 (at 25°C, in 10 mM Tris-HCl, pH 7.5) for neomycin. Analysis of the footprinting plots for neo-acridine and neo-guanidinium also indicated a primary binding site on SL1 having binding constants of 3.4 × 10 5 M -1 and 2.2 × 10 5 M -1 for each drug respectively. Both neo-acridine and neo-guanidinium also interacted with sites on SL2 and SL4. Neo-neo footprinting analysis indicated a single primary binding site on SL2, possibly formed by an intermolecular binding pocket, with a binding constant of 2.5 × 10 5 M -1 . At non-binding sites, each drug produced footprinting plots having an increase in intensity at high drug concentrations. Such effects are a result of structural changes, predominately found in the linker regions of the RNA. Using circular dichroism, the global effects of drug-induced structural change of the 176-mer RNA was measured for each drug. Analysis of plots of CD versus drug concentration revealed an increase in intensity for neomycin, paromomycin and neo-guanidinium at 208 nm and a slight red shift with no intensity change at the same wavelength for neo-neo and neo-acridine. In the drug concentration range of 11 to 20 μM, a decrease in the intensity of the 266 nm band was observed for all the drugs except paromomycin. The intensity changes observed for the 208 nm band are associated with the structural changes in the linker region. By plotting the change in extinction coefficient against drug concentration, CD derived binding constants were calculated for each drug. Results indicated that several binding sites were responsible for the structural changes measured in the CD analysis. This study demonstrates how footprinting and CD methods can be used to identify drug binding sites and drug-induced structural changes associated with drug binding to a large RNA target.

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