TY - JOUR
T1 - Footprinting and circular dichroism studies on paromomycin binding to the packaging region of human immunodeficiency virus type-1
AU - McPike, Mark P.
AU - Goodisman, Jerry
AU - Dabrowiak, James C.
N1 - Funding Information:
We wish to thank Professor Y. Tor (U. of California at San Diego) and P. N. Borer (Syracuse University) for helpful discussions. We also wish to thank Professor S. Loh (SUNY Upstate Medical University) for the use of the CD instrument. The research was supported in part by the NIH (GM32691 to P. N. Borer) and the Chemistry Department.
PY - 2002/11
Y1 - 2002/11
N2 - We have studied the interaction of the aminoglycoside drug, paromomycin, with a 171-mer from the packaging region of HIV-1 (ψ-RNA), using quantitative footprinting and circular dichroism spectroscopy. The footprinting autoradiographic data were obtained by cutting end-labeled RNA with RNase I or RNase T1 in the presence of varying paromomycin concentrations. Scanning the autoradiograms produced footprinting plots showing cleavage intensities for specific sites on the ψ-RNA as functions of drug concentration. Footprinting plots showing binding were analyzed using a two-state model to give apparent binding constants for specific sites of the ψ-RNA. These plots show that the highest-affinity paromomycin binding site involves nucleotides near bulges in the main stem and SL-1, and other nucleotides in SL-4 of the ψ-RNA. RNase I gives an apparent value of K for this drug site of ∼1.7×105 M-1 while RNase T1 reports a value of K of ∼8×104 M-1 (10 mM Tris HCl, pH 7). Footprinting shows that loading the highest affinity site with paromomycin causes structural changes in the single-stranded linker regions, between the stem-loops and main stem and the loops of SL-1 and SL-3. Drug-induced structural changes also affect the intensity of the 208 nm band in the circular dichroism spectrum of the ψ-RNA. Fitting the changes in CD band intensity to a two-state model yielded a binding constant for the highest-affinity drug site of 6×106 M-1. Thus, the binding constants from footprinting are lower than those obtained for the highest-affinity site from the circular dichroism spectrum, and lower than those earlier obtained using absorption spectroscopy (Sullivan, J. M.; Goodisman, J.; Dabrowiak, J. C., Bioorg. Med. Chem. Lett. 2002, 12, 615). The discrepancy may be due to competitive binding between drug and cleavage agent in the footprinting experiments, but other explanations are discussed. In addition to revealing sites of binding and regions of drug-induced structural change, footprinting showed that the loop regions of SL-1, SL-3 and SL-4 are exposed in the RNA, whereas the linker region between SL-1 and SL-2 is 'buried' and not accessible to cutting by RNase I or RNase T1.
AB - We have studied the interaction of the aminoglycoside drug, paromomycin, with a 171-mer from the packaging region of HIV-1 (ψ-RNA), using quantitative footprinting and circular dichroism spectroscopy. The footprinting autoradiographic data were obtained by cutting end-labeled RNA with RNase I or RNase T1 in the presence of varying paromomycin concentrations. Scanning the autoradiograms produced footprinting plots showing cleavage intensities for specific sites on the ψ-RNA as functions of drug concentration. Footprinting plots showing binding were analyzed using a two-state model to give apparent binding constants for specific sites of the ψ-RNA. These plots show that the highest-affinity paromomycin binding site involves nucleotides near bulges in the main stem and SL-1, and other nucleotides in SL-4 of the ψ-RNA. RNase I gives an apparent value of K for this drug site of ∼1.7×105 M-1 while RNase T1 reports a value of K of ∼8×104 M-1 (10 mM Tris HCl, pH 7). Footprinting shows that loading the highest affinity site with paromomycin causes structural changes in the single-stranded linker regions, between the stem-loops and main stem and the loops of SL-1 and SL-3. Drug-induced structural changes also affect the intensity of the 208 nm band in the circular dichroism spectrum of the ψ-RNA. Fitting the changes in CD band intensity to a two-state model yielded a binding constant for the highest-affinity drug site of 6×106 M-1. Thus, the binding constants from footprinting are lower than those obtained for the highest-affinity site from the circular dichroism spectrum, and lower than those earlier obtained using absorption spectroscopy (Sullivan, J. M.; Goodisman, J.; Dabrowiak, J. C., Bioorg. Med. Chem. Lett. 2002, 12, 615). The discrepancy may be due to competitive binding between drug and cleavage agent in the footprinting experiments, but other explanations are discussed. In addition to revealing sites of binding and regions of drug-induced structural change, footprinting showed that the loop regions of SL-1, SL-3 and SL-4 are exposed in the RNA, whereas the linker region between SL-1 and SL-2 is 'buried' and not accessible to cutting by RNase I or RNase T1.
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U2 - 10.1016/S0968-0896(02)00220-1
DO - 10.1016/S0968-0896(02)00220-1
M3 - Article
C2 - 12213482
AN - SCOPUS:0036837080
SN - 0968-0896
VL - 10
SP - 3663
EP - 3672
JO - Bioorganic and Medicinal Chemistry
JF - Bioorganic and Medicinal Chemistry
IS - 11
ER -