Using pyrrolo-deoxycytosine to probe RNA/DNA hybrids containing the human immunodeficiency virus type-1 3' polypurine tract

Abstract

Recent structural analyses indicate that localized regions of abnormal base pairing exist within RNA/DNA hybrids containing the HIV-1 polypurine tract (PPT) and that these distortions may play a role in PPT function. To examine this directly, we have introduced pyrrolo-deoxycytosine (pdC), a fluorescent, environmentally sensitive analog of deoxycytosine (dC), into the DNA strand of PPT-containing hybrids. Steady-state fluorescence analysis of these hybrids reveals that the DNA base 11 nt from the PPT-U3 junction is unpaired even in the absence of reverse transcriptase (RT). Unstable base pairing is also observed within the (rG:dC)6 tract in the downstream portion of the duplex, suggesting that HIV-1 RT may recognize multiple pre-existing distortions during PPT selection. HIV-1 RT hydrolyzes pdC-containing hybrids primarily at the PPT-U3 junction, indicating that the analog does not induce a gross structural deformation of the duplex. However, aberrant cleavage is frequently observed 3 bp from the site of pdC substitution, most likely reflecting a specific interaction between the analog and amino acid residues within the RNase H primer grip. pdC substitution within the template strand of a DNA duplex does not appear to significantly affect RT-catalyzed DNA synthesis. Implications of these findings on the use of pdC to examine nucleic acid structure are discussed.