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

摘要

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.