![]() ![]() Mutations in the thumb subdomain of T圓 also affected RNase H activity, suggesting a closer spatial relationship between its N- and C-terminal catalytic centers compared with HIV-1 RT. Substitution of Ala for Gln290 was well tolerated, despite the high degree of conservation at this position. ![]() Based on this combined strategy, our data suggest an interaction of T圓 RT Tyr298 with primer nucleotide -3, Gly294 with primer nucleotide -4, and Asn297 with template nucleotide -6. DNA-dependent DNA synthesis was evaluated on unmodified substrates and on duplexes containing targeted insertion of locked nucleic acid analogs and abasic lesions in either the template or primer. ![]() The consequences of substituting T圓 RT Gln290, Phe292, Gly294, Asn297, and Tyr298 (the counterparts of HIV-1 RT Gln258, Leu260, Gly262, Asn265, and Trp266, respectively) for both DNA polymerase and RNase H activities were examined. The counterpart to helix alphaH of human immunodeficiency virus type 1 (HIV-1) RT, which mediates important interactions with a duplex nucleic acid approximately 3-6 bp behind the DNA polymerase catalytic center, was identified between amino acids 290 and 298 of the T圓 enzyme. The lone exception is the alkaloid ellipticine, which intercalates into LNA-DNA and LNA-RNA duplexes with affinities comparable to unmodified DNA-DNA and RNA-DNA duplexes.Īmino acid sequence alignment was used to identify the putative thumb subdomain of reverse transcriptase (RT) from the Saccharomyces cerevisiae long terminal repeat-containing retrotransposon T圓. Using UV-vis, fluorescence and circular dichroism spectroscopies, we find that the minor groove binder as well as the intercalators exhibit significantly lower affinity for LNA-containing duplexes. This report describes the ability of several DNA-intercalating ligands and one minor groove binder to recognize LNA-DNA and LNA-RNA hybrid duplexes. Each of these factors will impact the ability of small molecules, proteins and other nucleic acids to recognize LNA-containing hybrids. In addition to changing the distribution of functional groups in the groove and the overall helical geometry relative to unmodified DNA, the bridge likely alters the hydration of the groove. The deoxyribose sugar is modified by a 2'-O, 4'-C oxymethylene bridge, which projects into the minor groove. Locked nucleic acid (LNA) is a conformationally constrained DNA analogue that exhibits exceptionally high affinity for complementary DNA and RNA strands. ![]()
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