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Structural probing of the HIV-1 polypurine tract RNA:DNA hybrid using classic nucleic acid ligands.

Turner KB, Brinson RG, Yi-Brunozzi HY, Rausch JW, Miller JT, Le Grice SF, Marino JP, Fabris D - Nucleic Acids Res. (2008)

Bottom Line: In contrast, neomycin bound preferentially and selectively to the PPT near the 5'(rA)(4):(dT)(4) tract and the 3' PPT-U3 junction.Nuclear magnetic resonance data from a complex between HIV-1 RT and the PPT indicate RT contacts within the same regions highlighted on the PPT by neomycin.These observations, together with the fact that the sites are correctly spaced to allow interaction with residues in the ribonuclease H (RNase H) active site and thumb subdomain of the p66 RT subunit, suggest that despite the long cleft employed by RT to make contact with nucleic acids substrates, these sites provide discrete binding units working in concert to determine not only specific PPT recognition, but also its orientation on the hybrid structure.

View Article: PubMed Central - PubMed

Affiliation: University of Maryland Baltimore County, Baltimore, MD, USA.

ABSTRACT
The interactions of archetypical nucleic acid ligands with the HIV-1 polypurine tract (PPT) RNA:DNA hybrid, as well as analogous DNA:DNA, RNA:RNA and swapped hybrid substrates, were used to probe structural features of the PPT that contribute to its specific recognition and processing by reverse transcriptase (RT). Results from intercalative and groove-binding ligands indicate that the wild-type PPT hybrid does not contain any strikingly unique groove geometries and/or stacking arrangements that might contribute to the specificity of its interaction with RT. In contrast, neomycin bound preferentially and selectively to the PPT near the 5'(rA)(4):(dT)(4) tract and the 3' PPT-U3 junction. Nuclear magnetic resonance data from a complex between HIV-1 RT and the PPT indicate RT contacts within the same regions highlighted on the PPT by neomycin. These observations, together with the fact that the sites are correctly spaced to allow interaction with residues in the ribonuclease H (RNase H) active site and thumb subdomain of the p66 RT subunit, suggest that despite the long cleft employed by RT to make contact with nucleic acids substrates, these sites provide discrete binding units working in concert to determine not only specific PPT recognition, but also its orientation on the hybrid structure.

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PPT-containing duplexes. PPTwt: wild-type hybrid from HIV-1 subtype A; PPTRNA: both strands made of RNA; PPTDNA: both strands DNA; PPTswp: sequences of the RNA and DNA strands swapped. Sequences are numbered from 5′ to 3′, and biological numbering relative to the scissile −1g/+1a phosphodiester bond is indicated. Nucleotides in uppercase denote DNA, and lowercase denotes RNA. The monoisotopic mass observed by nanospray-FTICR for each duplex construct is reported together with the respective mass calculated from sequence (see Materials and methods section).
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Figure 9: PPT-containing duplexes. PPTwt: wild-type hybrid from HIV-1 subtype A; PPTRNA: both strands made of RNA; PPTDNA: both strands DNA; PPTswp: sequences of the RNA and DNA strands swapped. Sequences are numbered from 5′ to 3′, and biological numbering relative to the scissile −1g/+1a phosphodiester bond is indicated. Nucleotides in uppercase denote DNA, and lowercase denotes RNA. The monoisotopic mass observed by nanospray-FTICR for each duplex construct is reported together with the respective mass calculated from sequence (see Materials and methods section).

Mentions: Nucleic acid ligands with well-characterized binding modes can serve as non-covalent probes for assaying the ability of target substrates to sustain specific interactions and for identifying the structural features necessary for stable binding (31,32). For this purpose, ligands employed here were selected among general intercalators (ethidium bromide), minor groove binders (distamycin A), mixed-mode intercalator/minor groove binders (mitoxantrone) and multifunctional polycationic aminoglycosides (neomycin B) (Scheme 1) (38–40). The extensive knowledge of the chemical properties of these ligands, as well as their binding preferences, allows for valid conclusions on the specific nature of target substrates. The effects of helical structure and pairing anomalies on binding mode were investigated by employing not only a consensus PPT RNA:DNA hybrid (PPTwt, Scheme 2), but also constructs in which both strands consisted exclusively of ribo- or deoxyribonucleotides (PPTRNA and PPTDNA, respectively). A control hybrid duplex designated PPTswp was also included in which the DNA and RNA sequences were interchanged (Scheme 2).Scheme 1.


Structural probing of the HIV-1 polypurine tract RNA:DNA hybrid using classic nucleic acid ligands.

Turner KB, Brinson RG, Yi-Brunozzi HY, Rausch JW, Miller JT, Le Grice SF, Marino JP, Fabris D - Nucleic Acids Res. (2008)

PPT-containing duplexes. PPTwt: wild-type hybrid from HIV-1 subtype A; PPTRNA: both strands made of RNA; PPTDNA: both strands DNA; PPTswp: sequences of the RNA and DNA strands swapped. Sequences are numbered from 5′ to 3′, and biological numbering relative to the scissile −1g/+1a phosphodiester bond is indicated. Nucleotides in uppercase denote DNA, and lowercase denotes RNA. The monoisotopic mass observed by nanospray-FTICR for each duplex construct is reported together with the respective mass calculated from sequence (see Materials and methods section).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2377446&req=5

Figure 9: PPT-containing duplexes. PPTwt: wild-type hybrid from HIV-1 subtype A; PPTRNA: both strands made of RNA; PPTDNA: both strands DNA; PPTswp: sequences of the RNA and DNA strands swapped. Sequences are numbered from 5′ to 3′, and biological numbering relative to the scissile −1g/+1a phosphodiester bond is indicated. Nucleotides in uppercase denote DNA, and lowercase denotes RNA. The monoisotopic mass observed by nanospray-FTICR for each duplex construct is reported together with the respective mass calculated from sequence (see Materials and methods section).
Mentions: Nucleic acid ligands with well-characterized binding modes can serve as non-covalent probes for assaying the ability of target substrates to sustain specific interactions and for identifying the structural features necessary for stable binding (31,32). For this purpose, ligands employed here were selected among general intercalators (ethidium bromide), minor groove binders (distamycin A), mixed-mode intercalator/minor groove binders (mitoxantrone) and multifunctional polycationic aminoglycosides (neomycin B) (Scheme 1) (38–40). The extensive knowledge of the chemical properties of these ligands, as well as their binding preferences, allows for valid conclusions on the specific nature of target substrates. The effects of helical structure and pairing anomalies on binding mode were investigated by employing not only a consensus PPT RNA:DNA hybrid (PPTwt, Scheme 2), but also constructs in which both strands consisted exclusively of ribo- or deoxyribonucleotides (PPTRNA and PPTDNA, respectively). A control hybrid duplex designated PPTswp was also included in which the DNA and RNA sequences were interchanged (Scheme 2).Scheme 1.

Bottom Line: In contrast, neomycin bound preferentially and selectively to the PPT near the 5'(rA)(4):(dT)(4) tract and the 3' PPT-U3 junction.Nuclear magnetic resonance data from a complex between HIV-1 RT and the PPT indicate RT contacts within the same regions highlighted on the PPT by neomycin.These observations, together with the fact that the sites are correctly spaced to allow interaction with residues in the ribonuclease H (RNase H) active site and thumb subdomain of the p66 RT subunit, suggest that despite the long cleft employed by RT to make contact with nucleic acids substrates, these sites provide discrete binding units working in concert to determine not only specific PPT recognition, but also its orientation on the hybrid structure.

View Article: PubMed Central - PubMed

Affiliation: University of Maryland Baltimore County, Baltimore, MD, USA.

ABSTRACT
The interactions of archetypical nucleic acid ligands with the HIV-1 polypurine tract (PPT) RNA:DNA hybrid, as well as analogous DNA:DNA, RNA:RNA and swapped hybrid substrates, were used to probe structural features of the PPT that contribute to its specific recognition and processing by reverse transcriptase (RT). Results from intercalative and groove-binding ligands indicate that the wild-type PPT hybrid does not contain any strikingly unique groove geometries and/or stacking arrangements that might contribute to the specificity of its interaction with RT. In contrast, neomycin bound preferentially and selectively to the PPT near the 5'(rA)(4):(dT)(4) tract and the 3' PPT-U3 junction. Nuclear magnetic resonance data from a complex between HIV-1 RT and the PPT indicate RT contacts within the same regions highlighted on the PPT by neomycin. These observations, together with the fact that the sites are correctly spaced to allow interaction with residues in the ribonuclease H (RNase H) active site and thumb subdomain of the p66 RT subunit, suggest that despite the long cleft employed by RT to make contact with nucleic acids substrates, these sites provide discrete binding units working in concert to determine not only specific PPT recognition, but also its orientation on the hybrid structure.

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