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

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.

Figure 1.

PPT-containing RNA:DNA hybrid. Indicated are regions for which high-resolution structures are available and their relationship with the sequence employed in this study.

Scheme 1.

Archetypical nucleic acid ligands used as non-covalent probes in this study: (a) ethidium bromide, representative of base intercalators; (b) distamycin A, a typical minor-groove binder; (c) mitoxantrone, a mixed-mode intercalator/groove binder; and (d) neomycin B, a polycationic aminoglycoside.

Scheme 2.

PPT-containing duplexes. PPT_wt: wild-type hybrid from HIV-1 subtype A; PPT_RNA: both strands made of RNA; PPT_DNA: both strands DNA; PPT_swp: 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).

Figure 2.

Nanospray-FTICR mass spectra of: (a) the initial equimolar mixture of the four PPT substrates (5 μM each); (b) the products obtained after a 1-fold per substrate addition of the mixed-mode binder mitoxantrone; (c) the assemblies after 10-fold ligand addition (see Materials and methods section). Red filled square: PPT_DNA; orange filled diamond: PPT_RNA; green filled triangle: PPT_wt; blue filled inverted triangle: PPT_sw and black filled circle: bound ligand. The high resolution and accuracy afforded by this analytical platform enabled unambiguous identification of the different species in solution according to their unique molecular masses (see also Scheme 2).

Figure 3.

Nanospray-FTICR mass spectra of the initial equimolar mixture of the four PPT substrates (5 μM each) after a 10-fold per substrate addition of: (a) ethidium bromide; (b) distamycin A; (c) neomycin B (see Materials and methods section). Red filled square: PPT_DNA; orange filled diamond: PPT_RNA; green filled triangle: PPT_wt; blue filled inverted triangle: PPT_sw; open circle: ethidium; open diamond: distamycin A; and asterisk: neomycin B.

Figure 4.

Tandem mass spectrum of the non-covalent 1:1 PPT_wt:neomycin B complex (observed mass 13148.74 Da, calculated from sequence 13148.26 Da, see Materials and methods section). For clarity, only some of the characteristic ion series are labeled on the spectrum, which contain either the 5′- or 3′-end (e.g. a_x−B and y_x, with x indicating the numeration from the respective end; −B indicates the loss of nucleobase at the site of cleavage). The asterisk symbol identifies products containing noncovalently bound neomycin B. All product ions observed for the DNA and RNA strands that constitute the substrate are reported on the respective sequences to highlight nucleotides prevented from undergoing fragmentation in the presence of bound ligand (32). The lines mark the phosphodiester bond cleaved by the gas-phase dissociation processes, pointing toward the respective end included in each fragment; asterisk indicates products containing noncovalently bound neomycin B. A clear gap in the fragmentation pattern exhibited by the DNA and RNA strands identifies the position of protected nucleotides.

Figure 5.

Tandem mass spectrum of the non-covalent 1:2 PPT_wt:neomycin B complex (observed mass 13762.43 Da, calculated from sequence 13762.57 Da, see Materials and methods section). For clarity, only some of the characteristic ion series are labeled on the spectrum, which contain either the 5′- or 3′-end (e.g. a_x−B and y_x, with x indicating the numeration from the respective end; −B indicates the loss of nucleobase at the site of cleavage). The asterisk symbol identifies products containing non-covalently bound neomycin B. All product ions observed for the DNA and RNA strands that constitute the substrate are reported on the respective sequences to highlight nucleotides prevented from undergoing fragmentation in the presence of bound ligand (32). The lines mark the phosphodiester bond cleaved by the gas-phase dissociation processes, pointing toward the respective end included in each fragment; asterisk indicates products containing noncovalently bound neomycin B. In this case, two distinct gaps are evident in the ion series observed for the DNA and RNA components of the construct, thus revealing the presence of two separate binding sites.

Figure 6.

One-dimensional water flip-back watergate ¹H NMR spectra of the imino region of the PPT_wt duplex after titration with neomycin B at 10°C, or E478Q RT at 30°C, in 80 mM NaCl and 10 mM NaH₂PO₄/Na₂HPO₄ at pH 7.0. (a) For the neomycin titration, the concentration of the PPT_wt duplex was ∼200 μM. 1D ¹H NMR spectra are shown in the absence (upper trace) and presence of 1.0 equivalents (middle trace) and 2.0 equivalents (lower trace) of neomycin B. The imino resonances for G(+2), T(+1), and g(−1) where chemical shift changes can be tracked are highlighted in bold. Dotted lines and arrows indicate the shift in position of the resonances. (b) For the E478Q RT titration, the concentration of PPT_wt was ∼40 μM. 1D ¹H NMR spectra are shown for PPT_wt in the absence (upper trace) and presence of 1.0 equivalent (lower trace) of E478Q RT. Imino protons were assigned using 2D NOESY experiments; assignments are listed above each peak.

Figure 7.

ITC titration of PPT_wt with neomycin B in 10 mM NaH₂PO₄/Na₂HPO₄ at pH 7.0 and 30°C. (a) ITC profile obtained at 80 mM NaCl; (b) integration of panel (a) and curve-fitting to a model including two sets of equivalents sites; (c) ITC profile obtained at 240 mM NaCl; (d) integration of panel (c) and curve-fitting to a model including one set of equivalent sites. The integrations in panels (b) and (d) were corrected for the heats of dilution from the titration of neomycin B into the respective buffer alone.