An interlocked dimeric parallel-stranded DNA quadruplex: A potent inhibitor of HIV-1 integrase

Abstract

We report on the NMR-based solution structure of the 93del d(GGGGTGGGAGGAGGGT) aptamer, a potent nanomolar inhibitor of HIV-1 integrase. This guanine-rich DNA sequence adopts an unusually stable dimeric quadruplex architecture in K ؉ solution. Within each 16-nt monomer subunit, which contains one A⅐(G⅐G⅐G⅐G) pentad sandwiched between two G⅐G⅐G⅐G tetrads, all G-stretches are parallel, and all guanines are anti with the exception of G1, which is syn. Dimer formation is achieved through mutual pairing of G1 of one monomer, with G2, G6, and G13 of the other monomer, to complete G⅐G⅐G⅐G tetrad formation. There are three single-nucleotide double-chain-reversal loops within each monomer fold, such that the first (T5) and third (A12) loops bridge three G-tetrad layers, whereas the second (A9) loop bridges two G-tetrad layers and participates in A⅐(G⅐G⅐G⅐G) pentad formation. Results of NMR and of integrase inhibition assays on loop-modified sequences allowed us to propose a strategy toward the potential design of improved HIV-1 integrase inhibitors. Finally, we propose a model, based on molecular docking approaches, for positioning the 93del dimeric DNA quadruplex within a basic channel͞canyon formed between subunits of a dimer of dimers of HIV-1 integrase. dimeric quadruplex ͉ DNA aptamer H IV-1 integrase catalyzes the integration of proviral DNA into the host-cell genome, a reaction critical for efficient viral replication. HIV-1 integrase is necessary and sufficient for the first two steps of DNA integration (1): (i) 3Ј-end processing, where two nucleotides are removed from each 3Ј-end of the viral DNA; (ii) strand transfer, where each 3Ј-processed viral DNA end is attached to the host-cell DNA. As a result, HIV-1 integrase constitutes an attractive target for drugs against AIDS (2, 3).Guanine-rich oligonucleotides have been identified as potent inhibitors of HIV-1 integrase (4, 5). In particular, a recent study (6) has identified a 16-nt guanine-rich sequence d(GGGGTGG-GAGGAGGGT), designated 93del, which inhibits both the processing and strand transfer functions of HIV-1 integrase at nanomolar concentrations.Guanine-rich oligonucleotides can form a variety of Gquadruplex architectures involving stacked planar G⅐G⅐G⅐G tetrads. This polymorphism of G-quadruplex topologies reflects the range of alternate strand directionalities, loop connectivities, and syn͞anti distribution of guanine bases around G-tetrads (7-16). Higher order pairing alignments involving G-tetrads have been recently identified, and these include pentads (17), hexads (18), and heptads (19) for sequences containing guanine stretches separated by adenine residues.Here, we present the NMR-based solution structure of 93del. We establish that this 16-nt DNA sequence adopts an unusually stable dimeric quadruplex architecture in K ϩ solution. Each monomer subunit contains two G⅐G⅐G⅐G tetrads and one A⅐(G⅐G⅐G⅐G) pentad, where all of the G-stretches are parallel and linked by three single-nucleotide double-chain-reversal loops. In our attempts to design potent...

Structure of an Unprecedented G-Quadruplex Scaffold in the Human c-kit Promoter

“…22 The present study has shown that this is not the case. The structure contains two single-residue double-chainreversal loops spanning three G-tetrad layers, a robust type of loop reported previously 15,28 that could play an important role in the folds of many G-quadruplexes 29 in K + solution. This quadruplex is remarkable in requiring the active participation of 18 out of the 22 nucleotides in the structural organization.…”

“…This was anticipated, as the structure of a single-residue double-chain-reversal loop bridging three G-tetrad layers has been shown to be independent of the nature of the base in it. 28 Formation of the Watson-Crick A1•T12 base pair appears to be important for the structure, as the A1T mutation in the ck16 sequence altered the general fold (Figure 6b). The ck19 sequence containing the C11T mutation showed a doublings of imino proton peaks ( Figure 6b).…”

(3 + 1) Assembly of Three Human Telomeric Repeats into an Asymmetric Dimeric G-Quadruplex

“…Residues of htel1U in the htel3I/htel1U quadruplex were numbered from 17 to 22 (Figure 1e). We refer to the htel3I/ htel1U quadruplex as a (3 + 1) quadruplex 13 assembly, because each G-tetrad contains three guanines from one strand and one guanine from the other strand. It should be noted that compared to the NMR spectra of the htel3I quadruplex the spectra of the htel3I/htel1U quadruplex showed the same number guanine and inosine imino protons, but fewer nonexchangeable protons (Figures 4, 5, and 7), consistent with the lack of an overhang tail in the htel3I/htel1U quadruplex (Figure 1d,e).…”

“…13 The calculation protocol started with initial structures that were generated as sets of two strands (16-nt htel3I and 6-nt htel1U), randomized for all dihedral angles, and separated by space intervals of 50 Å. A total of 100 structures were embedded and optimized using the distance geometry simulated annealing protocol.…”

Engineering the Quadruplex Fold: Nucleoside Conformation Determines Both Folding Topology and Molecularity in Guanine Quadruplexes

“…These two activities suggest that quadruplexes may be new chemotherapeutic targets, and indeed this is being pursued by a variety of research groups 4 . In addition to serving as potential drug targets, some quadruplexes have been observed to possess therapeutic activity 5,6 . While most of these studies involve DNA quadruplexes, there has also been interest in the biological roles played by RNA quadruplexes 7 .…”