Enhanced anti-HIV-1 activity of G-quadruplexes comprising locked nucleic acids and intercalating nucleic acids

Abstract

Two G-quadruplex forming sequences, 5′-TGGGAG and the 17-mer sequence T30177, which exhibit anti-HIV-1 activity on cell lines, were modified using either locked nucleic acids (LNA) or via insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (intercalating nucleic acid, INA) or (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol (twisted intercalating nucleic acid, TINA). Incorporation of LNA or INA/TINA monomers provide as much as 8-fold improvement of anti-HIV-1 activity. We demonstrate for the first time a detailed analysis of the effect the incorporation of INA/TINA monomers in quadruplex forming oligonucleotides (QFOs) and the effect of LNA monomers in the context of biologically active QFOs. In addition, recent literature reports and our own studies on the gel retardation of the phosphodiester analogue of T30177 led to the conclusion that this sequence forms a parallel, dimeric G-quadruplex. Introduction of the 5′-phosphate inhibits dimerisation of this G-quadruplex as a result of negative charge–charge repulsion. Contrary to that, we found that attachment of the 5′-O-DMT-group produced a more active 17-mer sequence that showed signs of aggregation—forming multimeric G-quadruplex species in solution. Many of the antiviral QFOs in the present study formed more thermally stable G-quadruplexes and also high-order G-quadruplex structures which might be responsible for the increased antiviral activity observed.

DNA Duplexes with Hydrophobic Modifications Inhibit Fusion between HIV-1 and Cell Membranes

“…Structural-activity relationship analysis showed that the presence of a DNA duplex and hydrophobic groups was crucial for the anti-HIV-1 activity observed, although the locations of the hydrophobic groups in the duplex and the sequence composition seemed not to be critical to the activity of the respective DNA duplexes tested. The modified DNA duplexes showed anti-HIV-1 activity at low M concentrations comparable to those of a previously described G-quadruplex HIV-1 entry inhibitor (17)(18)(19)(20)(21)(22)(23)(24). These duplex inhibitors were found to be interacting with the HIV-1 gp41 NHR deep pocket to prevent 6HB formation between the gp41 NHR and CHR.…”

“…In contrast to Hotoda's quadruplex inhibitors d(TGGGAG) 4 (17)(18)(19)(20)(21)(22)(23)(24), the duplex inhibitors reported in this paper can be extensively modified based on the location of the hydrophobic groups and skeleton length, thereby presenting more opportunities for modification.…”

“…These aptamers fold into defined three-dimensional (3D) structures and interact with HIV-1-associated proteins, such as HIV-1 reverse transcriptases (6,7), RNase H (8), Tat proteins (9), integrase (10)(11)(12)(13), and surface glycoproteins (14,15). A subcategory of aptamers possess an additional intermolecular assembly feature such as the quadruplex d(TGGGAG) 4 described by Hotoda et al, which was assembled by four 6-mer G-rich sequences in parallel with the 5=-end attaching to aromatic substituents of adequate size, such as tert-butyldiphenylsilyl (TBDPS) and 4=4-dimethoxytriphenyl (DMT) (16)(17)(18)(19)(20)(21)(22)(23)(24). These quadruplexes are aptamer-like since they interact with the V3 loop or the CD4 binding site on viral gp120, preventing viral adhesion and fusion with target cells (17,25,26).…”

DNA Triplex-Based Complexes Display Anti-HIV-1-Cell Fusion Activity

“…T1 contained three hydrophobic groups (one TBDPS and two DMTs) at each end of the helix. The IC 50 for T1 was 1.8 -0.7 mM, comparable to the previously reported IC 50 values of duplex and quadruplex inhibitors [3][4][5][6][7][8][9][10][11][12][13][14][15]. T2 had no hydrophobic group modifications and did not show the anti-HIV-1 fusion activity.…”

“…Nearly 20 years ago, researchers discovered a category of DNA G-quadruplexbased inhibitors that display an anti-HIV-1 fusion activity [2]. Among these inhibitors, the most representative structure is Hotoda's sequence, d(TGGGAG), which interacts with the V3 loop or CD4-binding site of viral glycoprotein 120 (gp120) [3][4][5][6][7][8][9][10][11][12][13]. Recently, we designed a novel class of DNA duplex-based HIV-1 fusion inhibitors, which have hydrophobic groups at several selected positions [14,15].…”