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

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

Selective determinants of inositol 1,4,5-trisphosphate and adenophostin A interactions with type 1 inositol 1,4,5-trisphosphate receptors

BACKGROUND AND PURPOSE: Adenophostin A (AdA) is a potent agonist of inositol 1,4,5-trisphosphate receptors (IP 3R). AdA shares with IP 3 the essential features of all IP 3R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP 3, but the basis of its increased affinity is unclear. Hitherto, the 2'-phosphate of AdA has been thought to provide a supra-optimal mimic of the 1-phosphate of IP 3. EXPERIMENTAL APPROACH: We examined the structural determinants of AdA binding to type 1 IP 3R (IP 3R1). Chemical synthesis and mutational analysis of IP 3R1 were combined with 3H-IP 3 binding to full-length IP 3R1 and its N-terminal fragments, and Ca 2+ release assays from recombinant IP 3R1 expressed in DT40 cells. KEY RESULTS: Adenophostin A is at least 12-fold more potent than IP 3 in functional assays, and the IP 3-binding core (IBC, residues 224-604 of IP 3R1) is sufficient for this high-affinity binding of AdA. Removal of the 2'-phosphate from AdA (to give 2'-dephospho-AdA) had significantly lesser effects on its affinity for the IBC than did removal of the 1-phosphate from IP 3 (to give inositol 4,5-bisphosphate). Mutation of the only residue (R568) that interacts directly with the 1-phosphate of IP 3 decreased similarly (by -30-fold) the affinity for IP 3 and AdA, but mutating R504, which has been proposed to form a cation-Π interaction with the adenine of AdA, more profoundly reduced the affinity of IP 3R for AdA (353-fold) than for IP 3 (13-fold). CONCLUSIONS AND IMPLICATIONS: The 2'-phosphate of AdA is not a major determinant of its high affinity. R504 in the receptor, most likely via a cation-Π interaction, contributes specifically to AdA binding