Purine twisted-intercalating nucleic acids: a new class of anti-gene molecules resistant to potassium-induced aggregation

Abstract

Sequence-specific targeting of genomic DNA by triplex-forming oligonucleotides (TFOs) is a promising strategy to modulate in vivo gene expression. Triplex formation involving G-rich oligonucleotides as third strand is, however, strongly inhibited by potassium-induced TFO self-association into G-quartet structures. We report here that G-rich TFOs with bulge insertions of (R)-1-O-[4-(1-pyrenylethynyl)-phenylmethyl] glycerol (called twisted intercalating nucleic acids, TINA) show a much lower tendency to aggregate in potassium than wild-type analogues do. We designed purine-motif TINA–TFOs for binding to a regulatory polypurine-polypyrimidine (pur/pyr) motif present in the promoter of the KRAS proto-oncogene. The binding of TINA–TFOs to the KRAS target has been analysed by electrophoresis mobility shift assays and DNase I footprinting experiments. We discovered that in the presence of potassium the wild-type TFOs did not bind to the KRAS target, differently from the TINA analogues, whose binding was observed up to 140 mM KCl. The designed TINA–TFOs were found to abrogate the formation of a DNA–protein complex at the pur/pyr site and to down-regulate the transcription of CAT driven by the murine KRAS promoter. Molecular modelling of the DNA/TINA–TFO triplexes are also reported. This study provides a new and promising approach to create TFOs to target in vivo the genome.

Triplex‐Forming Twisted Intercalating Nucleic Acids (TINAs): Design Rules, Stabilization of Antiparallel DNA Triplexes and Inhibition of G‐Quartet‐Dependent Self‐Association

“…[32] It was also established that Gcontaining oligonucleotides designed to bind to this duplex via a triplex motif instead existed as self-aggregated complexes that precluded their use as triplex forming oligonucleotides. [16,17] However, our recent results [26] inspired us to investigate the G-rich TFOs targeting HIV-1 PPT, as the duplex contains six adjacent guanines in its structure (target I, Table 2). …”

“…[26] The designed TINA-TFOs were found to inhibit the formation of a DNA-protein complex and to down-regulate the transcription of CAT driven by the murine KRAS promoter. It is important to mention that CT-TINA-TFOs designed to bind to the same target in a parallel orientation did not form triplexes at neutral pH.…”

“…The selected GT-TFO consists of a single run of six guanines and forms intermolecular G-quadruplex of high thermal stability, while in the previous study TINA monomers were inserted in the TFO in which several runs of 2-5 guanines were interrupted by adenines and were able to form intramolecular complexes. [26] Thus we advanced our knowledge of the design of TINA-TFOs for different types of TFO and genomic sequences. Bulged TINA monomers were incorporated into G 6 -tract of G-rich TFO and a series of TINA-TFOs forming stable triplexes with the target was obtained.…”

“…The remaining GA TINA-TFOs were able to bind to the DNA duplex in an antiparallel fashion even in the presence of K + . [26,27] We [21][22][23][28][29][30] and others [31] have undertaken several studies on the ability of TINA moiety to stabilize pH-dependent parallel CT-triplexes, while very little is known about the effects of TINA on antiparallel GA or GT-containing TFOs. Herein we synthesized several triplex-forming 16-mer GT oligonucleotides with 2-4 TINA insertions to target the polypurine tract of HIV-1 proviral DNA [32] in different orientations.…”

Enhancement of TFO Triplex Formation by Conjugation with Pyrene <i>via</i> Click Chemistry

“…These results indicate that the pyrene molecule intercalated into duplex DNA, assisting in the formation of triplex DNA. 31) Computational chemistry was used to predict the effect of this pyrene. The molecular modeling result is presented in Fig.…”