Effect of competing self-structure on triplex formation with purine-rich oligodeoxynucleotides containing GA repeats

Abstract

Competition between triplex formation with double-stranded DNA and oligonucleotide self-association was investigated in 23mer GA and GT oligonucleotides containing d(GA)5 or d(GT)5 repeats. Whereas triplex formation with GT oligonucleotides was diminished when temperature increased from 4 to 37 degrees C, triplex formation with GA oligonucleotides was enhanced when temperature increased within the same range due to the presence of competing intermolecular GA oligonucleotide self-structure. This self-structure was determined to be a homoduplex stabilized by the internal GA repeats. UV spectroscopy of these homoduplexes demonstrated a single sharp transition with rapid kinetics (Tm = 38.5-43.5 degrees C over strand concentrations of 0.5-4 microM, respectively, with transition enthalpy, delta H = -89 +/- 7 kcal/mol) in 10 mM MgCl2, 100 mM NaCl, pH 7.0. Homoduplex formation was strongly stabilized by multivalent cations (spermine > Mg2+ = Ca2+) and destabilized by low concentrations of monovalent cations (K+ = Li+ = Na+) in the presence of divalent cations. However, unlike GA or GT oligonucleotide-containing triplexes, the homoduplex formed even in the absence of multivalent cations, stabilized by only moderate concentrations of monovalent cations (Li+ > Na+ > K+). Through the development of multiple equilibrium states and the resulting depletion of free oligonucleotide, it was found that the presence of competing self-structure could decrease triplex formation under a variety of experimental conditions.

Specific Inhibition of ICAM-1 Expression Mediated by Gene Targeting with Triplex-forming Oligonucleotides

“…Inhibition was also observed when a buffer containing an additional 140 mM potassium mimicking more physiologic ionic conditions was used. However, a 6-fold higher amount of TFOgt was required for comparable inhibition (data not shown), suggesting that triplex formation is less efficient in the presence of potassium, as described (31).…”

Evaluation of effects of bivalent cations on the formation of purine-rich triple-helix DNA by ESI-FT-MS

“…Since self-association of G-rich oligonucleotides could decrease triplex formation, inhibitory effects of monovalent cations (especially K ϩ ) on purine-rich triplex formation might relate to the promotion of such competing self-structures [6,9]. In this work, Ba 2ϩ ions have been found to enhance the formation of homodimers, so it might inhibit triplex formation like K ϩ does.…”

“…For the purine motif, the triplex will be inhibited in the presence of monovalent cations, such as sodium and potassium, which serve to coordinate and stabilize the G-rich oligonucleotide dimmers and tetramers [1,4]. The stabilization of the bivalent cation to triplex is complicated-i.e., it is not a simple electrostatic phenomenon-and has been investigated using circular dichroism, UV absorption spectroscopy, footprinting techniques, electrophoresis mobility shift assay, and laser densitometry [5][6][7][8][9][10][11].…”

Bioconjugation of Oligonucleotides for Treating Liver Fibrosis

“…G-rich TFOs can easily form intra-or intermolecular four-stranded structures involving G-quartets (316). Other intermolecular structures, such as parallel homoduplexes involving AA, GG and GA base pairs can also be formed (317). In addition, monovalent cations (Na + , K + ) at physiological concentrations (140 mM) favor G-quartet formation and thus inhibit triplex formation (318).…”