Triplex Formation by Psoralen-Conjugated Chimeric Oligonucleoside Methylphosphonates

Abstract

Interactions between nuclease-resistant, 5'-psoralen-conjugated, chimeric methylphosphonate oligodeoxyribo- or oligo-2'-O-methylribo-triplex-forming oligomers (TFOs) and a purine tract found in the envelope gene of HIV proviral DNA (env-DNA) were investigated by gel mobility shift assays or by photo-cross-linking experiments. These chimeric TFOs contain mixtures of methylphosphonate and phosphodiester internucleotide bonds. A pyrimidine chimeric TFO composed of thymidine and 5-methyl-2'-deoxycytidine (C), d-PS-TpCpTpCpTpCpTpTpTpTpTpTpCpTpC (1mp) where PS is trimethylpsoralen and p is methylphosphonate, forms a stable triplex with env-DNA whose dissociation constant is 1. 3 microM at 22 degrees C and pH 7.0. The dissociation constant of chimeric TFO 2mp, d-PS-UpCpTpCpTpCpTpUpTpUpTpUpCpTpC, decreased to 400 nM when four of the thymidines in 1mp were replaced by 5-propynyl-2'-deoxyuridines (U), a result consistent with the increased stacking interactions and hydrophobic nature of 5-propynyl-U. An even greater decrease, 470 -50 nM, was observed for the all-phosphodiester versions of 1mp and 2mp. The differences in behavior of the chimeric versus the all-phosphodiester oligomers may be related to differences in the conformations between the propynyl-U-substituted versus the nonsubstituted TFOs. Thus, in the chimeric oligomer, the stabilizing effect of the propynyl-U's may be offset by the reduced ability of the methylphosphonate backbone to assume an A-type conformation, a conformation that appears to be preferred by propynyl-U-containing TFOs. A chimeric oligo-2'-O-methylribopyrimidine with the same sequence as 1mp also formed a stable triplex, K(d) = 1.4 microM, with env-DNA. In contrast to the behavior of the pyrimidine TFOs, antiparallel A/G oligomers and parallel or antiparallel T/G oligomers did not form triplexes with env-DNA, even at oligomer concentrations of 10 microM. This lack of binding may be a consequence of the low G content (33%) of the triplex binding site. Irradiation of triplexes formed between the pyrimidine TFOs and env-DNA resulted in formation of photoadducts with either the upper-strand C or the lower-strand T at the 5'-CpA-3' duplex/triplex junction. No interstrand cross-links were observed. The presence of a 5-propynyl-U at the 5'-end of the oligomer caused a reduction in the amount of upper-strand photoadduct but had no effect on photoadduct formation with the lower strand, suggesting that increased stacking interactions caused by the presence of the 5-propynyl-U change the orientation of psoralen with respect to the upper-strand C. The ability of chimeric methylphosphonate TFOs to bind to DNA, combined with their resistance to degradation by serum 3'-exonucleases, suggests that they may have utility in biological experiments.

Modified RNA triplexes: Thermodynamics, structure and biological potential

“…The EMSA data showed that the dissociation constant values of model triplexes fall in the range of low micromolar concentrations, under the conditions comparable to physiological ones. These K d values are in the range reported by other investigators for RNA and DNA triplexes 40 , 41 . Considering the specificity of LNA and 2-thioU modified TFOs, as well as hairpin interactions, we decided to test their potential in HeLa cells.…”

Newly characterized interaction stabilizes DNA structure: oligoethylene glycols stabilize G-quadruplexes CH–π interactions

“…The propynyl group stacks with bases in duplexes and triplexes (29,30). Therefore, we designed propynyl-modified deoxythymidine (Pro) (Supplementary Figure S5a) and incorporated this residue into duplex- and G-quadruplex-forming oligonucleotides.…”

Effect of DNA target sequence on triplex formation by oligo-2'-deoxy- and 2'-O-methylribonucleotides

“…The sequences and designations for each oligomer are shown in Tables 1 and 2. The psTFOs were synthesized on controlled pore glass (CPG) supports using an ABI model 392 DNA/ RNA synthesizer essentially as previously described (46). The protected nucleoside phosphoramidites and methylphosphonamidites were dissolved in anhydrous acetonitrile to a concentration of 0.15 M, and the nucleoside methylphosphonamidite solutions were stored for 2 h over 4 A°molecular sieves prior to use.…”