Selective Formation of Stable Triplexes Including a TA or a CG Interrupting Site with New Bicyclic Nucleoside Analogues (WNA)

Abstract

Triplex-forming oligonucleotides (TFOs) are potential DNA-targeting molecules and would become powerful tools for genomic research. As the stabilization of the TFO is partially provided by hydrogen bonds to purine bases, the most stable triplexes form with homopurine/homopyrimidine sequences, and a pyrimidine base in the purine strand of the duplex interrupts triplex formation. If a TFO can recognize sequences including such an interrupting site, the target regions in the genome would be expanded to a greater extent. However, this problem has not been generally solved despite extensive studies. We have previously reported a new base analogue (WNA) constructed of three parts, a benzene ring, a heterocyclic ring, and a bicyclic skeleton to hold these two parts. In this study, we have further investigated modification of WNA systematically and determined two useful WNA analogues, WNA-beta T and WNA-beta C, for selective stabilization of triplexes at a TA and a CG interrupting site, respectively. The triplexes with WNA analogues have exhibited an interesting property in that they are more stable than natural-type triplexes even at low Mg(2+) concentration. From comparison of the results with H-WNA-beta T lacking benzene and those with WNA-H without thymine, it has been suggested that benzene is a major contributor for triplex stability and thymine provides selectivity. Thus, it has been successfully demonstrated that WNA-beta T/TA and WNA-beta C/CG combinations may expand triplex recognition codes in addition to the natural A/AT and G/GC base triplet codes. The results of this study will provide useful information for the design of new WNA analogues to overcome inherent problems for further expansion of triplex recognition codes.

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

“…(1S,2R,3R,4R)-1-(2-Formylethyl)-2-Omethyl-1-phenyl-3,5-O-tetraisopropyldisiloxyribose (11) 14) was synthesized as described in our previous paper. The aldehyde group was converted to the alkyne unit using Ohira-Bestmann reagents, followed by treatment with tetrabutylammonium fluoride (TBAF) to obtain the diol compound (12). This diol compound was further transformed into the amidite compound (13).…”

“…Surprisingly, no triplex band was observed for TFO(ethyl(T)), therefore, we thought that the 14-mer TFO with the WNA derivative [12][13][14][15][16][17][18] would not form the triplex DNA for the region including one CG site. The results of the gel-shift assay and the K s value demonstrated that pyrene contributed to the stability of triplex formation with target duplex DNA.…”

“…To overcome the drawback of sequence specificity and enable the use of TFOs as genomic research tools, researchers have attempted to develop artificial nucleoside analogues able to recognize the CG or TA sites with high selectivity and/or stability. [6][7][8][9][10][11] We developed W-shaped nucleoside analogues (WNAs) able to recognize the TA site with high selectivity, [12][13][14][15][16][17][18] and demonstrated that the antigene TFO containing three WNA-βTs exerted an anti-proliferative effect on living cells. 19) Molecules with non-selective stabilizing effects would represent a powerful tool to promote triplex formation.…”

Synthesis and Triplex-Forming Ability of 2',4'-BNAs Bearing Imidazoles as a Nucleobase

“…To overcome this problem, extensive research on nucleic acid analogues has so far been carried out. [5][6][7][8] We have focused on the parallel type triplex, and synthesized 2Ј-O,4Ј-C-methylene-bridged nucleic acid (2Ј, [9][10][11][12][13] /locked nucleic acid (LNA) 14) ) bearing unnatural nucleobases to recognize pyrimidine-purine interruption in the target dsDNA. [15][16][17][18][19] Recently, it was found that the 2Ј,4Ј-BNA bearing oxazole 15,16) : Fig.…”

Chemical Tools for Targeted Mutagenesis of DNA Based on Triple Helix Formation

“…12) We have recently expanded in part the recognition codes of triplex DNA. 13,14) The binding characteristics and sequence specificity of TFOs give these oligonucleotides vast potential in gene modification, such as inhibition of gene expression, inhibition of replication, and induction of site-specific mutagenesis. [15][16][17][18][19][20][21][22][23][24][25] However, there are a number of obstacles to TFO activity under physiologic conditions.…”