Four base recognition by triplex-forming oligonucleotides at physiological pH

Abstract

We have achieved recognition of all 4 bp by triple helix formation at physiological pH, using triplex-forming oligonucleotides that contain four different synthetic nucleotides. BAU [2′-aminoethoxy-5-(3-aminoprop-1-ynyl)uridine] recognizes AT base pairs with high affinity, MeP (3-methyl-2 aminopyridine) binds to GC at higher pHs than cytosine, while APP (6-(3-aminopropyl)-7-methyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one) and S [N-(4-(3-acetamidophenyl)thiazol-2-yl-acetamide)] bind to CG and TA base pairs, respectively. Fluorescence melting and DNase I footprinting demonstrate successful triplex formation at a 19mer oligopurine sequence that contains two CG and two TA interruptions. The complexes are pH dependent, but are still stable at pH 7.0. BAU, MeP and APP retain considerable selectivity, and single base pair changes opposite these residues cause a large reduction in affinity. In contrast, S is less selective and tolerates CG pairs as well as TA.

The stability of triplex DNA is affected by the stability of the underlying duplex

“…Unless otherwise stated the concentration of the fluorescein-labelled strand was 0.25 µM, while the dabcyl-labelled strand was 3 µM, as in previous work [19,29,30]. Melting curves were measured using a Roche A C C E P T E D M A N U S C R I P T…”

“…Triplex melting curves are commonly used to assess the stabilising effects of nucleotide substitutions [16,19,23,[30][31][32][33][34][35]. Such modifications can produce complexes in which the triplex and duplex melts overlap, producing a single melting transition.…”

“…The formation of these complexes is also limited by the requirement for low pH (required for protonation of cytosine in the C + .GC triplet) and is usually restricted to oligopurine.oligopyrimidine tracts. A large number of nucleotide analogues have been devised in attempts to overcome these limitations [9][10][11][12][13][14][15][16][17][18][19].…”

“…In some experiments the duplex has been made longer than the triplex in order to separate the duplex and triplex melting transitions [21][22][23], while others have employed intramolecular duplexes in order to increase the stability of the target [24][25][26]. In contrast in most footprinting experiments and for in vivo applications the duplexes in which the target sequences are located will be much longer than the third strand [19,27,28].…”

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

“…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.…”