pH and Cation Effects on the Properties of Parallel Pyrimidine Motif DNA Triplexes

Abstract

The effects of cytosine protonation and various cations on the properties of parallel pyrimidine motif DNA triplexes were intensively investigated and characterized by several different techniques, such as circular dichroism (CD) conformation, ultraviolet (UV) melting, differential scanning calorimetry (DSC) thermal denaturation, and surface plasmon resonance (SPR) real-time dynamics. The comparative CD spectra of the triplex and the corresponding homoduplexes showed that the negative peak at approximately 218 nm would be the eigenpeak of the Hoogsteen paired strand, and moreover, the formation pathway of a triplex was significantly pH-dependent and fell into three groups: under acidic conditions, the triplex is formed by a one-step docking, under near physiological conditions, the Watson-Crick duplex is first structured and then accepts the Hoogsteen third strand into its major groove, and under basic conditions, the triplex is not formed. The pH-dependent thermodynamics of the global triplex, the Watson-Crick antiparallel duplex, and the Crick-Hoogsteen parallel duplex were comparatively discussed for the first time. These data revealed that the thermodynamic stabilities of the Watson-Crick-Hoogsteen triplex and the Crick-Hoogsteen duplex would be strongly dependent on cytosine protonation, but a low-pH environment somewhat destabilized the Watson-Crick duplex. The binding energy of triplex formation would be different from the unfolding energy of triplex melting under acidic conditions due to the disparity in the pathway between the formation and unfolding of a triplex. Real-time dynamic measurements showed that the association and dissociation rate constants of a duplex-to-triplex formation are (1.98 +/- 0.24) x 10(3) M(-1) s(-1) and (4.09 +/- 0.96) x 10(-4) s(-1) at 20 degrees C and pH 6.0, respectively. The formation energy of the duplex-to-triplex transition derived from SPR measurements was in agreement with the unfolding energy of the free Hoogsteen paired duplex derived from UV measurements. The calorimetric enthalpies of the triplex-to-duplex-to-single transition were 39.3 and 75.3 kcal/mol under near physiological conditions (pH 7.0), respectively, which were underestimated relative to the van't Hoff enthalpies. In addition, the effects of various cations, ionic strength, mixed-valent cations, and the position of the C(+)xG.C triplets on the thermodynamics of the triplexes were addressed under near physiological conditions. The interaction of metal ions with the triplexes clearly depended on the type and ionic strength of the cations, and the efficiency with which the cations stabilized the global triplex was in the order Mg(2+) > Mn(2+) > Ca(2+) > Ba(2+) >> Na(+). These observations would be useful for the design of triplex-forming oligonucleotides for antigene drugs and therapeutic purposes.

Triplexator: Detecting nucleic acid triple helices in genomic and transcriptomic data

“…Conversely, the pyrimidine motif (Fig. 2, ''TC''), which is not expected to be functional due to the nonphysiological pH required for the protonation of the cytosines (Sugimoto et al 2001), is substantially depleted. This may be an artifact of the particular sequencing protocol used to generate the data, which involves addition of cytosines to the 39 end of the RNA and poly(G) guided amplification.…”

“…Over the past decades, these rules have been scrutinized with respect to various determinants of triplex formation such as the chemistry of the nucleotides present in each strand (e.g., nucleotide backbone [Nielsen et al 1991;Roberts and Crothers 1992;Escude et al 1993;], sugars [Alam et al 2007], bases [Hogeland and Weller 1993], and modifications [Lee et al 1984]), the impact of pH (Sugimoto et al 2001), ionic environment (Wu et al 2002), sequence composition (Völker and Klump 1994), and base mismatches (Mergny et al 1991) using a multitude of complementary experimental setups (for more information, see the review Duca et al 2008). While each of the different determinants affects the triplex stability, these studies demonstrate that the rule set can be used to model triple-helix formation.…”

Programmable pH-Triggered DNA Nanoswitches

“…We created our switches by taking advantage of the wellcharacterized pH sensitivity of the parallel Hoogsteen (T,C)-motif in triplex DNA [34][35][36] . To do so we have designed a DNAbased triplex pH-triggered nanoswitch that consists in a double intramolecular hairpin stabilized with both Watson-Crick (W-C) and parallel Hoogsteen interactions (Fig.…”

“…1) 37 . Of note, while W-C base pairing is almost insensitive to pH 34 , Hoogsteen interactions show a strong and variable pH-dependence [34][35][36] . More specifically, the CGC parallel triplet requires the protonation of the N3 of cytosine in the third strand in order to form (average pK a of cytosines in triplex structure is ≈ 6.5 35,38 ) (Fig.…”

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

“…In the pyrimidine motif, the third strand consisting of cytosine and thymine binds parallel to the purine strand of duplex via Hoogsteen bonds and the N3 of cytosine is required to be protonated under acidic conditions. Because it is pH dependent, pyrimidine does not usually bind to DNA at physiologic pH without further modification [1][2][3]. In the purine motif, the third strand binds to the purine strand in duplex with binding of A to A:T, G to G:C, and T to A:T base pairs (Figure 1), requires no base protonation, and primarily exhibits pH-independent binding.…”