Influence of Sequence-Dependent Cytosine Protonation and Methylation on DNA Triplex Stability

Abstract

To investigate cytosine protonation and its influence on the sequence-dependent thermal stability of DNA triplexes in detail, we have employed homo- and heteronuclear NMR experiments on specifically (15)N-labeled oligodeoxynucleotides that were designed to fold into intramolecular triple helices of the pyrimidine motif under appropriate conditions. These experiments reveal that cytosines in central positions of the triplex are significantly protonated even at neutral pH. However, semiprotonation points for individual cytosine bases as determined from pH-dependent measurements show considerable differences depending on their position. Thus, protonation is disfavored for adjacent cytosines or for cytosines at the triplex termini, resulting in a smaller contribution to the overall free energy of the triple helical system. In contrast, protonation of the base upon substitution of 5-methylcytosine for cytosine in the triplex third strand is only affected to a minor extent, and triplex stabilization by the methyl substituent is shown to primarily arise from stacking energies and/or hydrophobic effects.

Mechanical Unfolding of the Beet Western Yellow Virus −1 Frameshift Signal

“…Cytosine in its protonated form is not unusual in and is known to stabilize DNA triplexes (58). The protonation of C8 as well as its orientation allow for the formation of more hydrogen bonds than would be predicted from normal, Watson-Crick base pairing (57).…”

“…The protonation on C8 is not an usual biological occurrence as cytosine in its protonated form is frequently found in stable DNA triplexes (58) Mutating C8 to any other base reduces the frameshifting to background levels, from 10.8 % for the wild-type pseudoknot to 0.6 % for C8A and 0.4 % for a C8U mutant (23). The C8A mutation disrupts the unique setup created by C8 both sterically and by reducing the number of favorable hydrogen bonding contacts.…”

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.…”

“…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. 1, top).…”

“…Here we designed programmable DNA-based triplex pHtriggered nanoswitches that form an intramolecular triplex structure through the formation of a Watson-Crick (dashed) pH-insensitive hairpin and a second Hoogsteen (dots) pH-sensitive hairpin. Because they require the protonation of the N3 of cytosine in the third strand (top, left), CGC triplets are only stable at acid pHs (average pKa of cytosines in triplex structure is ≈ 6.5 35 ). For this reason, triplex switches containing only CGC triplets should unfold into an open duplex conformation at slightly acidic pHs.…”