Conformational energetics of stable and metastable states formed by DNA triplet repeat oligonucleotides: Implications for triplet expansion diseases

Abstract

We have embedded the hexameric triplet repeats (CAG)6 and (CTG) 6 between two (GC)3 domains to produce two 30-mer hairpins with the sequences d[(GC) 3(CAG)6(GC)3] and d[(GC)3(CTG)6(GC)3]. This construct reduces the conformational space available to these repetitive DNA sequences. We find that the (CAG) 6 and (CTG)6 repeats form stable, ordered, single-stranded structures. These structures are stabilized at 62°C by an average enthalpy per base of 1.38 kcal⅐mol ؊1 for the CAG triplet and 2.87 kcal⅐mol ؊1 for the CTG triplet, while being entropically destabilized by 3.50 cal⅐K ؊1 ⅐mol ؊1 for the CAG triplet and 7.6 cal⅐K ؊1 ⅐mol ؊1 for the CTG triplet. Remarkably, these values correspond, respectively, to 1͞3 (for CAG) and 2͞3 (for CTG) of the enthalpy and entropy per base values associated with Watson-Crick base pairs. We show that the presence of the loop structure kinetically inhibits duplex formation from the two complementary 30-mer hairpins, even though the duplex is the thermodynamically more stable state. Duplex formation, however, does occur at elevated temperatures. We propose that this thermally induced formation of a more stable duplex results from thermal disruption of the single-stranded order, thereby allowing the complementary domains to associate (perhaps via "kissing hairpins"). Our melting profiles show that, once duplex formation has occurred, the hairpin intermediate state cannot be reformed, consistent with our interpretation of kinetically trapped hairpin structures. The duplex formed by the two complementary oligonucleotides does not have any unusual optical or thermodynamic properties. By contrast, the very stable structures formed by the individual single-stranded triplet repeat sequences are thermally and thermodynamically unusual. We discuss this stable, triplet repeat, single-stranded structure and its interconversion with duplex in terms of triplet expansion diseases.

Analysis of DNA Replication Intermediates Suggests Mechanisms of Repeat Sequence Expansion

“…Breslauer and co-workers (34) report that, once formed, a sufficiently long single stranded hairpin state is stable under physiological conditions and requires thermal disruption for it to revert to duplex DNA, despite the higher stability of the latter state. Thus, once formed, the hairpin flap intermediate may not equilibrate readily to other intermediates.…”

Mechanistic Studies of Hairpin to Duplex Conversion for Trinucleotide Repeat Sequences

“…Völker et al (17,19) also reported an irreversible conversion to duplex by ⍀-DNA structures that contain a (CAG) 6 or (CTG) 6 single-stranded hairpin embedded within duplex. Because the hairpin to duplex conversion process is irreversible, we conclude that the FL-(CAG) 10 -DCL/ (CTG) 10 duplex is the thermodynamically more stable state at low temperature.…”

Synthesis and photophysical evaluations of fluorescent quaternary bipyridyl-1,8-naphthalimide conjugates as nucleic acid targeting agents

“…39,40 In all three cases very strong binding was found, see Table 1, with binding constants in the range K = 1.7 × 10 7 (± 0.5) M strongly suggests that quenching of emission is due to electron transfer from the DNA bases to the diquat centre. 42 The efficiency of this process is improved when the oxidizing agent is held in close proximity to the bases. Hence the extent of quenching can be taken as a further indication of tight binding.…”