Length-dependent energetics of (CTG)n and (CAG)n trinucleotide repeats

Abstract

Trinucleotide repeats are involved in a number of debilitating diseases such as myotonic dystrophy. Twelve to seventy-five base-long (CTG)n oligodeoxynucleotides were analysed using a combination of biophysical [UV-absorbance, circular dichroism and differential scanning calorimetry (DSC)] and biochemical methods (non-denaturing gel electrophoresis and enzymatic footprinting). All oligomers formed stable intramolecular structures under near physiological conditions with a melting temperature that was only weakly dependent on oligomer length. Thermodynamic analysis of the denaturation process by UV-melting and calorimetric experiments revealed an unprecedented length-dependent discrepancy between the enthalpy values deduced from model-dependent (UV-melting) and model-independent (calorimetry) experiments. Evidence for non-zero molar heat capacity changes was also derived from the analysis of the Arrhenius plots and DSC profiles. Such behaviour is analysed in the framework of an intramolecular ‘branched-hairpin’ model, in which long CTG oligomers do not fold into a simple long hairpin–stem intramolecular structure, but allow the formation of several independent folding units of unequal stability. We demonstrate that, for sequences ranging from 12 to 25 CTG repeats, an intramolecular structure with two loops is formed which we will call ‘bis-hairpin’. Similar results were also found for CAG oligomers, suggesting that this observation may be extended to various trinucleotide repeats-containing sequences.

Comparative Genomics and Molecular Dynamics of DNA Repeats in Eukaryotes

“…The same repeats also form slipped structures on double-stranded DNA in which both DNA strands carry a hairpin (387). A more recent analysis of (CTG) n repeats shows that two nucleotides at the base of the stem are sensitive to single-strand-specific nucleases, suggesting that some sort of secondary hairpin arises from the stem base (10). In RNA, (CUG) n repeats are able to fold into a triangular tubelike structure that looks like the chocolate bar confection "Toblerone" and is more similar to a triplex than to a classical hairpin (395).…”

“…Several genes that could be directly involved in the tumorigenesis were subsequently found to be altered by this hypermutator phenotype. The type II transforming growth factor ␤ receptor gene, involved in epithelial cell growth, contains an insertion in a (GT) 3 dinucleotide repeat or a 1-or 2-nucleotide deletion in an (A) 10 mononucleotide repeat in cancerous cells (316). IGFIIR, the insulin-like growth factor II receptor, contains a 1-or 2-bp deletion in a (G) 8 mononucleotide repeat (473), and deletions and insertions in a (G) 8 mononucleotide repeat in the BAX gene, involved in apoptosis, were found in tumors (405).…”

“…However, it was unclear if the microsatellite by itself was able to act as a meiotic hot spot, hence inducing meiotic recombination, although some data suggested that it could be the case (212). This question was first addressed by Moore and colleagues (345), who showed that the presence of (CAG) 10 or (CGG) 10 trinucleotide repeat tracts did not increase recombination between two flanking markers and were not the preferential site of meiotic DSBs. Additional experiments using much longer trinucleotide repeat tracts, (CAG) 98 and (CAG) 255 , confirmed that even these long repeats did not act as meiotic recombination hot spots in yeast, the meiotic recombination rate being independent of the presence or absence of the microsatellite (412).…”

Identification of Trinucleotide Repeat Ligands with a FRET Melting Assay

“…The double-labeled (CTG) 7 DNA oligonucleotide had an apparent T1 = 2 of 55 8C, similar to that of the unmodified oligonucleotide as determined by absorbance; this indicates that the attachment of the fluorophores did not greatly perturb the stability of the hairpin. [6] Screening a panel of compounds We chose to carry out our test of the double-labeled (CTG) 7 on an initial panel of 33 different chemicals. Over a hundred compounds were actually tested, but the other molecules gave few positive results.…”

“…DNA molecules with more than twelve trinucleotide A C H T U N G T R E N N U N G repeats can fold into unusually long hairpins called broken hairpins. [5,6] As the length of the repeated array is a crucial feature in the onset of disease and the progression of symptoms, suppression of somatic expansion could be therapeutically beneficial. Different research teams have recently tried to trigger tripletrepeat contractions by using chemotherapeutical approaches.…”