A group of 15 debilitating human diseases have been traced to highly repetitive DNA sequences such as (CTG)n and (CAG)m. These DNA sequences do not show Mendelian inheritance patterns. The diseases are collectively referred to as triplet expansion diseases [cite num=1]. Proposed mechanisms for DNA expansion assume that the structure and dynamics of the repetitive DNA segments play critical roles in the development of the diseases. It is suggested that the repeat sequences adopt unusually stable alternative secondary structures involving single-stranded domains.
As models for the intermediate states we have proposed hairpin/loop structures that contain 6 repeats of either CTG or CAG in a single-stranded loop sequence with an alternating (CG)3 sequence as a stem.
In the first set of experiments the thermal stability of the hairpin/loop structures will be established as a function of the salt concentrations. Mixing and heating the two complementary sequences will rearrange the hairpin/loops into a linear double helix with 30 base pairs. The metastable hairpin/loops that can be thermally unfolded to random coils now have settled into the more stable linear double helix. Variations of stem sequences and or point mutations in the loops will give a detailed picture of the subtleties of metastable states and their conversation into the final stable state.
The group of Prof H Klump has the capacity of synthesise the sequences, and follow the thermal unfolding/rearrangements into double helices by UV and CD spectroscopy.
Goal of the project
The goal of the project will be to demonstrate that DNA sequences can fold into a variety of metastable states that can reversibly unfolded and refolded. Small variations of the sequence/ionic environment can shift the equilibrium drastically in the direction of an altogether different conformer. These shifts between conformers can be the course for debilitating diseases.
UV/CD spectroscopy. Gel chromatography. Mass spectroscopy. NMR/IR spectroscopy.
- Völker, J., Makube, N., Plum, G. E., Klump, H. H., & Breslauer, K. J. (2002). Conformational energetics of stable and metastable states formed by DNA triplet repeat oligonucleotides: implications for triplet expansion diseases. Proc Natl Acad Sci U S A, 99(23), 14700-5. PMID: 12417759 [cite num=”1″ return=TRUE]