Expansion of trinucleotide repeats (TNR) has been implicated in the emergence of neurodegenerative diseases. Formation of non-B conformations such as hairpins by these repeat sequences during DNA replication and/or repair has been proposed as a contributing factor to expansion. In this work we employed a combination of fluorescence, chemical probing, optical melting, and gel shift assays to characterize the structure of a series of (CTG)n sequences and the kinetic parameters describing their interaction with a complementary sequence. Our structure-based experiments using chemical probing reveal that sequences containing an even or odd number of CTG repeats adopt stem-loop hairpins that differ from one another by the absence or presence of a stem overhang. Furthermore, we find that this structural difference dictates the rate at which the TNR hairpins convert to duplex with a complementary CAG sequence. Indeed, the rate constant describing conversion to (CAG)10/(CTG)n duplex is slower for sequences containing an even number of CTG repeats than for sequences containing an odd number of repeats. Thus, when both the CAG and CTG hairpins have an even number of the repeats they display a longer lifetime relative to when the CTG hairpin has an odd number of repeats. The difference in lifetimes observed for these TNR hairpins has implications towards their persistence during DNA replication or repair events and could influence their predisposition towards expansion. Taken together these results contribute to our understanding of trinucleotide repeats and the factors that regulate persistence of hairpins in these repetitive sequences and conversion to canonical duplex.
The expansion of a trinucleotide repeat sequence, such as CAG/CTG, has been pinpointed as the molecular basis for a number of neurodegenerative disorders. It has been proposed that as part of the expansion process, these repetitive sequences adopt non-B conformations such as hairpins. However, the prevalence of these hairpins and their contributions to the DNA expansion have not been well defined. In this work, we utilized a molecular beacon strategy to examine the stability of the (CAG) 10 hairpin and also its behavior in the presence of the complementary (CTG) 10 hairpin. We find that the two hairpins represent kinetically trapped species that can coexist but irreversibly convert to duplex upon thermal induction. Furthermore, as monitored by fluorescence and optical analysis, modifications to the base composition of either the loop or stem region have a profound effect on the ability of the trinucleotide repeat hairpins to convert to duplex. Additionally, the rate of duplex formation is also reduced with these loop and stem-modified hairpins. These results demonstrate that the trinucleotide repeat hairpins can convert to duplex via two independent mechanisms as follows: the loop-loop interactions found in kissing hairpins or the stem-stem interactions of a cruciform.
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