Effect of Loop-Closing Residues on DNA Hairpin Stability

Abstract

Structure-prediction algorithms, such as DINAMelt (1), are often used to analyze hairpin formation in single-stranded DNA and RNA oligomers. In general, an oligomer and its complement are predicted to form hairpins with similar stabilities. Here, we have used capillary electrophoresis to analyze hairpin formation in two complementary 26-nucleotide DNA oligomers that exhibit significantly different free solution mobilities at 20°C. The free solution mobility is a useful indicator of the presence or absence of hairpins, because hairpins and random coils containing the same number of nucleotides have different frictional coefficients, leading to different mobilities (2). The two oligomers studied here are predicted to form molecular beacon-like hairpins with 5 base-pair stems and 16-nucleotide loops at 20°C. The oligomer with the higher free solution mobility forms a stable hairpin with melting temperatures that are reasonably well predicted by DINAMelt, especially at high salt concentrations. However, its complement, which migrates more slowly in free solution, exhibits melting temperatures that are significantly lower than predicted by DINAMelt. Stable hairpins are observed at 20°C only in solutions with Na+ concentrations greater than 200 mM. Since the two oligomers have the same predicted stem sequences, the results suggest that differences in the nucleotides closing the two loops are responsible for the differences in stability of the two hairpins. Hairpin formation in oligomers with different base pairs at the top of the stem and different nucleotides closing the loop are being investigated.1. N. R. Markham, M. Zuker, Nucleic Acids Res. 2005 33, W577.2. E. Stellwagen, A. Abdulla, Q. Dong, N. C. Stellwagen, Biochemistry 2007, 46 10931.