In this research supported by the Analytical and Surface Chemistry Program, Professor Stellwagen and her group will study the binding of monovalent cations to DNA hairpins, and the effect of monovalent cation binding on hairpin stability, using capillary electrophoresis. The current structure-prediction algorithms do not reliably predict DNA hairpin stability, indicating that the interactions leading to hairpin formation are not properly included in the folding routines. Since hairpin structures in single-stranded DNA oligomers are only marginally stable, they are difficult to study by conventional methods such as ultraviolet absorption or differential scanning calorimetry. By contrast, free solution capillary electrophoresis can detect hairpin formation and cation binding directly, because electrophoretic mobility depends on both the shape and the effective charge of the analyte.
Broader impact of the proposed research: An important aspect of the project is its contribution to the training of undergraduate students and recent graduates in the biochemical and/or biophysical sciences. There are also scientific and technological broader impacts. Unexpected hairpin formation in single-stranded DNA oligomers can interfere with the hybridization of DNA oligomers to their target DNA or RNA sequences, creating difficulties in the design of multiplex PCR reactions and the interpretation of microarray experiments. Hairpin formation can also interfere with the effectiveness of DNA oligonucleotides used as antisense gene therapy agents. Hence, it is important to be able to predict hairpin formation in DNA oligomers and to understand the factors contributing to hairpin stability. The capillary electrophoresis methods that we have developed to measure monovalent cation binding to DNA are of general utility and can be used to measure the binding of other ligands to other analytes. .
" was designed to measure the effect of different monovalent cations on the thermal stability of small DNA hairpins and duplexes. A second goal was to use the project to contribute to the education of a scientifically trained public by using young undergraduate students and recent college graduates to help carry out the experiments. DNA stability was measured by capillary electrophoresis, since the folded, native structures of DNA hairpins and duplexes have faster mobilities than their unfolded, denatured counterparts. Two papers related to the project have been published in the scientific literature; others are in preparation. The first published paper (E. Stellwagen, E., J.M. Muse, and N.C. Stellwagen, Biochemistry 2011, 50, 3084) showed that the melting temperature of a small DNA hairpin depended on the concentration and size of the cation. Small monovalent cations increased the thermal stability of the hairpin with increasing cation concentration, while large monovalent cations either had no effect or decreased the thermal stability with increasing cation concentration. The results suggest that small cations stabilize DNA hairpins and duplexes because they are more effective at shielding the negative charges of the relatively closely spaced phosphate residues in native DNA. By contrast, large cations are more effective at shielding the negative charges of the more widely spaced phosphate residues in denatured DNA, shifting the hairpin ↔ random coil equilibrium toward the coiled conformation. The second paper published in the scientific literature (C.Y. Chang and N.C. Stellwagen, Biochemistry 2011, 50, 9148) described the effect of DNA sequence on the thermal stability of hairpins with short stems and large loops. The melting temperatures observed for some of the hairpins were significantly lower than expected from popular structure-prediction programs in the literature. Analyzing the thermal stabilities of hairpins with different sequences indicated that the residues in the loops of the more stable hairpins form non-standard base pairs across the loop, effectively increasing the length of the stem and stabilizing the hairpin. Hairpins with relatively low thermal stabilities have unstructured loops without non-standard base pairs forming across the loop.
