Differential scanning calorimetry (DSC) melting analysis was performed on 27 short double stranded DNA duplexes containing 15 to 25 base pairs and short single stranded overhangs from one to 10 bases, on both ends. Molecules have two 5' dangling ends or one 5' and one 3' dangling end. For these molecules the duplex region was incrementally reduced from 25 to 15 base pairs with increased length of the dangling ends from one to 10 bases. A third set of molecules contained 21 base pair duplexes with a four base dangling end on either the 5' or 3' end. Blunt ended duplexes from 15 to 25 base pairs were also examined and served as control duplexes. DSC melting curves were measured in solution containing 85 mM, 300 mM or 1.0 M Na+. From these measurements, thermodynamic parameters for 5' and 3' dangling ends as a function of end length were evaluated. Results showed the 5' ends were slightly stabilizing, and this stability was essentially constant with end length, while the 3' ends were generally destabilizing with increasing length of the end. This finding of lower stability for the 3' ends is consistent with results of published studies that have found 5' dangling ends to be more than or equally as stabilizing as 3' dangling ends. Our finding that 3' dangling ends are actually destabilizing for duplex DNA contrasts with published results. The 3' ends also display a stronger dependence on the [Na+]. In the lower Na + environment the 3' ends are more destabilizing than at the higher salt environments. Analysis of the thermodynamic parameters of the dangling-ended duplexes as a function [Na+] indicated the 3' dangling end molecules behave differently compared to 5' dangling ended and blunt ended duplexes. The net counterion release per phosphate upon melting the molecules having one 5' and one 3' end was approximately 15% smaller as a function of end length compared to the duplex having two 5' ends. Further analysis of the DSC evaluated thermodynamic transition parameter, ΔH cal, and its relationship to the measured transition temperatures of the DNA molecules, provided an estimate on the excess heat capacity differences, Δ Cp, between duplex and melted single strands for the dangling-ended molecules. The analysis revealed the molecules with one 5' and one 3' dangling end had very different ΔCp values compared to the blunt-ended molecule; while the molecules with two 5' ends have ΔCp that are essentially the same as the blunt-ended duplex. These observations are interpreted as differences in the interactions with Na+, solvent and the terminal base pairs of the duplex for the 5' versus 3' dangling ends.
|Advisor:||Benight, Albert S.|
|Commitee:||Iwata-Reuyl, Dirk, Rice, Andrew|
|School:||Portland State University|
|School Location:||United States -- Oregon|
|Source:||MAI 49/03M, Masters Abstracts International|
|Subjects:||Biochemistry, Bioinformatics, Biophysics|
|Keywords:||DNA, Dangling end, Duplex formation, Microarray, Single strand, Thermodynamics|
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