Melting studies of short DNA hairpins: Influence of loop sequence and adjoining base pair identity on hairpin thermodynamic stability

Spectroscopic and calorimetric melting studies of 28 DNA hairpins were perf ormed. These hairpins form by intramolecular folding of 16 base self-comple mentary DNA oligomer sequences. Sequence design dictated that the hairpin s tructures have a six base pair duplex linked by a four base loop and that t he first five base pairs in the stem are the same in every molecule. Only l oop sequence and identity of the duplex base pair closing the loop vary for the set of hairpins. For these DNA samples, melting studies were carried o ut to investigate effects of the variables on hairpin stability. Stability of the 28 oligomers was ascertained fi om their temperature-induced melting transitions in buffered 115 mM Na+ solvent, monitored by ultraviolet absor bance and differential scanning calorimetry (DSC). Experiments revealed the melting temperatures of these molecules range from 32.4 to 60.5 degrees C and are concentration independent over strand concentrations of 0.5 to 260 mu M; thus, as expected for hairpins, the melting transitions are apparentl y unimolecular. Model independent thermodynamic transition parameters, Delt a H-cal, Delta S-cal, and Delta G(cal), were determined from DSC measuremen ts. Model dependent transition parameters, Delta H-vH, Delta S-vH, and Delt a G(vH) were estimated from a van't Hoff (two-state) analysis of optical me lting transitions. Results of these studies reveal a significant sequence d ependence to DNA hairpin stability. Thermodynamic parameters evaluated by e ither procedure reveal the transition enthalpy, Delta H-cal (Delta H-vH) ca n differ by as much as 20 kcal/mol depending on sequence. Similarly, values of the transition entropy Delta S-cal (Delta S-vH) can differ by as much a s 60 cal/Kmol (eu) for different molecules, Differences in fr ee energies D elta G(cal) (Delta G(vH)) are as large as 4 kcal/mol for hairpins,with diff erent sequences. Comparisons between the model independent calorimetric val ues and the thermodynamic parameters evaluated assuming a two-stare model r eveal that 10 of the 28 hairpins display non-two-state melting behavior. Th e database of sequence-dependent melting free energies obtained for the hai rpins was employed to extract a set of n-n (nearest-neighbor) sequence depe ndent loop parameters that were able to reproduce the input data within err or (with only two exceptions). Surprisingly, this suggests that the thermod ynamic stability of the DNA hairpins carl in large part be reasonably repre sented in terms of sums of appropriate nearest-neighbor loop sequence param eters. (C) 1999 John Wiley & Sons, Inc.