Thermodynamic and spectroscopic investigations of hairpin, duplex and dumbbell deoxyoligonucleotides.

摘要

Optical and DSC melting curves were collected for 31 by perfectly matched duplex and two single base (A/C) mismatched duplexes. The (A/C) mismatches resided in unique sequence context beyond nearest-neighbors. Thermodynamic analysis showed a clear effect of context on mismatch stability. More effective sequence design strategies are required to consider effects of sequence context on DNA stability calculations. Thus, we developed functional representations of DNA sequences and convenient algorithms to quantitatively characterize the effect of sequence context and oligoduplex stability.; Hairpin capture probes were studied by UV and DSC melting in 25 and 115 mM Na+. The effects of single strand dangling-end length, biotinylation position of loop bases and loop sequence identity on the thermal stability of DNA hairpin probes were measured. Stability of these dangling-ended DNA hairpins was not affected by dangling-end length, loop biotinylation or sequence and vary uniformly with salt concentration. Thus, considerable freedom is afforded designing DNA hairpins for use as probes in nucleic acid based detection assays.; UV and DSC melting curves were measured for perfect duplex and duplex with 3 single by mismatches at equal strand concentration and when one strand exists in a two-fold molar excess. Both duplexes displayed significant increases in thermal stability under excess strand conditions. Estimation of the nucleation entropy from DSC values suggested temperature dependent transition occurred in the single strand.; Melting and spectroscopic properties of dinucleotide repeat sequences were investigated as a function of sequence, length and [Na+]. CD spectra were not temperature dependent in the pre-transition region. Analysis of thermodynamic data with our optical melting database as a function of [Na +] indicated significant interactions beyond nearest-neighbors in certain sequences, particularly AG and GC sequences. Release of Na + ion was sequence dependent, indicating the salt dependent properties of duplex DNA are also sequence dependent.; Linear DNA molecules containing (AG)n and (RN)n, sequences, n = 5, 10, 20 and 30 were studied by melting and CD. Comparison for identical sequences in dumbbell and linear molecules reveals interesting features between sequence dependent structural variability, sequence length and effects of molecular environment.