THE THERMODYNAMIC ADVANTAGE OF DNA OLIGONUCLEOTIDE STACKING HYBRIDIZATION REACTIONS - ENERGETICS OF A DNA NICK

'Stacking hybridization reactions' wherein two or more short DNA oligo mers hybridize in a contiguous tandem orientation onto a longer comple mentary DNA single strand have been employed to enhance a variety of a nalytical oligonucleotide hybridization schemes. If the short oligomer s anneal in perfect head-to-tail register the resulting duplex contain s a nick at every boundary between hybridized oligomers. Alternatively , if the short oligomers do not hybridize precisely in register, i.e. single strand regions on the longer strand are left unbound, gaps are formed between regions where short oligomers bind. The resulting gappe d DNA duplexes are considerably less stable than their nicked duplex a nalogs. Formation of base pair stacking interactions between neighbori ng oligomers at the nicks that do not occur in gapped duplexes has bee n proposed as the source of the observed added stability. However, qua ntitative evidence supporting this hypothesis for DNA has not been rep orted. Until now, a direct comparison of the thermodynamics of DNA nic ks versus DNA gaps has not been performed. In this communication we re port such a comparison. Analysis of optical melting experiments in a w ell defined molecular context enabled quantitative evaluations of the relative thermodynamic difference between nicked and gapped DNA duplex es. Results of the analysis reveal that a nick may be energetically fa vored over a gap by at least 1.4 kcal/mol and perhaps as much as 2.4 k cal/mol. The presence of a 5' phosphate at a nick or gap fails to sign ificantly affect their stabilities.