A quantitative method for evaluating the stabilities of nucleic acids

We report a general method for screening, in solution, the impact of deviat ions from canonical Watson-Crick composition on the thermodynamic stability of nucleic acid duplexes. We demonstrate how fluorescence resonance energy transfer (FRET) can be used to detect directly free energy differences bet ween an initially formed "reference" duplex (usually a Watson-Crick duplex) and a related "test" duplex containing a lesion/alteration of interest (e. g., a mismatch, a modified, a deleted, or a bulged base, etc.). In one appl ication, one titrates into a solution containing a fluorescently labeled, F RET-active, reference duplex, an unlabeled, single-stranded nucleic acid (t est strand), which may or may not compete successfully to form a new duplex . When a new duplex forms by strand displacement, it will not exhibit FRET, The resultant titration curve (normalized fluorescence intensity vs. logar ithm of test strand concentration) yields a value for the difference in sta bility (free energy) between the newly formed, test strand-containing duple x and the initial reference duplex. The use of competitive equilibria in th is assay allows the measurement of equilibrium association constants that f ar exceed the magnitudes accessible by conventional titrimetric techniques. Additionally, because of the sensitivity of fluorescence, the method requi res several orders of magnitude less material than most other solution meth ods. We discuss the advantages of this method for detecting and characteriz ing any modification that alters duplex stability, including, but not limit ed to, mutagenic lesions. We underscore the wide range of accessible free e nergy values that can be defined by this method, the applicability of the m ethod in probing for a myriad of nucleic acid variations, such as single nu cleotide polymorphisms, and the potential of the method for high throughput screening.