Nonenzymatic autoligation in direct three-color detection of RNA and DNA point mutations

Enzymatic ligation methods are useful in diagnostic detection of DNA sequen ces. Here we describe the investigation of nonenzymatic phosphorothioate-io dide DNA autoligation chemistry as a method for detection and identificatio n of both RNA and DNA sequences. Combining ligation specificity with the hy bridization specificity of the ligated product is shown to yield discrimina tion of a point mutation as high as >10(4)-fold. Unlike enzymatic ligations , this reaction is found to be equally efficient on RNA or DNA templates. T he reaction is also shown to exhibit a significant level of self-amplificat ion, with the template acting in catalytic fashion to ligate multiple pairs of probes. A strategy for fluorescence labeling of three autoligating ener gy transfer (ALET) probes and directly competing them for autoligation on a target sequence is described. The method is tested in several formats, inc luding solution phase, gel, and blot assays. the ALET probe design offers d irect RNA detection, combining high sequence specificity with an easily det ectable color change by fluorescence resonance energy transfer (FRET).