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

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Abstract
Enzymatic ligation methods are useful in diagnostic detection of DNA sequences. Here we describe the investigation of nonenzymatic phosphorothioate–iodide DNA autoligation chemistry as a method for detection and identification of both RNA and DNA sequences. Combining ligation specificity with the hybridization specificity of the ligated product is shown to yield discrimination of a point mutation as high as >104-fold. Unlike enzymatic ligations, this reaction is found to be equally efficient on RNA or DNA templates. The reaction is also shown to exhibit a significant level of self-amplification, with the template acting in catalytic fashion to ligate multiple pairs of probes. A strategy for fluorescence labeling of three autoligating energy transfer (ALET) probes and directly competing them for autoligation on a target sequence is described. The method is tested in several formats, including solution phase, gel, and blot assays. The ALET probe design offers direct RNA detection, combining high sequence specificity with an easily detectable color change by fluorescence resonance energy transfer (FRET).