Single nucleotide specific detection of DNA by native chemical ligation of fluorescence labeled PNA-probes.

DNA-directed chemical ligations provide the opportunity to diagnose DNA sequences with very high sequence specificity. Fluorescent labels have been attached to reactive probes to enable the homogeneous detection of DNA and RNA. However, it has frequently been found that the attachment of fluorescent labels results in decreases of ligation fidelity. Herein we describe the development of a fluorogenic ligation reaction that provides for 10(2)-fold to perfect sequence selectivity. The reaction is based on the isocysteine-mediated native chemical PNA ligation. It is shown that DNA-induced rate accelerations of approximately 43.000-fold can be obtained through subtle variations of the ligation conditions. PNA-thioesters and isocysteine-PNA conjugates were labeled with FAM and TMR fluorophores, respectively. For gaining rapid synthetic access, a convenient on-resin labeling approach was developed. A new PNA monomer featuring an Alloc-protected lysine side chain was synthesized and coupled in solid-phase PNA synthesis. In the event of a ligation reaction the two fluorophores are brought into proximity. It is shown that fluorescence resonance energy transfer provides a positive fluorescence signal which is specific for product formation rather than for loss of starting materials. Single base mutations can be detected within minutes and with very high sequence selectivity at optimized conditions.