Convergent strategies for the attachment of fluorescing reporter groups topeptide nucleic acids in solution and on solid phase

The site-selective conjugation of peptide nucleic acids (PNA) with fluoresc ent reporter groups is essential for the construction of hybridisation prob es that can report the presence of a particular DNA sequence. This paper de scribes convergent methods for the solution- and solid-phase synthesis of m ultiply labelled PNA oligomers. The solid-phase synthesis of protected PNA enabled the selective attachment of fluorescent labels at the C-terminal en d (3' in DNA) which demonstrated that further manipulations on protected PN A fragments are feasible. For the conjugation to internal sites, a method i s introduced that allows for the on-resin assembly of modified monomers the reby omitting the need to synthesise an entire monomer in solution. Further more, it is shown that the application of a highly orthogonal protecting gr oup strategy in combination with chemoselective conjugation reactions provi des access to a rapid and automatable solid-phase synthesis of dual labelle d PNA probes. Real-time measurements of nucleic acid hybridisation were pos sible by taking advantage of the fluorescence resonance energy transfer (FR ET) between suitably appended fluorophoric groups. Analogously to DNA-based molecular beacons, the dual labelled PNA probes were only weakly fluoresci ng in the single-stranded state. Hybridisation to a complementary oligonucl eotide, however, induced a structural reorganisation and conferred a vivid fluorescence enhancement.