Convergent Strategies for the Attachment of Fluorescing Reporter Groups to Peptide Nucleic Acids in Solution and on Solid Phase

摘要

The site-selective conjugation of peptide nucleic acids (PNA) with fluorescent reporter groups is essential for the construction of hybridization probes that can report the presence of a particular DNA sequence. This paper describes convergent methods for the solution-and solid-phase synthesis of multiply labeled PNA oligomers. The solid-phase synthesis of protected PNA enabled the selective attachment of fluorescent labels at the C-terminal end (3' in DNA) which demonstrated that further manipulations on protected PNA fragments are feasible. For the conjugation to internal sites, a method is introduced that allows for the on-resin assembly of modified monomers thereby omitting the need to synthesise an entire monomer in solution. Furthermore, it is shown that the application of a highly orthogonal protecting group strategy in combination with chemoselective conjugation reactions provides access to a rapid and automatable solid-phase synthesis of dual labeled PNA probes. Real-time measurements of nucleic acid hybridisation were possible by taking advantage of the fluorescence resonance energy transfer (FRET) between suitably appended fluorophoric groups. Analogously to DNA-based molecular beacons, the dual labeled PNA probes were only weakly fluorescing in the single-stranded state. Hybridization to a complementary oligonucleotide, however, induced a structural reorganization and conferred a vivid fluorescence enhancement.