title: Convergent strategies for the attachment of fluorescing reporter groups to peptide nucleic acids in solution and on solid phase.
abstract: The site-selective conjugation of peptide nucleic acids (PNA) with fluorescent reporter groups is essential for the construction of hybridisation 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 labelled 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 labelled 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 labelled PNA probes were only weakly fluorescing in the single-stranded state. Hybridisation to a complementary oligonucleotide, however, induced a structural reorganisation and conferred a vivid fluorescence enhancement.
authors.affiliation: Department of Chemical Biology and Institut für Organische Chemie, Universität Dortmund, Germany. oliver.seitz@mpi-dortmund.mpg.de
authors.forename: O
authors.initials: O
authors.lastname: Seitz
authors.forename: O
authors.initials: O
authors.lastname: Köhler
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D005456:Fluorescent Dyes; D008958:Models, Molecular; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D020135:Peptide Nucleic Acids / Q000138:chemical synthesis / Q000737:chemistry*; D011485:Protein Binding; D012996:Solutions; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D020135:Peptide Nucleic Acids; D012996:Solutions; D004247:DNA