PNA-Ligand Bioconjugates as Potential Building Blocks for Sequence-Specific, Metal-Mediated DNA-/RNA-Cleavage

Abstract

In this Thesis, the development of new nuclease mimics on the basis of PNA-metal bioconjugates is described. The design of artificial enzymes for sequence-specific DNA and RNA cleavage is one of the most challenging problems in modern biotechnology, since the commercially available biotools are restricted to a limited number of promoter sequences. Possible nuclease mimics consist of a recognition- and a cleavage domain, with the latter one in this case being chosen to be a metal complex. Complexes of several chelating nitrogen ligands are proved to catalyze the phosphodiester cleavage of oligonucleotides. In the course of this Thesis, functionalized nitrogen ligands (terpyridine, bis-picolylamine and phenanthroline) were synthesized, and their metal binding behaviour in unsubstituted form and as pseudoneurotensin conjugates were investigated, revealing different binding modes depending on the type of ligand and its substitution. As a recognition domain, PNA (peptide nucleic acid), a DNA mimic with a pseudopeptide backbone was chosen, and the synthesis of monomer building blocks was optimized. PNA oligomers with terminal ligand substitution were developed, and their DNA hybridization behaviour was examined by UV melting experiments, revealing the intercalating effect of a terminal terpyridine ligand. A new criterion for the significance of PNA DNA melting curves was introduced. The influence of the replacement of one internal PNA monomer with two unsubstituted amino acids on hybridization is described. Presumably, a bulge structure is formed by the modified PNA, involving the insight that the substituted PNA nucleotide does not have to be omitted for optimum attraction. A new and versatile method for the internal derivatization of PNA oligomers was developed by the introduction of a p-nitro-phenylalanine residue which is reduced on resin and is accessible for peptide bond formation. A large variety of ligands, organometallic moieties or fluorescent markers can thus be coupled to PNA oligomers at any position.