Intelligent Nucleic Acid Interactions with Peptide Nucleic Acid (PNA) and Recombination Enzyme RecA
Doctoral thesis, 1996
The work presented in this thesis focuses on interactions between nucleic-acids in biological systems, particularly interactions of the promising drug-candidate peptide nucleic acid (PNA), and on protein-DNA intermediates during recombination. Several experimental techniques, mainly circular dichroism and fluorescence spectroscopy, have been used to characterize the interactions of interest.
Various DNA analogs are currently investigated in the search for a new type of drugs that can target genes directly. PNA is a recent DNA analog which differs from DNA in that the backbone of DNA has been replaced by a neutral, achiral peptide-like backbone. PNA shows several positive features as a promising candidate, both as a drug and as a diagnostic tool. PNA is also interesting from a fundamental point of view, as a novel mimic of DNA.
As described in Papers 1 and 2, we discovered that two PNA molecules can form a base-paired helical duplex, with the preferred helical sense induced by a terminal chiral amino acid. The propagation of helicity depends on the base-pairs closest to the chiral center, and the choice of amino acid is crucial for the sense of helicity. These results suggest that the ordinary DNA backbone is not essential for formation of helical duplexes. Moreover, we observed that DNA-binding ligands have strongly reduced affinities for PNA-containing complexes compared to their affinities for normal DNA, indicating electrostatic attraction to be a major component of the binding affinity of these ligands to DNA, discussed more in Paper 3. PNA binds to double-stranded DNA by forming a triplex with the complementary DNA strand and displacing the other. In Paper 4 we report the rate-limiting step for this reaction to involve two PNA molecules and a transient opening of a few base-pairs of the DNA. Furthermore, DNA-binding ligands were able to modulate the PNA-binding rate.
The second part of the thesis concerns the Escherichia coli protein RecA which is involved in the cellular processes of DNA repair and homologous recombination. RecA binds to single-stranded DNA and forms long helical protein-DNA filaments. Such complexes can bind another DNA molecule and search for homology between the DNA sequences.