The interaction of peptide nucleic acids with free and histone bound DNA

Abstract

This thesis studies the interaction of several polypyrimidine PNAs with both free and nucleosomal DNA under different ionic conditions.  These different PNAs explore the effects of base sequence, length and polarity on the interaction.  This work used variants of the tyrT DNA sequence, which contain PNA binding sites located a different positions with respect to the end of the fragments, thereby affecting their translational positioning when reconstituted onto histone octamers.  Footprinting studies with DNaseI and S1 nuclease, together with gel retardation assays and fluorescence melting studies were used to examine the sequence specificity and binding affinity of these PNAs to free double strand DNA.  At low pHs (5.0) DNaseI footprints were observed at submicromolar PNA concentrations.  No binding was detected at higher pHs, consistent with the involvement of C⁺•GC triplets in this interaction.  Higher PNA concentrations (typically 10μM) were required to induce S1 cleavage and to produce bandshifts, suggesting strand invasion (generating 2:1 PNA:DNA complexes) is a slower and less stable process.  The most stable complexes were formed when the PNA was of increased length and orientated parallel to the duplex purine strand.

Within all eukaryotic cells, DNA is associated with histone proteins in the form of chromatin.  Therefore, similar studies were performed on these DNA fragments, which have been reconstituted into nucleosome core particles, thereby assessing the accessibility of each of the target sites, when located in different positions.  These complexes were prepared by either challenging reconstituted nucleosomes with PNA, or by attempting to wrap pre-formed PNA-DNA complexes around the histone octamer.  PNA is unable to bind to the central nucleosomal DNA core particle, though some interaction with the peripheral regions is detected.

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