Exploring interactions of peptide nucleic acids with quadruplex forming DNA sequences

The ability of G-rich sequences to form quadruplexes has been known for close to twenty years. However, research in the past decade has provided much evidence that such sequences are important in regulation of a variety of processes such as transcription, mRNA processing and telomere degradation. In addition many diseases caused by genomic instability such as cancer, Bloom's syndrome and Wemer's syndrome show correlation with the occurrence of quadruplex forming sequences (QFS). However, mechanistic details as well as the cellular networks and pathways involving quadruplexes remain largely obscure. In order to obtain such information there is a need to develop synthetic agents that can bind to quadruplex targets in a specific manner and modify their biological functions. These quadruplex binding agents are also envisioned as potential therapeutics.; In this thesis we have studied the ability of a complementary and homologous PNA sequence to invade quadruplex structures. PNA is a DNA and RNA analog that is comprised of the natural nucleobases attached to an anminoethyl glycine backbone. Initially we demonstrated that an extremely stable quadruplex adopted by a sequence from the MYC promoter element is invaded by an 11-mer complementary PNA. Subsequently, it was also shown that this PNA is able to bind to its target even when the quadruplex forming element is part of a double stranded construct with an association constant of 7.6 x 107 M-1. Moreover introduction of 2 mismatches in the PNA sequence led to a 50 fold reduction in binding affinity. This study pointed out that complementary PNA probes are likely to be valuable probes for targeting quadruplex forming regions throughout the genome due to their high affinity and sequence specificity.; Further, we also evaluated the ability of G-rich homologous peptide nucleic acid sequences to bind to quadruplex forming DNA sequences leading to PNA-DNA hybrid quadruplexes. Binding of an 8-mer G-rich PNA to a DNA sequence derived from the MYC promoter region was characterized using UV melting analysis, fluorescence assays, and surface plasmon resonance experiments which revealed that this PNA binds to the MYC QFS in a 2:1 stoichiometry and with an average binding constant Ka = (2.0 +/- 0.2) x 108 M-1 or K d = 5.0 nM. In addition, experiments carried out with short DNA targets revealed a dependence of the affinity on the sequence of bases in the loop region of the DNA. Based on this and DMS footprinting analysis a structural model of the PNA-DNA hybrid was proposed.; Subsequently, we also discovered that the same PNA show striking difference in binding to the MYC sequence when compared to a quadruplex forming sequence derived from the human telomeric region using surface plasmon resonance. Experiments revealed that this PNA is able to discriminate between the two quadruplexes due to differences in kinetics of association. In addition a 2.5 fold difference in thermodynamic affinity was also observed. The kinetic discrimination was found to be related to the syn or anti arrangement of guanines in the target quadruplex. The binding to a quadruplex target within a cell in a selective manner is critical. Therefore the high affinity coupled with the discrimination displayed by these PNA probes make then useful probes for targeting quadruplex targets within cells.