G Quadruplex Research Paper

Project Proposal

Project Title: Isolation of G-quadruplex structures from human cells using small molecular probes.

Introduction

Guanine rich nucleotide sequences can fold into four stranded, non-canonical secondary structures called G-quadruplexes. These are self-assembled structures of guanines from a single strand or multiple strands. The structural unit of a G-quadruplex is called a G-quadret which is a planar structure formed from four guanines connected together by Hoogsteen hydrogen bonding. In a typical G-quadruplex structure, two or more G-quadrets are stacked on top of each other by π-π stacking interaction (Figure 1). These structures are shown to be stable under near physiological conditions and are stabilized by monovalent cations

Four Guanines connected together by hoogsteen hydrogen binding forms a planar structure which is the structural unit of a G-quadruplex called a G-quadret. Two or more G-quadrets stacked on top of each other by π- π stacking forms a G-quadruplex. Monovalent cations like K+ coordinate to the O6 Oxygen atom of guanine and stabilize these structures.

Depending upon the number of strands involved in the formation and the orientation of the strands, there are different classes of G-quadruplex structures. Quadruplexes formed from four different strands all of them pointing in the same direction are called as a tetra molecular parallel quadruplex. Similarly, quadruplex formed from two different strands oriented in the same direction is called a bi-molecular parallel G-quadruplex (figure 2). Intramolecular G-quadruplexes are formed from a single strand of DNA. Based on the orientation of strands there can be different classes of intramolecular quadruplexes too.

Figure 2: G-quadruplex DNA structures. Intramolecular G-quadruplexes (left) and intermolecular G-quadruplexes

1. Isolation of G-quadruplex structures from human cancer cells using biotinylated quadruplex ligands and mapping their positions in the chromosome.

(a) Binding and selectivity of these ligands to G-quadruplex structure will be checked by G4- fluorescent intercalator displacement assay (G4-FID). For this, G-quadruplex structures will be prepared by heating wild type and G4 mutant telomere repeat oligonucleotides in 100mM Potassium Chloride and 10mM Sodium Cacodylate buffer at 950C for 5 minutes followed by slow cooling to room temperature. G-quadruplex structure formation will be confirmed by Circular Dichroism Spectroscopy and UV melting experiments. G4-FID will be performed as mentioned in (ref)

(b) In order to check the ability of these ligands to pull down structurally different G4s, pull down using different G-quadruplex oligonucleotides will be performed. Different concentrations of the biotinylated ligands will be incubated with annealed G4 oligos. Then ligand bound oligos will be pulled down using streptavidin beads. UV absorbance of the samples before and after the pull down will be measured to check the pull down