Title

Studies on the binding affinity and selectivity of phenanthroline-neomycin conjugates towards different G-quadruplex conformations

Poster Number

6

Lead Author Affiliation

Chemical synthesis, Drug discovery and design

Lead Author Status

Doctoral Student

Second Author Affiliation

Chemical synthesis, Drug discovery and design

Second Author Status

Doctoral Student

Third Author Affiliation

Chemistry

Third Author Status

Faculty

Introduction

G-quadruplexes are dynamic, polymorphic, four stranded non-canonical structures of the nucleic acids formed by the stacking of the planar G-tetrads, stabilized by monovalent ions. There are about 370,000 putative G-quadruplex (pG4) forming sequences in the human genome. The distribution of the G4 in the genome is non-random and they are highly abundant in the promoter regions of the oncogenes, replication origins, and ends of the chromosomes. G4 are also associated with genomic instability. The region which is particularly rich in pG4 sequences is the telomeres of the chromosomes, which contain about 5-10000 tandems repeats of the TTAGGG sequence. This region gets progressively short in normal cells during each cycle of cell replication, which ultimately triggers cellular apoptosis. Cancer cells avoid this problem and possible apoptosis, due to the over-activation of the enzyme named telomerase. The induction and stabilization of the G-quadruplex can inhibit telomerase from binding and extending ends of the chromosomes, and this can be a promising strategy for cancer remediation.

Purpose

G-quadruplexes are polymorphic in terms of conformation, loop length, composition, and orientation. They share one common feature of planar G-tetrads, which required the G4-ligands to have the extended planar aromatic surface. This required feature in the ligands makes it harder to target specifically different conformations of the G-quadruplexes. We designed ligands to have dual recognition mode by combining planar 1,10-phenanthroline to the structured polyamine neomycin to selectively target different G4 conformations

Method

The compounds were synthesized and characterized using 1H NMR, 1D TOCSY, mass spectrometry, IR. The ligand’s conformation selectivity and binding affinity were analyzed using mass spectrometry and isothermal titration calorimetry.

Mass spectrometry analysis:- 20 nM G4 DNA was precipitated using ice-cold alcohol to desalt it and which is resuspended in 150 mM ammonium acetate solution. The G4 DNA is annealed at 90 oC for 5 mins, slowly cooled to room temperature and finally stored at 4 oC before further use. G4 and ligands are mixed in different molar ratios 1h before analysis. The final solutions are analyzed using Thermo Fisher Orbitrap Fusion Tribrid Mass Spectrometer with 50% methanol-water.

ITC analysis:- G4 DNA and ligand solutions were prepared in 10 mM potassium phosphate and 150 mM potassium chloride buffer. The binding affinity of the ligands to the different G4 conformations was analyzed using TA instruments affinity ITC instrument.

Results

The length of the linker found to have a profound effect on the binding affinity and conformation selectivity of the ligands. The ligand with a shorter linker between two binding moieties binds with nM affinity to the hybrid G4 conformation of telomeric DNA. This is also confirmed with MS analysis. Whereas, the ligand with longer linker binds with very high affinity to the parallel G4 conformation of c-myc DNA

Significance

The study showed insights into the ligand development for the synthesis of more potent and selective G4-DNA binding ligands. We found that the conformation selective binding of the ligand depends highly on the linker length between the two binding moieties in the ligand.

Location

DeRosa University Center

Format

Poster Presentation

Poster Session

Morning

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Apr 27th, 10:00 AM Apr 27th, 12:00 PM

Studies on the binding affinity and selectivity of phenanthroline-neomycin conjugates towards different G-quadruplex conformations

DeRosa University Center

G-quadruplexes are dynamic, polymorphic, four stranded non-canonical structures of the nucleic acids formed by the stacking of the planar G-tetrads, stabilized by monovalent ions. There are about 370,000 putative G-quadruplex (pG4) forming sequences in the human genome. The distribution of the G4 in the genome is non-random and they are highly abundant in the promoter regions of the oncogenes, replication origins, and ends of the chromosomes. G4 are also associated with genomic instability. The region which is particularly rich in pG4 sequences is the telomeres of the chromosomes, which contain about 5-10000 tandems repeats of the TTAGGG sequence. This region gets progressively short in normal cells during each cycle of cell replication, which ultimately triggers cellular apoptosis. Cancer cells avoid this problem and possible apoptosis, due to the over-activation of the enzyme named telomerase. The induction and stabilization of the G-quadruplex can inhibit telomerase from binding and extending ends of the chromosomes, and this can be a promising strategy for cancer remediation.