Quadruplex melting

Abstract

Melting curves are commonly used to determine the stability of folded nucleic acid structures and their interaction with ligands. This paper describes how the technique can be applied to study the properties of four-stranded nucleic acid structures that are formed by G-rich oligonucleotides. Changes in the absorbance (at 295nm), circular dichroism (at 260 or 295nm) or fluorescence of appropriately labelled oligonucleotides, can be used to measure the stability and kinetics of folding. This paper focuses on a fluorescence melting technique, and explains how this can be used to determine the T(m) (T((1/2))) of intramolecular quadruplexes and the effects of quadruplex-binding ligands. Quantitative analysis of these melting curves can be used to determine the thermodynamic (DeltaH, DeltaG, and DeltaS) and kinetic (k(1), k(-1)) parameters. The method can also be adapted to investigate the equilibrium between quadruplex and duplex DNA and to explore the selectivity of ligands for one or other structure.

How to study G-quadruplex structures

“…However, for DNA and RNA quadruplexes those differences are not that evident as they barely amount to 4%. Optionally, it is possible to record the melting profile at different wavelengths, preferably at 295 nm, where the absorbance is significantly lower, but denaturation of the sample is exhibited in an over 50% change in absorbance amplitude (Mergny et al, 1998, Rachwal et al, 2007. A detailed protocol for UV melting studies of quadruplexes was prepared by J.L.…”

A G‐Quadruplex Aptamer Inhibits the Phosphatase Activity of Oncogenic Protein Shp2 in vitro

“…These quadruplexes can form through the intermolecular association of four or two DNA molecules, or by the intramolecular folding of a single strand containing four blocks of guanines. [42] CD experiments were performed to investigate the secondary structure of HJ24. The CD spectrum of the HJ24 aptamer showed a negative peak near 240 nm and a positive peak near 260 nm (Figure 4 A), a typical spectrum of a parallel G-quadruplex.…”

Platinum(II) Phenanthroimidazoles for Targeting Telomeric G‐Quadruplexes

“…Therefore, caution should be taken when interpreting FRET melting temperature results, which can be complicated by changes in dye fluorescence brought about by the typically extended aromatic G4 binders. [25] We also attempted to measure the binding affinity by monitoring the UV/Vis spectrum of the platinum complex while titrating in the G-quadruplex. For these extended aromatic complexes, the experiment was complicated by aggregation of the platinum complexes in solution at the start of the experiment and the consequent difficulty in obtaining the initial absorbance value for the free, unstacked complex.…”