The Binding of G‐Quadruplex DNA to Insulin: From Single Molecule to Bulk‐Methods

Abstract

The formation of guanine (G)‐quadruplex structures in the guanine‐rich tandem repeats of the insulin‐linked polymorphic region (ILPR) is linked to transcriptional effects on the insulin gene. Recent studies demonstrate that the ILPR G‐quadruplexes can bind to insulin, and while this may impact the transcription of insulin, little is known about the binding mechanism. We have performed single molecule atomic force microscopy (AFM), differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC) and fluorescence quenching spectroscopy to characterize the binding interaction between G‐quadruplex DNA and insulin. In this presentation, I will describe the bulk thermodynamic measurements performed at various temperatures from 10 to 37 °C. The experimental results based on KD demonstrate a moderate affinity by the ILPR consensus sequence to insulin. In addition to thermodynamic measurements, kinetic off‐rates were measured by single molecule force‐pulling measurements and these results will be discussed in relation to kinetic off‐rates measured from the unfolding of G‐quadruplex DNA.