A Single Molecule Force Spectroscopy Study of the Binding Interaction Between Insulin and G-Quadruplex DNA

Abstract

The formation of guanine (G)-quadruplex 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 these G-quadruplexes can bind insulin, and while this may impact the transcription of insulin, little is known about the binding mechanism. A single molecule force spectroscopy study was performed to examine the selective binding of insulin to the ILPR G-quadruplex DNA. In this study, the insulin was covalently attached to a flat gold surface while the quadruplex DNA was attached to an AFM probe. The rupture force between insulin and quadruplex DNA was measured at various force loading rates. To confirm the specificity of the binding, control studies were performed by blocking the tethered G-quadruplex with “free insulin” in solution. Additional control studies were performed with a shorter DNA sequence incapable of forming a G-quadruplex on the AFM tip and a scrambled DNA sequence of the ILPR sequence. Results from the dynamic force-pulling studies are described based on the Bell-Evans and Dudko-Hummer models.