Intercalative DNA binding governs fluorescence enhancement of SYBR Gold

Abstract

ABSTRACTSYBR Gold is a commonly used and particularly bright fluorescent DNA stain, however, its binding mode to DNA remains controversial. Here, we quantitate SYBR Gold binding to DNA using two complementary approaches. We use mechanical micromanipulation with magnetic tweezers (MT) to determine the effects of SYBR Gold binding on DNA length, twist, and mechanical properties. The MT assay reveals systematic lengthening and unwinding of DNA upon SYBR Gold binding, consistent with an intercalative binding mode where every SYBR Gold molecule unwinds DNA by 19.1° ± 0.7°. We complement the MT data with a spectroscopic characterization of SYBR Gold fluorescence upon addition to DNA. The data are well described by a global binding model for dye concentrations ≤1 μM, with binding parameters that quantitatively agree with the MT results. The fluorescence signal increases linearly with the number of intercalated SYBR Gold molecules. At dye concentrations >1 μM, fluorescence quenching and inner filter effects become relevant and it is required to correct the SYBR Gold fluorescence signals for quantitative assessment of DNA concentrations. In summary, we provide a mechanistic understanding of DNA-SYBR Gold interactions and present practical guidelines for optimal DNA detection and quantitative DNA sensing applications using SYBR Gold.