Abstract
SYBR Gold is a commonly used and particularly bright fluorescent DNA stain, however, its chemical structure is unknown and its binding mode to DNA remains controversial. Here, we solve the structure of SYBR Gold by NMR and mass spectrometry to be [2-(4-{[diethyl(methyl)ammonio]methyl}phenyl)-6-methoxy-1-methyl-4-{[(2Z)-3-methyl-1,3-benzoxazol-2-ylidene]methyl}quinolin-1-ium] and determine its extinction coefficient. We quantitate SYBR Gold binding to DNA using two complementary approaches. First, we use single-molecule magnetic tweezers (MT) to determine the effects of SYBR Gold binding on DNA length and twist. The MT assay reveals systematic lengthening and unwinding of DNA by 19.1° ± 0.7° per molecule upon binding, consistent with intercalation, similar to the related dye SYBR Green I. We complement the MT data with spectroscopic characterization of SYBR Gold. The data are well described by a global binding model for dye concentrations ≤2.5 μM, with parameters that quantitatively agree with the MT results. The fluorescence increases linearly with the number of intercalated SYBR Gold molecules up to dye concentrations of ∼2.5 μM, where quenching and inner filter effects become relevant. 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.
Highlights
The interaction of DNA with ligands is fundamental for many cellular processes as well as biotechnological applications
We determined the molecular structure of SYBR Gold and its extinction coefficient to calibrate the concentration of the stock solution
Using NMR analysis and mass spectrometry, we determined the molecular structure of SYBR Gold to be [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1phenyl-quinolinium] (Figure 1A)
Summary
The interaction of DNA with ligands is fundamental for many cellular processes as well as biotechnological applications. SYBR Gold is a popular stain with very high sensitivity owing to the >1000-fold increase in fluorescence quantum yield on binding to DNA [1,2]. Its structure is unknown and there is disagreement whether SYBR Gold binds in an intercalative [2,3] or in a minor-groove binding mode [4,5,6]. The binding mode of DNA dyes impacts how binding depends on environmental conditions, DNA chain topology, or sequence context. Smallmolecule binding to DNA can alter its structure and mechanical properties. Minor groove binding has only much smaller effects, if any, on DNA length and winding angle [24]
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