SYBR 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 degrees. 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 uM, 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 uM, 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.

中文翻译：

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嵌入DNA结合控制SYBR Gold的荧光增强

SYBR Gold是一种常用且特别明亮的荧光DNA染色剂，但是，其与DNA的结合方式仍存在争议。在这里，我们使用两种互补方法对SYBR Gold与DNA的结合进行定量。我们使用机械镊子和磁性镊子（MT）来确定SYBR Gold结合对DNA长度，扭曲和机械性能的影响。MT分析揭示了SYBR Gold结合后DNA的系统延长和解链，这与插入结合模式一致，其中每个SYBR Gold分子将DNA解链19.1 +/- 0.7度。当添加到DNA上时，我们用SYBR Gold荧光光谱特征对MT数据进行了补充。染料浓度<1 uM的全局结合模型很好地描述了数据，其结合参数与MT结果定量一致。荧光信号随插入的SYBR Gold分子的数量线性增加。在染料浓度> 1 uM时，荧光猝灭和内部过滤效果变得很重要，因此需要校正SYBR Gold荧光信号以定量评估DNA浓度。总之，我们提供了对DNA-SYBR Gold相互作用的机械理解，并提供了使用SYBR Gold进行最佳DNA检测和定量DNA传感应用的实用指南。