Abstract
The oxazole yellow dye, YOYO-1 (a symmetric homodimer), is a commonly used molecule for staining DNA. We applied the brightness analysis to study the intercalation of YOYO-1 into the DNA. We distinguished two binding modes of the dye to dsDNA: mono-intercalation and bis-intercalation. Bis-intercalation consists of two consecutive mono-intercalation steps, characterised by two distinct equilibrium constants (with the average number of base pair per binding site equals 3.5): and , respectively. Mono-intercalation dominates at high concentrations of YOYO-1. Bis-intercalation occurs at low concentrations.
Highlights
The reactions in living cells occur in nanomolar or even picomolar concentrations
Our brightness analysis method (BAM) is applicable even for reactions characterised by changes in molecular brightness (MB) as small as 5% [6]
We monitored the MB change of DNA-YOYO-1 complex depending on YOYO-1 to DNA concentrations
Summary
Quantitative analysis of reactions at such small concentrations requires understanding the reaction mechanism and appropriate methods to determine the equilibrium constants, K. We developed a method for precise and accurate determination of equilibrium constants of biochemical complex formations at the subnanomolar concentration range [5]. The method is based on a change of intrinsic molecular brightness (MB) of fluorescent molecules upon complex formation. Our brightness analysis method (BAM) is applicable even for reactions characterised by changes in MB as small as 5% [6]. We use this method to study DNA staining, providing a much higher sensitivity than microscopic imaging, which requires a submicromolar-micromolar dye concentration regime
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