The Butterfly Structure of Leucomethylene Blue Results in Weak Binding to Single- or Double-Stranded DNA

The DNA binding properties of methylene blue (MB) have been well characterized, but analysis of the DNA binding behavior of the reduced form, leucomethylene blue (LMB), has been minimal. We demonstrate here, via modeling and X-ray crystallography, that LMB is significantly bent into a "butterfly" conformation with an angle of ∼150 ° at the central sulfur and nitrogen atoms. Binding studies with single- and double-stranded DNA constructs using isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, UV-vis absorbance spectroscopy, and fluorescence confirm binding by MB and show essentially no binding by LMB. As MB redox chemistry has become an integral component in many electrochemical DNA aptamer-based (E-AB) applications, better understanding of the presence and impact of this conformational change in LMB will inform future developments of this exciting biosensor class.