The popularity of carbocyanine dyes in single molecule spectroscopy of nucleic acids is unbroken . Studying the dynamics of large RNA constructs and the binding kinetics such as the exon/intron binding side interaction of the group II intron in S. Cerevisiae [2,3] have motivated a thorough photophysical characterization of the FRET pair Cy3/Cy5 in context of nucleic acids and RNA in particular. We show that Mg2+ as a mediator of RNA-dye interactions enhances the cyanine fluorescence lifetime. The increasing window for depolarization as monitored by time-resolved anisotropy further revealed a dynamic equilibrium between free tumbling and stacking on the RNA backbone, with the stacked conformation preventing photoisomerization . Tracking fluorophore mobility covalently bound to the RNA on an atomistic level by means of molecular dynamics  allow to disentangle different types of dye-dye and dye-RNA interactions. Our hybrid approach combining time-correlated single photon counting and computer simulations will benefit the interpretation of absolute distance measurement by smFRET.
 M.Levitus and S.Ranjit, Q. Rev. Biophys 2011, 44, 123-151.
 D.Kowerko, S.L.B.König, M.Skilandat, D.Kruschel, M.C.A.S.Hadzic, L.Cardo, R.K.O.Sigel, PNAS 2015, 112, 3403-3408.
 M. Khier, D. Kowerko, F. Steffen, R. Börner and R.K.O.Sigel, in preparation.
 F.Steffen, R.K.O. Sigel, R.Börner, in preparation.
 R.Best, H. Hofmann, D. Nettels, B. Schuler, Biophys J 2015, 11,2721-2731.