Temperature dependence of interaction between double stranded DNA and Cy3 or Cy5

Fluorescence resonance energy transfer (FRET) has been widely used as molecular rulers to measure the distance in biomolecules. In this Letter, double stranded DNA (dsDNA) with Cy3 and Cy5 labeled on the 5′-terminals of each strand is used to investigate how temperature influences the interaction between dsDNA and Cy3/Cy5. By applying the orientation-dependent model to fit our FRET data, we found that the interaction between dsDNA and Cy3/Cy5 is so strong that Cy3 and Cy5 are mostly capped on the end of dsDNA duplex in temperature range of 4–50 °C. So, the orientation factor has to be cautiously considered when using FRET to measure distance. Meanwhile, with the increase of temperature, the orientational randomization of the dyes becomes more prominent.