The motions of a dye-labeled DNA hairpin loop (Cy5-5′-GGGTT-(A) 30 -AACCC-3′-TMR) have been investigated through the fluctuations in proximity ratio from fluorescence resonance energy transfer (FRET). We examine three solution conditions: (1) MilliQ water, (2) Tris-EDTA buffer, and (3) Tris-EDTA buffer plus an excess of DNA complementary to the loop sequence, (T) 30 . Correlations in proximity ratio show submillisecond dynamics. Static heterogeneity is revealed from the distribution of proximity ratio amplitudes. The observed stretched exponential kinetics are consistent with a model based on the transition between two states over a complex energy landscape.

Single-molecule fluorescence resonance energy transfer (FRET) combined with bulk fluorescence lifetimes, anisotropy, and spectra have been used to study a donor-acceptor labeled model DNA system (Cy5-5′-ACCTGCCGACGC-3′-TMR). A general ratiometric analysis method using independent donor and acceptor thresholding has been developed. Use of two logical combinations of thresholding criteria provides more information than either method alone, revealing heterogeneity within this system. Conditions yielding similar bulk fluorescence spectra can be readily distinguished by this single-molecule method. Fluorescence lifetimes and anisotropy measurements also suggest nonnegligible fluorophore-DNA interaction.