Ratiometric analysis of single-molecule fluorescence resonance energy transfer using logical combinations of threshold criteria: A study of 12-mer DNA

Single-molecule fluorescence resonance energy transfer (FRET) combined with bulk fluorescence lifetimes, anisotropy, and spectra have been used to stu dy 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 thresho lding criteria provides more information than either method alone, revealin g heterogeneity within this system. Conditions yielding similar bulk fluore scence spectra can be readily distinguished by this single-molecule method. Fluorescence lifetimes and anisotropy measurements also suggest nonnegligi ble fluorophore-DNA interaction.