Ratiometric Analysis of Single-Molecule Fluorescence Resonance Energy Transfer Using Logical Combinations of Threshold Criteria: A Study of 12-mer DNA Liming Ying, Mark I. Wallace, Shankar Balasubramanian,* and David Klenerman* The Department of Chemistry, UniVersity of Cambridge, Lensfield Road, Cambridge, CB2 1EW United Kingdom ReceiVed: NoVember 5, 1999; In Final Form: March 13, 2000 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. Introduction Recent advances in single-molecule spectroscopy and mi- croscopy allow one to study the molecular properties and dynamic processes of biomolecules on an individual basis. 1-10 In contrast to conventional experiments, which measure en- semble averaged behavior, single-molecule measurements are able to probe the differences between individual molecules. The static heterogeneity and dynamic fluctuations in the conforma- tions of DNA, protein, and other biological macromolecules demonstrate one such property that is difficult, if not impossible to detect at the bulk level, but can be potentially probed using single-molecule techniques. 11-16 Fluorescence resonance energy transfer (FRET) is a widely used tool in biochemistry and structural biology. 17,18 In FRET, energy is nonradiatively transferred from an excited donor to an acceptor fluorophore with an efficiency that varies as the inverse sixth power of the distance between donor and acceptor. By attaching donor and acceptor dyes to two sites of a bio- molecule, FRET can be used as a sensitive probe of intra- molecular distance. Recently, FRET has been introduced into the single-molecule regime. 4,15 Weiss and co-workers have established a ratiometric method to study subpopulations of single DNA molecules. 19,20 The fluctuations in energy transfer have been also used by several groups; for examples, studying the conformational dynamics of single SNase protein molecules during catalysis, 3 the ligand-induced conformational changes in single RNA three- way junctions, 9 and the folding dynamics of single GCN-4 peptides. 10 Although single-molecule FRET has great potential as a tool for the investigation of biomolecular dynamics, care must be taken in attributing the change of FRET signal to the distance change between donor and acceptor. In single-molecule mea- surements, reasons for the fluctuation in energy transfer include: (1) reversible transitions to dark states; 6,21,22 (2) irreversible photobleaching; 1,15,23 (3) intersystem crossing to the triplet state; 24 (4) spectral diffusion; 25 (5) rotational dynamics of the dyes; 26,27 and (6) distance change between two dyes. 28 Therefore, it is essential to choose suitable control samples and examine both single-molecule and bulk measurements to fully understand the contributions from nondistance-change processes. Well-defined sequences of DNA oligomers can now be routinely synthesized and labeled at specific sites with one or more fluorescent molecules. Consequently, FRET has been extensively applied in determining the structure and conforma- tion of DNA molecules at an ensemble level. 29-35 However, there is evidence to suggest that when many of these systems are examined in greater detail, a more complex picture emerges. For example, Clegg and co-workers have shown that for carboxytetramethylrhodamine (TMR) linked to DNA, multiple conformations exist with different quantum yields. 36 In addition, single-molecule lifetime measurements by Rigler et al. on TMR-labeled double-stranded DNA show fluctuation between two distinct conformations, one in which the TMR fluorophore is interacting with a guanine base and the other in which it is not. 37 Conformational fluctuations of single-TMR-labeled DNA molecules have also found to be nonergodic. 38-40 These results suggest that fluorescent hetero- geneity may be an intrinsic property of dye-labeled DNA. Therefore, characterization of the fluorescence properties of dyes attached to DNA is important to ensure accurate interpretation of FRET data. Motivated by these observations, we set out to assess the capabilities and limitations of single-molecule FRET in a study of DNA. A Cy5-12mer-TMR DNA system was chosen as a simple model. Changes in the FRET of single DNA molecules diffusing in solution were monitored, and a general method for identifying single-molecule FRET events has been developed. The single-molecule experiments are accompanied by bulk FRET, lifetime, and anisotropy measurements to justify the assumptions needed to perform the single-molecule analysis. Experimental Section Materials. The 12-base oligonucleotides whose sequence is 5′-ACCTGCCGACGC-3′, with either carboxytetramethyl- * To whom correspondence should be addressed. (E-mail: dk10012@cam.ac.uk and sb10031@cam.ac.uk). 5171 J. Phys. Chem. B 2000, 104, 5171-5178 10.1021/jp993914k CCC: $19.00 © 2000 American Chemical Society Published on Web 05/03/2000