DNA bubbles kinetics using single-molecule Fluorescence Resonance Energy Transfer (FRET)
Initiations of RNA transcription or DNA replication is associated with the local melting, or "bubble formation" in the DNA duplex. The initiation occurs at DNA promoter sites that usually contain conserved sequences (such as "TATA" box in eukaryotes) and has been the subject of a number of theoretical studies. Bubble formation and its closure extend over a wide range of timescales from microseconds to seconds.
We use single molecule Fluorescence Resonance Energy Transfer (sm-FRET) to characterize and quantify DNA bubble kinetics (Figure 1). Surface immobilized DNA molecules internally labeled with donor-acceptor FRET pairs are interrogated using a custom-made confocal microscope with temporal resolution of ~10 µs (ref. 1 and 2). Figure 2 displays a 20 µm x 20 µm scan of a coverslip, where each red spot is due to an individual FRET pair. Our instrument is equipped with two lasers (green and red) which are used to excite the donor or the acceptor fluorophores simultaneously, or separately. This feature is used to distinguish dark state due to fluorophore blinking from real FRET variation due to distance fluctuations (read more about cyanine blinking in Ref 3). In addition, our setup is equipped with a precise sample temperature control (0.2 °C), and a continuous buffer exchange capability throughout the measurement period (Figure 3). Once an experiment has been set up, its execution is fully controlled by a custom LabView code.
DNA bubble formation is signaled by a change in the FRET efficiency. We show that by taking into account a number of experimental factors, mainly the relative quantum efficiencies of the donor to acceptor for each pair, and the channel-to-channel leakage, the FRET efficiency obtained at the single molecule level can be translated to the time-dependent distance between the donor and the acceptor. A typical trace is shown in Figure 4. We are currently studying the bubble kinetics as a function of DNA sequence, ionic strength and temperature.
- Sabanayagam, C.R., J.S. Eid and A. Meller. 2004. High-throughput scanning confocal microscope for single molecule analysis. Appl. Phys. Lett. 84 (7),1216-1218.
- Sabanayagam, C.R., J.S. Eid and A. Meller. 2005. Using fluorescence resonance energy transfer to measure distances along individual DNA molecules: Corrections due to nonideal transfer. J. Chem. Phys. 122 (6), 061103.
- Sabanayagam, C.R., J.S. Eid and A. Meller. 2005. Long time scale blinking kinetics of cyanine fluorophores conjugated to DNA and its effect on Forster resonance energy transfer. J. Chem. Phys. 123 (22), 224708.