M. J. Levene, W.W. Webb et al. Science (2003). Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations M. J. Levene, W.W. Webb et al. Science (2003). Julia Hoscheit March 11, 2004

Brief Outline of Presentation Introduction Describing zero-mode waveguides Simulations Applying the technology to DNA synthesis Conclusion

Reasons for studying single molecules Can reveal information about kinetic processes not normally accessible by ensemble measurements such as variances in kinetic rates Measurements can reveal the distribution of molecular properties in inhomogeneous systems Can visualize real-time observations of chemical reactions of biomolecules

Common Approaches Fluorescence correlation spectroscopy (FCS) – Based on the measurement of fluctuations of the fluorescence Direct observation of sparse molecules using diffraction-limited optics

What is a zero-mode waveguide? In this case, a waveguide is a hollow metal conductor (hole) that provides a path to guide a 488 nm laser. Single and multimode vs. zero-mode waveguide More biologically relevant

Apparatus for single-molecule enzymology using zero-mode waveguide

What the coverslip looks like

Simulations

Autocorrelation analysis Autocorrelation analysis is a very clever way of analyzing the measuring fluorescence data. G(τ) contains information about equilibrium concentrations, reaction kinetics and diffusion rates of molecules in the sample .

FCS Using Zero-mode Waveguides

Applying this Technology They used zero-mode waveguides to observe the enzymatic synthesis of dsDNA by DNA polymerase with the use of fluorescently tagged nucleotide analog coumarin-dCTP. They immobilized mutant T7 DNA polymerase in the waveguides

Applying This Technology cont.

Conclusions Studying single molecules can provide insight into the individual properties of the molecules that can be masked by ensemble averaging in experiments performed on several molecules. This technology will find wide applicability in FCS and other spectroscopic techniques and will allow the techniques to be performed at higher, more biologically relevant concentrations.

Discussion What other biochemical assays can be assessed with zero-mode waveguides? What are the limitations of this technology?

Observation Volume in a waveguide