In recent years, fluorescence-based single-molecule imaging techniques have been used to follow the action of macromolecular machines along a DNA substrate. Direct observations of DNA replication (1
), transcription (5
), and repair (8
)atthe single-molecule level are continuing to offer fresh insights into these complex, multi-step reactions. The knowledge gained from these studies typically could not be accessed using traditional biochemical or biophysical approaches. Single-molecule experiments offer the advantage of being able to study rare or short-lived intermediates that can be obfuscated among the often-heterogenous populations of molecules that are studied in traditional biochemical assays.
Despite decades of intense technique development, single-molecule observations of protein–DNA interactions continue to be experimentally challenging. The relatively short fluorescent lifetimes of most organic dyes significantly limit the accessible reaction timescales. The molecules under investigation usually must be anchored to a surface that is inherently different from the normal environments encountered within cells. In addition, experimental platforms that manipulate the target DNA via optical or magnetic tweezers are typically carried out on single DNA molecules in a serial fashion (i.e., one molecule at a time), and this low data throughout often limit the scope of the experimental results. In the protocol presented here, we describe a method for rapid, real-time imaging of hundreds of individual protein–DNA complexes over extended, biological timescales within a biologically friendly microenvironment. The method is flexible and can be used to address a number of different biological problems. We have successfully applied this experimental approach to observe the diffusion and translocation of DNA repair proteins (10
), the localization of nucleosomes along an intrinsic DNA-binding energy landscape (12
), and to follow the polymerization activity of recombinases on double-stranded DNA (13
In this protocol, we describe a nanofabricated, micro-fluidic system for simultaneous imaging of hundreds of DNA molecules in real time (). The DNA molecules are organized into “DNA curtains” on the surface of a micro-fluidic sample chamber that is otherwise coated with a fluid lipid bilayer. Various aspects of the DNA curtains technology have been presented previously (16
). Briefly, the experimental system consists of a total internal reflection fluorescence (TIRF) microscope builtaround an inverted Nikon TE2000 microscope. Laser illumination is provided by a ~200 mW 488 nm diode laser. The laser beam impinges on a DOVE prism atop a flowcell constructed from a silica microscope slide containing nanofabricated barriers to lipid diffusion (). An evanescent wave is generated at the water–silica interface, illuminating a shallow observation volume at the flowcell surface. Fluorescence from molecules immobilized at the surface (see below) is collected by a 60×, N.A.1.2 water immersion objective. The signal is passed through a holographic 488 nm notch filter and imaged on a back-thinned 512 × 512 pixel EM-CCD. For multi-color fluorescence imaging, the signal is passed through a DualView beam splitter and each color imaged on one half of the CCD chip.
Fig. 26.1 Schematic of the fluorescence microscope setup. The flowcell is placed on a microscope stage in an inverted configuration. A 488 nm laser impinges on a DOVE prism that rests atop the flowcell. Fluorescent signal is collected by a high N.A. objective and (more ...)
Fig. 26.2 Overview of electron beam lithography. (a) For e-beam lithography, the slide is first coated with PMMA, and a layer of Aquasave, and an electron beam rastered across the surface to burn through these layers creating a pattern that defines the shapes of (more ...)
The surface of the flowcell is passivated by a fluid lipid bilayer (20
). DNA is immobilized at the lipid bilayer by a streptavidin–biotin linkage and extended into the evanescent wave via shear buffer flow delivered by a syringe pump. The fluidity of the lipid bilayer permits organization of individual DNA molecules at nanofabricated diffusion barriers (). The spacing, density, and orientation of DNA molecules relative to one another may be controlled by appropriately designed diffusion barriers (17
). Recently, we have also extended the DNA curtain technology to generate DNA arrays that are immobilized at both ends (16
Fig. 26.3 Assembly of DNA curtains. (a) A schematic illustration of DNA molecules assembled into DNA curtains on a fluid lipid bilayer. DNA is tethered to the bilayer by a streptavidin–biotin linkage. In the presence of buffer flow, individual DNA molecules (more ...)
For our studies we label the proteins with highly fluorescent semiconductor nanocrystal quantum dots (QDs). Quantum dotsare relatively small (~10–20 nm diameter) nanoparticles that display broad excitation spectra, narrow emission peaks, large Stokes shifts, large absorbance cross sections, and very high quantum yields (23
). Individual QDs can be readily visualized at data collection rates of 100 frames/s and the QDs do not bleach even after prolonged illumination (23
). This allows imaging for extended periods (up to hours) without risk of photobleaching the sample. To specifically label a protein of interest, an epitope tag is engineered into the protein. Antibodies raised against the epitope tag are chemically linked to QDs and the QD–antibody complex is conjugated with the protein prior to visualization on the DNA curtain.
The DNA can be viewed by staining with very low concentrations (1–2 nM) of the intercalating dye YOYO1. To avoid rapid photobleaching of YOYO1 and concomitant DNA damage due to the reactivity of excited fluorophores with molecular oxygen, we employ an enzymatic oxygen scavenging system. The coupled activity of glucose oxidase and catalase in a buffer containing millimolar amounts of glucose significantly reduces DNA breaks and permits observations of individual molecules for tens of minutes (27
). Although this approach does not inhibit the biochemical activity of many enzymes (9
), care should be taken to biochemically assay all the protein–DNA interactions in the presence of YOYO1, as well as all additional buffer components. If necessary, YOYO1 may be used to stain the DNA at the beginning of an experiment and subsequently flushed out by washing the flowcell with a high-salt (500 mM NaCl or 10 mM MgCl2
) buffer. In addition, alternative labeling procedures that employ recognition of digoxigenin (DIG)-labeled DNA by anti-DIG antibody–QD conjugates have also been developed (13
). These labeling methods leave the duplex almost completely unperturbed and do not require intercalating DNA dyes or an oxygen scavenging system to visualize the DNA curtains.