Direct Visualization of Single-Molecule DNA-Binding Proteins Along DNA to Understand DNA–Protein Interactions

protein molecules along visualized. Some of studies investigated the effect of DNA tension on DNA–protein interactions. and incorporated magnetic tweezers another apparatus for single-molecule DNA manipulation, into the single-molecule fluorescence set-up with my research which has allowed us to visualize single-molecule DNA helicase interacting with single dsDNA that is tethered by the magnetic tweezers.


Fluorophores and fluorescence labeling methods
Direct visualization of protein dynamics by single-molecule fluorescence imaging requires labeling of target protein molecules with a fluorophore. To be used in single-molecule imaging, fluorophores must be (1) bright (have high extinction coefficients and high quantum yield), (2) photostable, and (3) relatively small so as not to perturb the functions of target protein molecules. With regard to photostability, fluorophores in single-molecule imaging tend to undergo photobleaching and blinking (repetitive fluorescence turning-on and -off) because of the high-power excitation. Agents that minimize such photophysical events have been reported (Aitken et al., 2008;Dave et al., 2009;Harada et al., 1990;Rasnik et al., 2006). In general, two classes of fluorophores are used for single-molecule fluorescence imaging: organic small-molecule fluorophores and quantum dots (Qdots) ( Table 1). Despite the advantage of the labeling capability with genetic engineering, fluorescent proteins are not popularly applied to single-molecule fluorescence imaging because of their lower intensity and instability of their fluorescence emission. This section briefly describes fluorescence properties of dyes and Qdots and their fluorescence labeling methods. www.intechopen.com

Organic small-molecule fluorophores (dyes)
Organic small-molecule fluorophores or dyes with a molecular weight < 1 kDa are mainly used for covalent labeling of protein molecules. Frequently used dyes in single-molecule imaging are Cy3, Cy5, and Alexa derivatives. They emit sufficeint photons to be detected, but subsequently photobleach in approximately 1 minute, and often exhibit blinking (Table 1).
The most popular site-specific labeling method with dyes involves labeling the sulfhydryl group of the cysteine residues with a dye containing a maleimide group. The double bond of the maleimide group may undergo an alkylation reaction with the sulfhydryl group to form a stable thioester bond. The reaction is specific for thiols in the physiological pH range of 6.5-7.5. At pH 7.0, the reaction proceeds 1,000 times faster than its reaction with amines (Hermanson, 2008). Another popular labeling method, which is less site-specific, involves labeling the amine group of amino acids in protein molecules with a dye containing an Nhydroxysuccinimide ester (NHS). NHS ester reacts principally with the ε-amines of the lysine side chains and α-amines at the N-terminals (Hermanson, 2008).

Quantum dots (Qdots)
Qdots are inorganic semiconductor nanocrystals, typically composed of a cadmium selenide (CdSe) core and a zinc sulphide (ZnS) shell measuring 10-20 nm in diameter (Michalet et al., 2005). They are commonly used in single-molecule imaging owing to their resistance to photobleaching and extreme brightness (Table 1). They are characterized by broad absorption profiles, high extinction coefficients, and narrow and spectrally tunable emission profiles, depending on their sizes. Although they are resistant to photobleaching, they often exhibit blinking, which seems to be related to charging of the nanocrystal upon excitation (Table 1). However, recently, the problem of blinking was reported to be overcome by the use of a nanocrystalline CdZnSe core capped with a ZnSe semiconductor shell, in which the transition between ZnSe and CdSe is not abrupt, but radially graded (Wang et al., 2009).
Qdots are usually labeled to protein molecules via immunolabeling. The most popular method is based on an avidin-biotin interaction, an antigen-antibody reaction with the highest affinity. In this method, target protein molecules, which are conjugated with a biotin by chemical crosslinking or genetic engineering, can be labeled with avidin-coated Qdots that are commercially available.

Advances in site-specific labeling methods
Recent advances in tagging technologies based on genetic have engineering enabled sitespecific targeting of fluorophores to protein molecules. One approach is to fuse the target protein molecule to a peptide or a protein recognition sequence, which then recruits a fluorophore coated with the proteins that have affinity for the specific peptide or the protein recognition sequence. For labeling of a Qdot used in single-molecule visualization, HA tag (Kad et al., 2010), Flag tag and HA tag , and His tag (Dunn et al., 2011) have been used. Enzyme-mediated covalent protein labeling is also used, in which a recognition peptide is fused to the protein of interest and a natural or engineered enzyme ligates the small-molecule probe to the recognition peptide. This approach can confer highly specific and rapid labeling, with the benefit of a small directing peptide sequence. This www.intechopen.com approach involved the following pairs: Halo tag and dehalogenase, biotin ligase acceptor peptide and biotin ligase, and SNAP tag and O 6 -alkylguanine-DNA alkyltransferase (Fernández-Suárez & Ting, 2008

Surface-coating methods
Minimization of the background noise arising from non-specific adsorption of the fluorescently labeled protein molecules on glass substrates is essential for their singlemolecule fluorescence visualization. The non-specifically adsorbed protein molecules severely interfere with the visualization because the fluorescence associated with these molecules confounds the fluorescence signal from the target protein molecules. Poly (ethylene glycol) (PEG) and lipid are commonly used for reducing the non-specific protein adsorption on the glass substrate.

Poly (Ethylene Glycol) (PEG)
PEG is a biocompatible polymer that exhibits protein and cell resistance when immobilized onto metal (Prime & Whitesides, 1991), plastic (Ito et al., 2007), and glass surfaces (Cuvelier et al., 2003), which serves many applications especially in biosensors and medical devices. The feature of PEG comes from its high hydrophilicity and appreciable chain flexibility that induce an effective exclusion volume effect (Harris, 1992). To my knowledge, Ha et al. first used PEG coated glass substrates for singlemolecule fluorescence imaging (Ha et al., 2002). The key factors affecting the suppression of nonspecific adsorption by PEG are its length and density (Harris, 1992), both of which are trade-offs. An increase in the chain length of PEG to construct a defined tethered chain layer results in a decrease in the density of the PEG chain due to the exclusion volume effect. PEG with a molecular weight of 5,000 appears to find a compromise between the trade-offs to maximize the suppression of non-specific adsorption of protein molecules on a glass surface (Heyes et al., 2004;Malmstena et al., 1998;McNamee et al., 2007;Pasche et www.intechopen.com al., 2003;Yang et al., 1999). A general approach to the immobilization of PEG onto a glass surface involves coupling of PEG to amine groups that have been conjugated on the surface by silanization (Figure 1a). Another approach involves coupling of PEG through poly (L-lysine) adsorption on the surface (Figure 1b). The inclusion of a small fraction of biotinylated PEG provides anchor points for tethering DNA (Section 4).

PEGylation through aminosilanization
The standard PEG coating method on quartz slides and silicate coverslips for singlemolecule fluorescence imaging is performed by silanization with N-2-(aminoethyl)-3aminopropyl-triethoxysilane in methanol containing acetic acid for few hours (Figure 1 (a)) (Joo et al., 2006). The aminosilanization treatment yields a surface that is densely coated with exposed primary amines. A covalently attached PEG layer is formed on the surfaces by a PEGylation reaction with the amine reactive N-hydroxy-succinimidyl (NHS)-PEG (M.W. = 5,000 Da) dissolved commonly in freshly prepared 0.1 M sodium bicarbonate buffer (pH 8.3) for a couple of hours.

Improvement of non-specific adsorption suppression capability on silicate coverslips
I found that the standard PEG coating method mentioned above did not suppress nonspecific adsorption as effectively on silicate coverslips as it did on quartz (Yokota et al., 2009). Therefore, an improved method for efficient PEG coating on silicates is required to reduce their non-specific adsorption. Then, I found that performing the PEGylation reaction in 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS, pH 7.5) instead of the usual 0.1 M sodium bicarbonate (pH 8.3) reduced non-specific adsorption on silicate surfaces by up to an order of magnitude. Figure 2 shows the single-molecule fluorescence images of a cyanine dye-labeled protein (the E. coli helicase UvrD (Lohman et al., 2008) labeled with Cy5: Cy5-UvrD) non-specifically adsorbed on a silicate coverslip that was PEGylated in MOPS buffer as compared to one with no treatment and one coated following the standard method. The improvement is crucial since silicate coverslips are extensively used for single-molecule fluorescence microscopy with epi-illumination (Funatsu et al., 1995), highly inclined thin illumination (Tokunaga et al., 2008), and totalinternal reflection fluorescence (Tokunaga et al., 1997) microscopies (sometimes combined with other techniques such as optical tweezers (Hohng et al., 2007;Lang et al., 2004;Zhou et al., 2011)). The improvement in non-specific adsorption using the new buffer condition for PEGylation was also observed on quartz, though to a lesser extent (Table 2) and with a Qdot-labeled protein molecule (UvrD labeled with Qdot655:Qdot655-UvrD) ( Table 3). Figure 3 shows the single-molecule fluorescence images of a Qdot-labeled protein (Qdot-UvrD) non-specifically adsorbed on a silicate coverslip that was PEGylated in MOPS buffer as compared to one with no treatment. While PEGylation is required to reduce the non-specific adsorption of Qdot-labeled protein on both silicate and quartz surfaces, PEGylation in MOPS buffer reduced non-specific adsorption of Qdot-UvrD on glass substrates by a factor of approximately 2 as compared to PEG coating in sodium bicarbonate buffer (Table 3). The key issue determining the non-adsorption properties of PEG-coated glass surfaces is their coverage by the polymer (Harris, 1992), which is improved with the new method. PEG coating at pH 7.5 may be enhanced with respect to coating at higher pH owing to better stability of the NHS ester. Its lifetime is in the order of hours at physiological pH and decreases steeply at higher pH owing to increased hydrolysis (Hermanson, 2008).       The results on the reduction of non-specific protein adsorption on glass surfaces indicate that our PEGylation method facilitates direct visualization of single-molecule interactions between fluorescently labeled protein molecules and DNA. Indeed, we could visualize how Cy5-UvrD interacts with a 18-bp dsDNA with or without an ssDNA tail immobilized on PEG-coated surfaces. The number of Cy5-UvrD fluorescent spots observed can be compared in Figure 4; this figure clearly shows that non-specific adsorption of Cy5-UvrD on the surface was effectively suppressed enough to conclude that Cy5-UvrD has higher affinity for the dsDNA with an ssDNA tail than for that without one; this finding is in agreement with that of a previous report (Maluf et al., 2003).

Single-molecule visualization of binding mode of helicase to DNA on silicate coverslips
Using microscopy, we could compare the number of UvrD molecules bound to 18-nt ssDNA, 4.7-kbp dsDNA, and 4.7-knt ssDNA immobilized on a PEG-coated silicate coverslip. Figure 5 shows a comparison of fluorescence intensity distributions of Cy5-UvrD bound to 18-nt ssDNA, 4.7-kbp dsDNA, or 4.7-knt ssDNA. In agreement with the results in Figure 4, Cy5-UvrD had low affinity for dsDNA, and thus, the fluorescence intensity distribution for 4.7-kbp dsDNA peaked at a fluorescence intensity that corresponds to that www.intechopen.com of single Cy5-UvrD, which was validated by the observation that most of the fluorescent spots photobleached in a single step. This was also the case with the experiment using 18-nt ssDNA. For the experiment using 4.7-knt ssDNA, the fluorescence intensity distribution shifted to a larger value, indicating that multiple Cy5-UvrD attached to the ssDNA.

Stability of PEG-coated surfaces
The long-term stability of PEG has been examined in several studies. On examination of a PEG-coated coverslip surface for up to 1 month, the surface, immersed in 0.1 M sodium phosphate buffer (pH 7.4), has been found to degrade after 25 days (Branch et al., 2001). Another study examined the stability of PEG-modified silicon substrates that were www.intechopen.com incubated in PBS (37 °C, pH 7.4, 5% CO 2 ) for different periods of time up to 28 days. This study also concluded that PEG-modified surfaces retain their protein and cell repulsive nature for the period of investigation, i.e., 28 days (Sharma et al., 2004). With regard to PEG stability in the air, no degradation was reported after 75 days (Anderson et al., 2008). In contrast to these macroscopic experimental studies, PEG-coated surfaces used for singlemolecule imaging imposes several challenges. The protein-non-adsorption capability of PEG-coated glass surfaces sharply deteriorated with time (incubation > 24 h) when incubated in a buffer (Yokota et al., 2009). Moreover, PEG-coated coverslips and quartz slides exhibited increasing non-specific adsorption with time when stored, exposed to the air, at room temperature (Yokota et al., 2009). PEG is known to degrade by oxidation (Han et al., 1997), and thus, to prevent the oxidative degradation, PEG-coated slides/coverslips can be stored in the dark under dry conditions at -20 °C until use (Joo et al., 2006).

PEGylation through poly(L-lysine) adsorption
PEGylation is also feasible through poly(L-lysine) adsorption on the glass surface ( Figure 1b). The amino groups of the side chain of poly(L-lysine) are positively charged at pH values below 10, and are therefore easily attached to the negatively charged glass surface (Iler, 1979) through an electrostatic interaction at physiological pH. The protein non-adsorption capability is comparable to that of PEG-coated surfaces through aminosilanization (unpublished data). A similar strategy for PEGylation, which is not used for single-molecule fluorescence imaging, involves using a poly(L-lysine) grafted with PEG (PLL-g-PEG), a polycationic copolymer that is positively charged at neutral pH. The compound spontaneously adsorbs from aqueous solution onto negatively charged surfaces, resulting in the formation of stable polymeric monolayers and rendering the surfaces protein-resistant to a degree relative to the PEG surface density (Huang et al., 2001;Pasche et al., 2003).

Lipid bilayer
Artificial lipid membranes deposited on solid supports have proven to be useful for many types of biochemical studies (Chan & Boxer, 2007). The membranes provide a surface environment that is similar to that inside a living cell and can prevent any non-specific interactions of biomolecules on the surface. Lipid bilayers that are spontaneously formed on quartz surfaces have been used for single-molecule fluorescence imaging. The bilayers can be modified through the incorporation of lipids with various functional groups such as biotin or PEG. A mixture of 1,2-dioleoyl-sn-glycerophosphocholine dioleoylphosphatidylcholine (DOPC) with a small amount of 1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N-[methoxy(polyethylene glycol)-550] (mPEG 550-DOPE) was reported to help minimize non-specific binding of Qdot-tagged proteins to the lipid bilayer . For experiments that require tethering DNA that is biotinylated at the end(s), avidin is attached either via non-specific interaction with the glass surface or via interaction with biotin head groups of the lipids present.

Platforms
A common feature among direct visualization of single-molecule DNA-binding protein molecules is the need for DNA to be tethered and extended. A pioneering platform www.intechopen.com published in 1993 employs dielectrophoresis between aluminium electrodes to extend DNA (Fig. 6g) (Kabata et al., 1993). DNA is a bacteriophage DNA that consists of 48,490 base pairs of double-stranded linear DNA with 12-nucleotide single-stranded segments at both 5′ ends (approximately 16.5 µm in total length). The platform can form "DNA belts" and has uncovered RNA polymerase dynamics along DNA, including sliding and jumping. Another pioneering platform published in 1998 employs optical tweezers, which was incorporated into a single-molecule fluorescence microscope, to extend DNA (Fig. 6d) (Harada et al., 1999), which revealed strain-dependent DNA-binding kinetics of RNA polymerase. At that time, casein, a conventional blocking agent that is found in milk in large quantities, was used to block non-specific protein adsorption. To minimize non-specific protein adsorption, most of the visualization has been performed on glass surfaces modified by the coating methods discussed in the previous section. Many platforms capable of real-time visualization of single-molecule DNA-binding protein molecules along tethered DNA, most of which are used in combination with total-internal fluorescence microscopy (Funatsu et al., 1995;Tokunaga et al., 1997), have been developed to understand protein dynamics. Among many methods for DNA tethering by non-specific adsorption, or specific attachments mediated by a non-covalent interaction, the most popular DNA tethering method used for the platforms is tethering biotinylated DNA on glass surfaces via an avidin-biotin interaction. The two single-stranded segments at both 5′ ends of DNA, termed the cos site, are the sticky ends and can be easily biotinylated via ligation with corresponding the complimentary biotinylated oligos. The biotinylated DNA can be tethered on glass surfaces at either one or both end(s) via an avidin-biotin interaction. In the following sections, I briefly review some of the modern platforms and present their target DNA protein molecules.

Platforms with continuous buffer flow
Tethering DNA at one end on the surface via an avidin-biotin interaction is the simplest method for tethering DNA. However, owing to the flexible nature of DNA, tethering alone cannot make the DNA extend sufficiently enough for use in single-molecule visualization with high spatial resolution. To stretch such DNA, some investigators use shear force generated by hydrodynamic flow (Fig. 6a) (Brewer & Bianco, 2008;Graneli et al., 2006;Greene & Mizuuchi, 2002;S. Kim et al., 2007;Lee et al., 2006;van Oijen et al., 2003). In this platform, buffer is continuously input into a flow cell made of sandwiched glass substrates. The glass substrates are coated with PEG (Blainey et al., 2006;Blainey et al., 2009;S. Kim et al., 2007) or a lipid bilayer (Graneli et al., 2006) to reduce non-specific adsorption of protein molecules. With this platform, dynamics of many single-molecule proteins have been uncovered. These include dynamics of single-protein polymers along DNA (Greene & Mizuuchi, 2002;Han & Mizuuchi, 2010;Tan et al., 2007) and one-dimensional diffusion of many DNA-binding protein molecules, for example, a base-excision DNA-repair protein along DNA (Blainey et al., 2006;Etson et al., 2010;Kochaniak et al., 2009;Komazin-Meredith et al., 2008;Tafvizi et al., 2011). A comparison of one-dimensional diffusion constants as a function of protein size with theoretical predictions indicates that DNA-binding proteins undergo rotation-coupled sliding along the DNA helix (Blainey et al., 2009).

Tethering DNA at multiple points on glass surfaces
As mentioned above, tethering DNA at one end on the surface alone is not sufficient for the single-molecule visualization. Several papers report methods of DNA tethering at many points via non-specific interaction or at both ends (Fig. 6b) on the surface via an avidinbiotin interaction. These include DNA immobilized on a polystyrene-coated coverslip via a non-specific interaction (Kim & Larson, 2007) or on a lipid bilayer-deposited fused silica slide via an avidin-biotin interaction (Graneli et al., 2006), and T7 bacteriophage DNA immobilized on a silanized coverslip via an avidin-biotin interaction (Bonnet et al., 2008). Using these platforms, one-dimensional diffusion and transcription of single T7 RNA polymerases along DNA (Kim & Larson, 2007) and sliding and jumping of EcoRV restriction enzyme (Bonnet et al., 2008) along DNA were visualized.

Single-tethered DNA curtains
To tether many DNA strands on the surface for a high-throughput single-molecule analysis, the Greene group has developed a new platform referred to as ''DNA curtains'' (Fig. 6e) (Fazio www.intechopen.com et al., 2008;Graneli et al., 2006;Visnapuu et al., 2008). This assay allows simultaneous study of up to hundreds of individual DNA strands anchored to a lipid bilayer and aligned with respect to one another within a single field-of-view with TIRFM. The DNA curtains were constructed in the following manner: (i) the surface of a glass slide was first mechanically etched to form lipid-diffusion barriers perpendicular to the direction of buffer flow. (ii) The flow cell was coated with a lipid bilayer by injecting lipid vesicles comprising DOPC, biotinylated lipids, and PEGylated lipids into the sample chamber (discussed in 3.2). (iii) After removing the excess vesicles by a buffer flush, neutravidin was injected into the flow cell. (iv) DNA that was biotinylated at one end was injected into the flow cell. The PEGylated lipid enhanced protein non-specific adsorption suppression capability on the surface. The barriers formed on the flow cell could not be traversed by the lipids, and thus, buffer flow extended the lipid-tethered DNA in the flow direction and confined the DNA within the evanescent field generated by total-internal reflection. This platform has been used to study a broad range of DNA-binding proteins that function in homologous recombination Prasad et al., 2007) and mismatch repair (Gorman et al., 2007).

Double-tethered DNA curtains
The DNA curtains require buffer flow to extend DNA for single-molecule visualization of protein dynamics along the DNA. The hydrodynamic force exerted by the buffer flow can potentially influence the behavior of protein molecules; this influence is expected to be proportional to the hydrodynamic radius of the protein molecules under observation (Gorman & Greene, 2008;Tafvizi et al., 2008). To circumvent such an issue, the Greene group has developed double-tethered DNA curtains without the need of buffer flow during the visualization (Fig. 6f)  . The group used electron beam lithography to fabricate diffusion barriers with nanoscale dimensions, which allows for much more precise control over both the location and lateral distribution of the DNA within the curtains. These patterns, which the group termed "DNA racks," comprise linear barriers to lipid diffusion along with arrays of metallic pentagons, which are used for the scaffold of antibodies to anchor DNA at one end. The lipid-tethered DNA is first aligned along the linear barriers; subsequently, the DNA is anchored on the pentagons positioned at a defined distance downstream from the linear barriers. Once aligned and anchored, the "doubletethered" DNA curtains maintain their extended form. This sophisticated platform has been applied to biological systems such as nucleosomes and chromatin remodeling (Fig. 7) (Finkelstein et al., 2010;.

DNA tightropes
Another platform that did not require buffer flow was developed for single-molecule visualization of protein dynamics along extended DNA. In this platform, DNA is suspended between poly (L-lysine)-coated microspheres non-specifically attached on a PEGylated surface to form "DNA tightropes" in a flow cell, allowing the DNA to maintain its extended form during the visualization (Fig. 6h) (Kad et al., 2010). Since DNA is negatively charged at physiological pH, DNA can attach to the positively charged poly (Llysine) that is coated on the microspheres via an electrostatic interaction. Injection of high concentration of DNA (1.6 nM) forms many DNA tightropes, allowing a high-throughput single-molecule analysis by simultaneous observation of multiple DNA-binding protein www.intechopen.com molecules. Since the DNA tightropes formed on the microspheres are off the glass surface, there is no interaction between the DNA and the glass surface, which may not interfere with the interactions with the protein molecules. Furthermore, the no buffer-flow condition enables the detection of various dynamic mechanisms of DNA repair proteins, including not only diffusion along DNA (Dunn et al., 2011) but also jumping from one DNA to another DNA (Fig. 8) (Kad et al., 2010).  (Kad et al., 2010), Copyright (2010, with permission from Elsevier).

Minimization of protein non-specific adsorption on microspheres by PEGylaton
One of the drawbacks of the DNA tightrope platform with poly (L-lysine)-coated microspheres is that many protein molecules are non-specifically adsorbed on the microspheres. Fluorescence from the Qdots that are labeled to the adsorbed protein www.intechopen.com molecules interferes with single-molecule fluorescence imaging. Kad et al. circumvented the interference by manually masking the bead portions on screen using an image analysis software before analysis (Kad et al., 2010). Here, I describe another tightrope platform developed to overcome the issue by PEGylation, in which the glass surface as well as microspheres are coated with biotinylated PEG. The excellent resistance of PEG to protein adsorption (discussed in 3.1) efficiently suppresses the non-specific adsorption of Qdotlabeled protein molecules on the microspheres. The biotin of the PEG allows the microspheres to be immobilized on the glass surface and biotinylated DNA to be tethered between the microspheres via an avidin-biotin interaction (Fig. 9). With this improved platform, dynamics of a DNA repair protein is currently being investigated.

DNA extended by single-molecule DNA manipulation
Technical advances in combining single-molecule fluorescence visualization with singlemolecule manipulation provide deeper insights into DNA-protein interactions. Some groups incorporated optical tweezers into a single-molecule fluorescence set-up (Fig. 6c,d) for studying DNA dynamics or DNA-protein interactions Comstock et al., 2011;Galletto et al., 2006;Harada et al., 1999;Hohng et al., 2007;Lang et al., 2004;van Mameren et al., 2009;Zhou et al., 2011). With such platforms, translocation , association and dissociation (Harada et al., 1999;Zhou et al., 2011), filament assembly (Galletto et al., 2006), and disassembly (van Mameren et al., 2009) of protein molecules along DNA were visualized. Some of these studies investigated the effect of DNA tension on DNA-protein interactions. Recently, my colleagues and I have incorporated magnetic tweezers (Allemand et al., 2007), another apparatus for single-molecule DNA manipulation, into the single-molecule fluorescence set-up through collaboration with my research colleagues, which has allowed us to visualize single-molecule DNA helicase interacting with single dsDNA that is tethered by the magnetic tweezers.

Outlook
I have provided an overview recent of advances in direct visualization of single-molecule DNA-binding proteins, especially of key techniques used for the visualization. Fluorescence labeling methods as well as modern platforms employing biocompatible surface-coating methods described here have allowed the dynamics of single-molecule DNA-binding proteins to be elucidated. Frequently used fluorophores, dyes and Qdots have allowed visualization to a certain extent. However, the fluorophores have not satisfied all the requirements related to optimum size and photophysical properties, for example, dyes are small but short lived whereas Qdots are bright and resistant to photobleaching, but relatively large in size. Thus, it is highly desirable that new all-round fluorophores be synthesized or extracted from materials. One such promising fluorophore is the nitrogen-vacancy center, a crystal defect in diamonds (Aharonovich et al., 2011). The defect that emits near-infrared fluorescence exhibits neither blinking nor photobleaching, which is distinct from the common fluorophores.
Technical advances in combining single-molecule fluorescence visualization with singlemolecule manipulation (discussed in 4.5) will provide deeper insights into DNA-protein interactions. Zero-mode waveguides (Levene et al., 2003), a nano-hole (<100 nm in diameter) array, is another promising microscopy for visualizing DNA-protein interactions. The waveguides attract increasing attention because compared to TIRFM, zero-mode waveguides make it possible to perform single-molecule fluorescence imaging under higher ligand concentration (µM range) conditions due to its extremely small excitation volume. And thus, a few biological processes were visualized at single-molecule resolution by zeromode waveguides (Miyake et al., 2008;Sameshima et al., 2010;Uemura et al., 2010).
In a recent study (Kad et al., 2010), single-molecule interaction of two different DNA protein molecules along DNA was visualized by labeling them with different fluorophores, although most studies typically focused on the dynamics of single DNA binding-proteins. In the future, simultaneous visualization of different DNA-binding protein molecules may be possible by labeling the molecules with different fluorophores. This will help elucidate fundamental processes of DNA metabolism. In fact, biological processes in DNA metabolism involve various protein molecules as a form of macromolecular complexes. Moreover, the processes can be associated with chromatin for eukaryotes. To approach the protein dynamics in such an environment, a recent study investigated single-molecule interaction between a protein complex and chromatin . Future developments in various fields, including platforms, fluorophores, surface coating, and fluorescence labelling, will provide fruitful information on complex DNA-protein interactions that cannot otherwise be explored.

Acknowledgments
I would like to thank the Harada group at Kyoto University and the Bensimon & Croquette group at École Normale Supérieure for supporting my research. The presented work performed in the groups was partly funded by JSPS and JST.