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In this study, we specifically visualized DNA molecules at their AT base pairs after in vitro phage ejection. Our AT-specific visualization revealed that either end of the DNA molecule could be ejected first with a nearly 50% probability. This observation challenges the generally accepted theory of Last In First Out (LIFO), which states that the end of the phage λ DNA that enters the capsid last during phage packaging is the first to be ejected, and that both ends of the DNA are unable to move within the extremely condensed phage capsid. To support our observations, we conducted computer simulations that revealed that both ends of the DNA molecule are randomized, resulting in the observed near 50% probability. Additionally, we found that the length of the ejected DNA by LIFO was consistently longer than that by First In First Out (FIFO) during in vitro phage ejection. Our simulations attributed this difference in length to the stiffness difference of the remaining DNA within the phage capsid. In conclusion, this study demonstrates that a DNA molecule within an extremely dense phage capsid exhibits a degree of mobility, allowing it to switch ends during ejection.

Streptavidin-fluorescent proteins (SA-FPs) are a versatile tool to visualize a broad range of biochemical applications on a fluorescence microscope. Although the avidin-biotin interaction is widely used, the use of SA-FPs has not been applied to single-molecule DNA visualization. Here, we constructed 12 bright SA-FPs for DNA staining or labeling reagents. To date, 810 FPs are available, many of which are brighter than organic dyes. In this study, 12 bright FPs were selected to construct SA-FP plasmids covering green to red colors. Their brightness ranges from 40 to 165 mM

DNA binding fluorescent proteins are a powerful tool for single-molecule visualization. In this chapter, we discuss a protocol for the synthesis of DNA binding fluorescent proteins and visualization of single DNA molecules. This chapter includes stepwise methods for molecular cloning, reversible staining, two-color staining, sequence-specific staining, and microscopic visualization of single DNA molecules in a microfluidic device. This content will be useful for DNA characterization using DNA binding fluorescent proteins and its visualization at the single-molecule level.

DNA curtain is a high-throughput system, integrating a lipid bilayer, fluorescence imaging, and microfluidics to probe protein-DNA interactions in real-time and has provided in-depth understanding of DNA metabolism. Especially, the microfluidic platform of a DNA curtain is highly suitable for a biochip. In the DNA curtain, DNA molecules are aligned along chromium nanobarriers, which are fabricated on a slide surface, and visualized using an intercalating dye, YOYO-1. Although the chromium barriers confer precise geometric alignment of DNA, reuse of the slides is limited by wear of the barriers during cleaning. YOYO-1 is rapidly photobleached and causes photocleavage of DNA under continuous laser illumination, restricting DNA observation to a brief time window. To address these challenges, we developed a new nanopatterned slide, upon which carved nanotrenches serve as diffusion barriers. The nanotrenches were robust under harsh cleaning conditions, facilitating the maintenance of surface cleanliness that is essential to slide reuse. We also stained DNA with a fluorescent protein with a DNA-binding motif, fluorescent protein-DNA binding peptide (FP-DBP). FP-DBP was slowly photobleached and did not cause DNA photocleavage. This new DNA curtain system enables a more stable and repeatable investigation of real-time protein-DNA interactions and will serve as a good platform for lab-on-a-chip.

Microscopic visualization of DNA molecules is a simple, intuitive, and powerful method. Nonetheless, DNA-molecule quantification methods that employ microscopic visualization have not been reported so far. In this study, a new quantitative approach is presented that enables the counting of individual DNA molecules that have been rendered visible by fluorescence microscopy. Toward this, a microfluidic device was employed that directed DNA molecules into microchannels and deposited the molecules onto a positively charged surface. This microfluidic device had a vertically tapered channel inlet structure that prevented the accumulation of excess DNA molecules in the channel inlet while creating a tapering flow, thereby ensuring the even distribution of the DNA molecules in the microchannels. The channel heights and the density of positive charges on the surface were optimized for analysis. The linearity of this method with respect to the determination of the concentration of DNA in solutions was subsequently determined. The limit of detection was 0.48 fg/μL, which corresponds to 64 molecules of 7.25 kbp DNA in 1 μL of sample. This quantitative approach was finally used to count two types of plasmids co-transformed in an E. coli cell; a measurement that is typically considered challenging with gel electrophoresis.

DNA binding fluorescent proteins are useful probes for a broad range of biological applications. Fluorescent protein (FP)-tagging allows DNA binding proteins expressed within a living cell to be directly visualised, in real-time, to study DNA binding patterns and dynamics. Moreover, FP-tagged DNA binding proteins (FP-DBP) have allowed the imaging of single proteins bound to large elongated DNA molecules with a fluorescence microscope. Although there are numerous DNA binding proteins, only a small portion of them have been exploited to construct FP-DBPs to study molecular motion in a cell or in vitro single-molecule visualisation. Therefore, it would be informative to review FP-DBP for further development. Here, we summarise the design of FP-DBPs and their brightness, photostability, pKa, maturation rate, and binding affinity (Kd) characteristics. Then, we review the applications of FP-DBP in cells to study chromosome dynamics, DNA replication, transcription factors, DNA damage, and repair. Finally, we focus on single DNA molecule visualisation using FP-DBP.

Significance Repeated sequences make up approximately two-thirds of the human genome, which become fully accountable when very large DNA molecules are analyzed. Long, single DNA molecules are problematic using common experimental techniques and fluidic devices because of mechanical considerations that include breakage, dealing with the massive size of these coils, or the huge length of stretched DNAs. Accordingly, we harness analyte “issues” as exploitable advantages by invention and characterization of the “molecular gate,” which controls and synchronizes formation of stretched molecules as DNA dumbbells within nanoslit geometries that may also offer new routes to separation. This was accomplished by theoretical studies and experiments leveraging a series of electrical forces acting on DNA molecules, device walls, and the fluid flows within our devices., Very large DNA molecules enable comprehensive analysis of complex genomes, such as human, cancer, and plants because they span across sequence repeats and complex somatic events. When physically manipulated, or analyzed as single molecules, long polyelectrolytes are problematic because of mechanical considerations that include shear-mediated breakage, dealing with the massive size of these coils, or the length of stretched DNAs using common experimental techniques and fluidic devices. Accordingly, we harness analyte “issues” as exploitable advantages by our invention and characterization of the “molecular gate,” which controls and synchronizes formation of stretched DNA molecules as DNA dumbbells within nanoslit geometries. Molecular gate geometries comprise micro- and nanoscale features designed to synergize very low ionic strength conditions in ways we show effectively create an “electrostatic bottle.” This effect greatly enhances molecular confinement within large slit geometries and supports facile, synchronized electrokinetic loading of nanoslits, even without dumbbell formation. Device geometries were considered at the molecular and continuum scales through computer simulations, which also guided our efforts to optimize design and functionalities. In addition, we show that the molecular gate may govern DNA separations because DNA molecules can be electrokinetically triggered, by varying applied voltage, to enter slits in a size-dependent manner. Lastly, mapping the Mesoplasma florum genome, via synchronized dumbbell formation, validates our nascent approach as a viable starting point for advanced development that will build an integrated system capable of large-scale genome analysis.

Large DNA molecules are a promising platform for in vitro single-molecule biochemical analysis to investigate DNA-protein interactions by fluorescence microscopy. For many studies, intercalating fluorescent dyes have been primary DNA staining reagents, but they often cause photo-induced DNA breakage as well as structural deformation. As a solution, we previously developed several fluorescent-protein DNA-binding peptides or proteins (FP-DBP) for reversibly staining DNA molecules without structural deformation or photo-induced damage. However, they cannot stain DNA in a condition similar to a physiological salt concentration that most biochemical reactions require. Given these concerns, here we developed a salt-tolerant FP-DBP: truncated transcription activator-like effector (tTALE-FP), which can stain DNA up to 100 mM NaCl. Moreover, we found an interesting phenomenon that the tTALE-FP stained DNA evenly in 1 × TE buffer but showed AT-rich specific patterns from 40 mM to 100 mM NaCl. Using an assay based on fluorescence resonance energy transfer, we demonstrated that this binding pattern is caused by a higher DNA binding affinity of tTALE-FP for AT-rich compared to GC-rich regions. Finally, we used tTALE-FP in a single molecule fluorescence assay to monitor real-time restriction enzyme digestion of single DNA molecules. Altogether, our results demonstrate that this protein can provide a useful alternative as a DNA stain over intercalators.

Large DNA molecules have been utilized as a model system to investigate polymer physics. However, DNA visualization via intercalating dyes has generated equivocal results due to dye-induced structural deformation, particularly unwanted unwinding of the double helix. Thus, the contour length increases and the persistence length changes so unpredictably that there has been a controversy. In this paper, we used TAMRA-polypyrrole to stain single DNA molecules. Since this staining did not change the contour length of B-form DNA, we utilized TAMRA-polypyrrole stained DNA as a tool to measure the persistence length by changing the ionic strength. Then, we investigated DNA stretching in nanochannels by varying the ionic strength from 0.06 mM to 47 mM to evaluate several polymer physics theories proposed by Odijk, de Gennes and recent papers to deal with these regimes.

The recent advances in the single cell genome analysis are generating a considerable amount of novel insights into complex biological systems. However, there are still technical challenges because each cell has a single copy of DNA to be amplified in most single cell genome analytical methods. In this paper, we present a novel approach to directly visualize a genomic map on a large DNA molecule instantly stained with red and green DNA-binding fluorescent proteins without DNA amplification. For this visualization, we constructed a few types of fluorescent protein-fused DNA-binding proteins: H-NS (histone-like nucleoid-structuring protein), DNA-binding domain of BRCA1 (breast cancer 1), high mobility group-1 (HMG), and lysine tryptophan (KW) repeat motif. Because H-NS and HMG preferentially bind A/T-rich regions, we combined A/T specific binder (H-NS-mCherry and HMG-mCherry as red color) and a non-specific complementary DNA binder (BRCA1-eGFP and 2(KW)2-eGFP repeat as green color) to produce a sequence-specific two-color DNA physical map for efficient optical identification of single DNA molecules.

Large DNA molecules were visualized with novel fluorescent protein–DNA binding peptides (FP–DBPs). We constructed FP–DBPs by linking fluorescent protein to the N- or C-terminal of one or two peptides. We designed these DNA binding peptides from various DNA binding motifs such as oligo-lysine (K) and Lys-Trp (KW) repeats, TPKRPRGRPKK from high mobility group (HMG) chromosomal protein, and Ser-Pro-Arg-Lys (SPRK) from histone protein. We demonstrated the use of FP–DBP to stain large DNA molecules, and then analysed the fluorescence brightness and their binding affinity to double stranded DNA. This investigation provided HMG-tagged FP–DBP as the best DNA staining reagent in terms of fluorescence intensity, signal-to-noise ratio, and DNA binding affinity (Kd = 586 nM). Furthermore, we measured elongation of FP–DBP-stained DNA molecules tethered on the surface in order to evaluate FP–DBP-induced structural deformation.

FRET sensors based on fluorescent proteins have been powerful tools for probing protein–protein interactions and structural changes within proteins. However, they are intrinsically limited by their large size and the requirement for N- or C-terminal fusions. In this report, a FRET-based sensor was developed by incorporating a fluorescent unnatural amino acid into glutamine-binding protein (GlnBP), which formed a FRET pair with green fluorescent protein (GFP). GFP was fused to the N-terminus of GlnBP, and the fluorescent unnatural amino acid was incorporated into the site for N138 in GlnBP. FRET sensors were tested that contained linkers of different length between GFP and GlnBP, and assay conditions were optimized by changing the pH and salt concentration of the assay buffer. Under optimal conditions, the best sensor protein produced a 1.9-fold increase in the FRET ratio upon L-Gln binding, whereas either no change or minimal change was seen using other amino acids, including the other 19 natural amino acids and D-Gln. This novel design strategy for FRET sensors overcomes the limitations of current FRET sensors, which require the use of two fluorescent proteins. Consequently, our strategy may prove useful for investigating protein–protein interactions and for probing changes in protein conformation.

Here, we demonstrate the use of MALDI-TOF as a fast and simple analytical approach to evaluate the DNA-binding capability of various peptides. Specifically, by varying the amino acid sequence of the peptides consisting of lysine (K) and tryptophan (W), we identified peptides with strong DNA-binding capabilities using MALDI-TOF. Mass spectrometric analysis reveals an interesting novel finding that lysine residues show sequence selective preference, which used to be considered as mediator of electrostatic interactions with DNA phosphate backbones. Moreover, tryptophan residues show higher affinity to DNA than lysine residues. Since there are numerous possible combinations to make peptide oligomers, it is valuable to introduce a simple and reliable analytical approach in order to quickly identify DNA-binding peptides.

Long and linear DNA molecules are the mainstream single-molecule analytes for a variety of biochemical analysis within microfluidic devices, including functionalized surfaces and nanostructures. However, for biochemical analysis, large DNA molecules have to be unraveled, elongated, and visualized to obtain biochemical and genomic information. To date, elongated DNA molecules have been exploited in the development of a number of genome analysis systems as well as for the study of polymer physics due to the advantage of direct visualization of single DNA molecule. Moreover, each single DNA molecule provides individual information, which makes it useful for stochastic event analysis. Therefore, numerous studies of enzymatic random motions have been performed on a large elongated DNA molecule. In this review, we introduce mechanisms to elongate DNA molecules using microfluidics and nanostructures in the beginning. Secondly, we discuss how elongated DNA molecules have been utilized to obtain biochemical and genomic information by direct visualization of DNA molecules. Finally, we reviewed the approaches used to study the interaction of proteins and large DNA molecules. Although DNA-protein interactions have been investigated for many decades, it is noticeable that there have been significant achievements for the last five years. Therefore, we focus mainly on recent developments for monitoring enzymatic activity on large elongated DNA molecules.

In the present study, we proposed a simple ESI-MS model for determining Zn 2+ binding (or dissociation) constants forzinc finger peptides (ZFPs) with a unique ββα fold consensus. The ionization efficiency (response) factors for this model, i.e., α andβ, could be determined for ZiCo ZFP with a known Zn 2+ binding constant. We could determine the binding constants for other ZFPsassuming those with a ββα consensus conformation have the same α/β response ratio. In general, the ZPF dissociation constantsexhibited K d values of 10 -7 ~10 -9 M, while K d values for a negative control non-specific Zn 2+ peptides were high, e.g., 5.5 ×10 -6 M and4.3×10 -4 M for BBA1 and melittin, respectively.Key words : zinc finger, binding constant, electrospray-mass spectrometry, zinc ion Introduction Since its discovery in transcription factor IIIA (TFIIIA)from Xenopuslaevis, zinc finger proteins (ZFPs) have beenextensively researched due to their implications foreukaryotic protein-nucleic acid interactions.

Bioorthogonal metabolic DNA labelling with fluorochromes is a powerful strategy to visualize DNA molecules and their functions. Herein, we report the development of a new DNA metabolic labelling strategy enabled by the catalyst-free bioorthogonal ligation using vinyl thioether modified thymidine and o-quinolinone quinone methide. With the newly designed vinyl thioether-modified thymidine (VTdT), we added labelling tags on cellular DNA, which could further be linked to fluorochromes in cells. Therefore, we successfully visualized the DNA localization within cells as well as single DNA molecules without other staining reagents. In addition, we further characterized this bioorthogonal DNA metabolic labelling using DNase I digestion, MS characterization of VTdT as well as VTdT-oQQF conjugate in cell nuclei or mitochondria. This technique provides a powerful strategy to study DNA in cells, which paves the way to achieve future spatiotemporal deciphering of DNA synthesis and functions.

Although carbon nanotubes (CNTs) are remarkable materials with many exceptional characteristics, their poor chemical functionality limits their potential applications. Herein, a strategy is proposed for functionalizing CNTs, which can be achieved with any functional group (FG) without degrading their intrinsic structure by using a deoxyribonucleic acid (DNA)-binding peptide (DBP) anchor. By employing a DBP tagged with a certain FG, such as thiol, biotin, and carboxyl acid, it is possible to introduce any FG with a controlled density on DNA-wrapped CNTs. Additionally, different types of FGs can be introduced on CNTs simultaneously, using DBPs tagged with different FGs. This method can be used to prepare CNT nanocomposites containing different types of nanoparticles (NPs), such as Au NPs, magnetic NPs, and quantum dots. The CNT nanocomposites decorated with these NPs can be used as reusable catalase-like nanocomposites with exceptional catalytic activities, owing to the synergistic effects of all the components. Additionally, the unique DBP-DNA interaction allows the on-demand detachment of the NPs attached to the CNT surface; further, it facilitates a CNT chirality-specific NP attachment and separation using the sequence-specific programmable characteristics of oligonucleotides. The proposed method provides a novel chemistry platform for constructing new functional CNTs suitable for diverse applications.

We present nanoslit confined DNA conformations at very low ionic strengths and a theory to explain most measurements for single DNA molecule size under strong nanoslit confinement. Very low ionic strength conditions not only increase the DNA persistence length dramatically, but also cause DNA molecules to swell to the extent that the effective diameter of DNA becomes larger than the nanoslit height. By accounting for these effects, our results and theory provide a reasonable clue for a current controversy regarding the dependence of the DNA conformation on slit height (h), persistence length (p), and effective diameter (w).

Various recent experimental observations indicate that growing cells on engineered materials can alter their physiology, function, and fate. This finding suggests that better molecular-level understanding of the interactions between cells and materials may guide the design and construction of sophisticated artificial substrates, potentially enabling control of cells for use in various biomedical applications. In this review, we introduce recent research results that shed light on molecular events and mechanisms involved in the interactions between cells and materials. We discuss the development of materials with distinct physical, chemical, and biological features, cellular sensing of the engineered materials, transfer of the sensing information to the cell nucleus, subsequent changes in physical and chemical states of genomic DNA, and finally the resulting cellular behavior changes. Ongoing efforts to advance materials engineering and the cell–material interface will eventually expand the cell-based applications in therapies and tissue regenerations.

A vertically aligned anatase TiO2 (A-TiO2) nanotube array has been fabricated by coating a ZnO nanorod (NR) template with a TiO2 precursor solution. After coating, the ZnO NR cores were selectively etched in an acidic environment to form TiO2 nanotubes (NTs). More specifically, after growing the ZnO NRs via a hydrothermal method, one drop of the TiO2 precursor solution was cast to coat the ZnO NRs, the tops of which were previously covered with chemical capping materials by electrostatic interaction, and then the sample was sintered. Finally, the sample was immersed in an acidic solution resulting in selective etching of the ZnO NR cores. Thus, only TiO2 NTs remained on the substrate. The capping material is effectively used to create a perfect, hexagonal open-ended TiO2 NT array, which interestingly extends onset absorption towards the visible region.

Arabidopsis thaliana gene At5g06450 encodes a putative DnaQ-like 3'-5' exonuclease domain-containing protein (AtDECP). The DnaQ-like 3'-5' exonuclease domain is often found as a proofreading domain of DNA polymerases. The overall structure of AtDECP adopts an RNase H fold that consists of a mixed β-sheet flanked by α-helices. Interestingly, AtDECP forms a homohexameric assembly with a central six fold symmetry, generating a central cavity. The ring-shaped structure and comparison with WRN-exo, the best structural homologue of AtDECP, suggest a possible mechanism for implementing its exonuclease activity using positively charged patch on the N-terminal side of the homohexameric assembly. The homohexameric structure of AtDECP provides unique information about the interaction between the DnaQ-like 3'-5' exonuclease and its substrate nucleic acids.

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

Synthesis of smooth and continuous DNA nanowires, preserving the original structure of native DNA, and allowing its analysis by scanning electron microscope (SEM), is demonstrated. Gold nanoparticles densely assembled on the DNA backbone via thiol-tagged DNA binding peptides work as seeds for metallization of DNA. This method allows whole analysis of DNA molecules with entangled 3D features.

Consumption of alcohol injures DNA, and such damage is considered to be a primary cause for the development of cancer and many other diseases essentially due to reactive oxygen species generated from alcohol. To sensitively detect alcohol-induced DNA lesions in a biological system, we introduced a novel analytical platform for visualization of single genomic DNA molecules using E. coli. By fluorescently labelling the DNA lesions, our approach demonstrated, with the highest sensitivity, that we could count the number of DNA lesions induced by alcohol metabolism in a single bacterial cell. Moreover, our results showed a linear relationship between ethanol concentration and the number of DNA lesions: 0.88 lesions per 1% ethanol. Using this approach, we quantitatively analysed the DNA damage induced by exposure to alcoholic beverages such as beer (5% ethanol), rice wine (13%), soju (20%), and whisky (40%).

We present a molecular dynamics simulation study that focuses on the formation and growth of nanoscale droplets inside polymer networks. Droplet formation and growth are investigated by the liquid-vapor phase separation of a dilute Lennard-Jones (LJ) fluid inside regularly crosslinked, polymer networks with varying mesh sizes. In a polymer network with small mesh sizes, droplet formation can be suppressed, the extent of which is dependent on the attraction strength between the LJ particles. When droplets form in a polymer network with intermediate mesh sizes, subsequent growth is significantly slower when compared with that in bulk without a polymer network. Interestingly, droplet growth beyond the initial nucleation stage occurs by different mechanisms depending on the mesh size: droplets grow mainly by diffusion and coalescence inside polymer networks with large mesh sizes (as observed in bulk), whereas Ostwald ripening becomes a more dominant mechanism for droplet growth for small mesh sizes. The analysis of droplet trajectories clearly reveals the obstruction effect of the polymer network on the movement of growing droplets, which leads to Ostwald ripening of droplets. This study suggests how polymer networks can be used to control the growth of nanoscale droplets.

The formation of zinc finger peptideZn 2+ complexes in electrospray ionization mass spectrometry (ESI-MS) was examined using three different metal ion sources: ZnCl2, Zn(CH3COO)2, and Zn(OOC(CHOH)2COO). For the four zinc finger peptides (Sp1-1, Sp1-3, CF2II-4, and CF2II-6) that bind only a single Zn 2+ in the native condition, electrospray of apo-zinc fin- ger in solution containing ZnCl2 or Zn(CH3COO)2 resulted in the formation of zinc fingerZn 2+ complexes with multiple zinc ions. This result suggests the formation of nonspecific zinc fingerZn 2+ complexes. Zn(tartrate), Zn(OOC(CHOH)2COO), mainly produced specific zinc fingerZn 2+ complexes with a single zinc ion. This study clearly indicates that tartrate is an excel- lent counter ion in ESI-MS studies of zinc fingerZn 2+ complexes, which prevents the formation of nonspecific zinc fingerZn 2+ complexes.

Soft lithography of polydimethyl-siloxane (PDMS), an elastomeric polymer, has enabled rapid and inexpensive fabrications of microfluidic devices for various biochemical and bioanalytical applications. However, fabrications of nanostructured PDMS components such as nanoslits remain extremely challenging because of deformation of PDMS material. One of the well-known issues is the unwanted contact between the surfaces of PDMS roof and bottom substrate, called 'roof collapse'. Here we have developed a novel approach for the facile stabilization of PDMS nanoslits in the low height (130 nm)/width (100 μm) ratio without roof-collapse. Within 130 nm high nanoslits, we demonstrate the confinement of single DNA molecules. We believe that this approach will serve as a key to utilize PDMS as nanoslits for integrated microfluidic devices.

Molecular confinement offers new routes for arraying large DNA molecules, enabling single-molecule schemes aimed at the acquisition of sequence information. Such schemes can rapidly advance to become platforms capable of genome analysis if elements of a nascent system can be integrated at an early stage of development. Integrated strategies are needed for surmounting the stringent experimental requirements of nanoscale devices regarding fabrication, sample loading, biochemical labeling, and detection. We demonstrate that disposable devices featuring both micro- and nanoscale features can greatly elongate DNA molecules when buffer conditions are controlled to alter DNA stiffness. Furthermore, we present analytical calculations that describe this elongation. We also developed a complementary enzymatic labeling scheme that tags specific sequences on elongated molecules within described nanoslit devices that are imaged via fluorescence resonance energy transfer. Collectively, these developments enable scaleable molecular confinement approaches for genome analysis.

We present a single molecule visualization approach for the quantitative analysis of reactive oxygen species (ROS) induced DNA damage, such as base oxidation and single stranded breaks in large DNA molecules. We utilized the Fenton reaction to generate DNA damage with subsequent enzymatic treatment using a mixture of three types of glycosylases to remove oxidized bases, and then fluorescent labeling on damaged lesions via nick translation. This single molecule analytical platform provided the capability to count one or two damaged sites per λ DNA molecule (48.5 kb), which were reliably dependent on the concentrations of hydrogen peroxide and ferrous ion at the micromolar level. More importantly, the labeled damaged sites that were visualized under a microscope provided positional information, which offered the capability of comparing DNA damaged sites with the in silico genomic map to reveal sequence specificity that GTGR is more sensitive to oxidative damage. Consequently, single DNA molecule analysis provides a sensitive analytical platform for ROS-induced DNA damage and suggests an interesting biochemical insight that the genome primarily active during the lysogenic cycle may have less probability for oxidative DNA damage.

The genetic incorporation of unnatural amino acids (UAAs) into proteins has been a useful tool for protein engineering. However, most UAAs are expensive, and the method requires a high concentration of UAAs, which has been a drawback of the technology, especially for large-scale applications. To address this problem, a method to recycle cultured UAAs was developed. The method is based on recycling a culture medium containing the UAA, in which some of essential nutrients were resupplemented after each culture cycle, and induction of protein expression was controlled with glucose. Under optimal conditions, five UAAs were recycled for up to seven rounds of expression without a decrease in expression level, cell density, or incorporation fidelity. This method can generally be applied to other UAAs; therefore, it is useful for reducing the cost of UAAs for genetic incorporation and helpful for expanding the use of the technology to industrial applications.

Single DNA Molecule Swollen and Trapped in NanoslitDirect visualization of individual DNA molecules confined within a nano/microfluidic geometry has provided a new route for the study of polymer physics. Molecular confinement has been utilized to evaluate theoretical predictions developed over several decades. Particularly, nanoslit confined DNA molecules are an interesting topic to show the conformational transition from 3D to 2D depending on nanoslit heights. Although several experiments have recently reported the dependence of DNA conformation on nanoslit confinement, a controversy still remains because the results have not been consistent with one another nor with theoretical predictions. Here we present nanoslit confined DNA conformations at various low ionic strength conditions down to 0.18 mM, which increase the repulsion between DNA−DNA and DNA−nanoslit. Moreover, we derived a new theoretical explanation for most measurements of single DNA molecule size under strong nanoslit confinement. Our findings demonstrates that very low ionic strength conditions not only increase the DNA persistence length dramatically, but also cause DNA molecules to swell to the extent that the effective diameter of DNA becomes larger than the nanoslit height (h < w), which can be considered as electrostatic trapping to confine molecules to geometry smaller than the physical boundaries. Consequently, by accounting for these effects, our results and theory provide a reasonable solution for a current controversy regarding the dependence of the DNA conformation on slit height (h), persistence length (p), and effective diameter (w).1. Lee J, Kim S, Jeong H, Jung GY, Chang R, Chen YL, Jo, K (2014) Nanoslit Confined DNA at Low Ionic Strengths. ACS Macro Letters 3(9): 926-930

Long and linear DNA molecules are the mainstream single-molecule analytes for a variety of biochemical analysis within microfluidic devices, including functionalized surfaces and nanostructures. However, for biochemical analysis, large DNA molecules have to be unraveled, elongated, and visualized to obtain biochemical and genomic information. To date, elongated DNA molecules have been exploited in the development of a number of genome analysis systems as well as for the study of polymer physics due to the advantage of direct visualization of single DNA molecule. Moreover, each single DNA molecule provides individual information, which makes it useful for stochastic event analysis. Therefore, numerous studies of enzymatic random motions have been performed on a large elongated DNA molecule. In this review, we introduce mechanisms to elongate DNA molecules using microfluidics and nanostructures in the beginning. Secondly, we discuss how elongated DNA molecules have been utilized to obtain biochemical and genomic information by direct visualization of DNA molecules. Finally, we reviewed the approaches used to study the interaction of proteins and large DNA molecules. Although DNA-protein interactions have been investigated for many decades, it is noticeable that there have been significant achievements for the last five years. Therefore, we focus mainly on recent developments for monitoring enzymatic activity on large elongated DNA molecules.

Protein–nucleic acid interaction is an important process in many biological phenomena. In this study, a fluorescence resonance energy transfer (FRET)-based protein–DNA binding assay has been developed, in which a fluorescent amino acid is genetically incorporated into a DNA-binding protein. A coumarin-containing amino acid was incorporated into a DNA-binding protein, and the mutant protein specifically produced a FRET signal upon binding to its cognate DNA labeled with a fluorophore. The protein–DNA binding affinity was then measured under equilibrium conditions. This method is advantageous for studying protein-nucleic acid interactions, because it is performed under equilibrium conditions, technically easy, and applicable to any nucleic acid-binding protein.

DNA polymerase I offers great promise for a wide range of biotechnological applications due to its capability to add labeled nucleotides into double-stranded large DNA molecules by using both polymerase and nuclease domains. Accordingly, it is crucially important to thoroughly characterize this enzyme for further developments. Although the enzyme has been thus far characterized using mainly traditional analytical instruments, here we utilized an advanced and convenient means of mass spectrometry to elucidate enzymatic functions and mechanisms by measuring DNA oligomers generated by polymerase and nuclease reactions. Our analysis revealed several novel enzymatic features, including the observation that polymerase readily dissociates from the DNA molecules containing a wide single-stranded section. From this finding, we reasoned a serious situation of DNA break because polymerase domains cannot efficiently repair the wide single-stranded section, which is susceptible to DNA breaks. Furthermore, we deduced a plausible explanation for a paradoxical question as to why two domains of polymerase and 5′-nuclease are linked by a small and flexible polypeptide in polymerase I. The polypeptide link seems to prevent a 5′-nuclease from causing DNA breaks by locating a polymerase domain closely for immediate repair reaction. Here we present experimental evidence to prove our hypothesis via a set of mass spectrometric analyses as well as single DNA molecule observation and bacterial cell growth assay. Consequently, mass spectrometric analysis for DNA polymerase I provides a meaningful biological insight that a polypeptide link can be a molecular leash to control an aggressive domain in order to prevent unmanageable damages.

Fluorescent proteins that also bind DNA molecules are promising reagents for a broad range of biological applications because they can be optically localized and tracked within cells, or provide versatile labels for in vitro experiments. We report a novel design for a fluorescent, DNA-binding protein (FP-DBP) that completely “paints” entire DNA molecules, whereby sequence-independent DNA binding is accomplished by linking a fluorescent protein to two small peptides (KWKWKKA) using lysine for binding to the DNA phosphates, and tryptophan for partial intercalating between DNA bases (1). Importantly, this ubiquitous binding motif enables fluorescent proteins (Kd =14.7 µM, 7-8 bp/FP-DBP of base occupancies per dye) to confluently stain DNA molecules and such binding is reversible via pH shifts of buffer solution. These proteins offer robust advantages for lack of fluorophore mediated photocleavage, which makes them ideal staining reagents for imaging of DNA molecules over extended time periods even without anti-bleaching agent because the DNA binding moiety of the FP is separated from the fluorophore moiety. Further, the staining with FP-DBPs does not perturb polymer contour lengths, showing that the λ DNA monomer is about 48 502 bp × 0.34 nm/bp = 16.5 μm long, while most of the intercalating dyes, such as EtBr and YOYO-1, are known to distort the DNA structure and increase its full contour length. Moreover, the most significant advantage of FP-DBPs for DNA staining is its application to live cells, or entire living organisms. In a unique fashion, the FP-DBP fluorescence localized in three or four discrete regions within the bacterial cells: one, or two spots in the middle, and some at both ends of cells, as well as DAPI stained bacterial cells.1. Lee, S. et al., 2015, Nucleic Acids Research http://dx.doi.org/10.1093/nar/gkv834

We investigate experimentally the effect of ionic strength on λ DNA molecule in glass/PDMS nanoslit by using fluorescence microscope. The radius of gyration Rg was measured as a function of slit height (250 ∼ 1049 nm), and ionic strength (0.183 ∼ 1.75 mM) as a result, we observed the Rg values increases as channel heights and ionic strengths decrease. Furthermore, based on Odijk's and de Gennes polymer physics theory, we interpret our experimental findings as a function of channel heights and ionic strengths. We also study the effect of EDTA concentration, radius of gyration of λ DNA change drastically in response to the varying EDTA concentration at low ionic strength condition. Our results are useful for understanding the manipulation of biomolecules in nanofluidic devices.