Scanning Electron Microscopy Imaging of Large DNA Molecules Using a Metal-Free Electro-Stain Composed of DNA-Binding Proteins and Synthetic Polymers.

This paper presents the first scanning electron microscopy (SEM)-based DNA imaging in biological samples. This novel approach incorporates a metal-free electro-stain reagent, formulated by combining DNA-binding proteins and synthetic polymers to enhance the visibility of 2-nm-thick DNA under SEM. Notably, DNA molecules stain with proteins and polymers appear as dark lines under SEM. The resulting DNA images exhibit a thickness of 15.0±4.0 nm. As SEM is the primary platform, it integrates seamlessly with various chemically functionalized large surfaces with the aid of microfluidic devices. The approach allows high-resolution imaging of various DNA structures including linear, circular, single-stranded DNA and RNA, originating from nuclear and mitochondrial genomes. Furthermore, quantum dots are successfully visualized as bright labels that are sequence-specifically incorporated into DNA molecules, which highlights the potential for SEM-based optical DNA mapping. In conclusion, DNA imaging using SEM with the novel electro-stain offers electron microscopic resolution with the ease of optical microscopy.

Utilizing peptide-anchored DNA templates for novel programmable nanoparticle assemblies in biological macromolecules: A review.

Biological macromolecules such as proteins and DNA are known to self-assemble into various structural moieties with distinct functions. While nucleic acids are the structural building blocks, peptides exemplify diversity as tailorable biochemical units. Thus, combining the scaffold properties of the biomacromolecule DNA and the functionality of peptides could evolve into a powerful method to obtain tailorable nano assemblies. In this review, we discuss the assembly of non-DNA-coated colloidal NPs on DNA/peptide templates using functional anchors. We begin with strategies for directly attaching metallic NPs to DNA templates to ascertain the functional role of DNA as a scaffold. Followed by methods to assemble peptides onto DNA templates to emphasize the functional versatility of biologically abundant DNA-binding peptides. Next, we focus on studies corroborating peptide self-assembling into macromolecular templates onto which NPs can attach to emphasize the properties of NP-binding peptides. Finally, we discuss the assembly of NPs on a DNA template with a focus on the bifunctional DNA-binding peptides with NP-binding affinity (peptide anchors). This review aims to highlight the immense potential of combining the functional power of DNA scaffolds and tailorable functionalities of peptides for NP assembly and the need to utilize them effectively to obtain tailorable hierarchical NP assemblies.

Negative nanopore sequencing for mapping biochemical processes on DNA molecules.

Nanopore sequencing maps biochemical processes on DNA by detecting negative peaks in the sequence alignment profile. Protein-bound DNA and single-strand broken DNA cannot pass through nanopores, resulting in unaligned regions in the genome MAP. This novel approach provides a clear representation of genomic biochemical events.

Origin of minicircular mitochondrial genomes in red algae.

Eukaryotic organelle genomes are generally of conserved size and gene content within phylogenetic groups. However, significant variation in genome structure may occur. Here, we report that the Stylonematophyceae red algae contain multipartite circular mitochondrial genomes (i.e., minicircles) which encode one or two genes bounded by a specific cassette and a conserved constant region. These minicircles are visualized using fluorescence microscope and scanning electron microscope, proving the circularity. Mitochondrial gene sets are reduced in these highly divergent mitogenomes. Newly generated chromosome-level nuclear genome assembly of Rhodosorus marinus reveals that most mitochondrial ribosomal subunit genes are transferred to the nuclear genome. Hetero-concatemers that resulted from recombination between minicircles and unique gene inventory that is responsible for mitochondrial genome stability may explain how the transition from typical mitochondrial genome to minicircles occurs. Our results offer inspiration on minicircular organelle genome formation and highlight an extreme case of mitochondrial gene inventory reduction.

AT-specific DNA visualization revisits the directionality of bacteriophage λ DNA ejection.

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.

DNA Visualization Using Fluorescent Proteins.

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.

Counting DNA molecules on a microchannel surface for quantitative analysis.

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.

Twelve Colors of Streptavidin-Fluorescent Proteins (SA-FPs): A Versatile Tool to Visualize Genetic Information in Single-Molecule DNA.

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-1 cm-1. Moreover, SA-FP is brighter than FP itself because streptavidin forms a tetramer complex; thus, four FPs are in a single complex. In addition, FPs often form a dimer or a tetramer, resulting in multiple FPs in a single spot on a microscopic image. This feature is advantageous because multiple fluorescent ß-barrels on a single biotin tag provide enough brightness to be easily visualized by epifluorescence microscopy. Using SA-FPs, we visualized DNA backbones, nickase-based optical mapping, and AT-frequency profiling. Finally, we demonstrated the combination of nickase-based optical mapping using SA-FP and AT-frequency profiling.

21 Fluorescent Protein-Based DNA Staining Dyes.

Fluorescent protein-DNA-binding peptides or proteins (FP-DBP) are a powerful means to stain and visualize large DNA molecules on a fluorescence microscope. Here, we constructed 21 kinds of FP-DBPs using various colors of fluorescent proteins and two DNA-binding motifs. From the database of fluorescent proteins (FPbase.org), we chose bright FPs, such as RRvT, tdTomato, mNeonGreen, mClover3, YPet, and mScarlet, which are four to eight times brighter than original wild-type GFP. Additionally, we chose other FPs, such as mOrange2, Emerald, mTurquoise2, mStrawberry, and mCherry, for variations in emitting wavelengths. For DNA-binding motifs, we used HMG (high mobility group) as an 11-mer peptide or a 36 kDa tTALE (truncated transcription activator-like effector). Using 21 FP-DBPs, we attempted to stain DNA molecules and then analyzed fluorescence intensities. Most FP-DBPs successfully visualized DNA molecules. Even with the same DNA-binding motif, the order of FP and DBP affected DNA staining in terms of brightness and DNA stretching. The DNA staining pattern by FP-DBPs was also affected by the FP types. The data from 21 FP-DBPs provided a guideline to develop novel DNA-binding fluorescent proteins.

DNA binding fluorescent proteins as single-molecule probes.

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.

High-throughput single-molecule imaging system using nanofabricated trenches and fluorescent DNA-binding proteins.

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.

Multifunctional Heterogeneous Carbon Nanotube Nanocomposites Assembled by DNA-Binding Peptide Anchors.

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.

Truncated TALE-FP as DNA Staining Dye in a High-salt Buffer.

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.

Molecular-Level Interactions between Engineered Materials and Cells.

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.

Single-molecule DNA visualization using AT-specific red and non-specific green DNA-binding fluorescent proteins.

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.

TAMRA-polypyrrole for A/T sequence visualization on DNA molecules.

Fluorophore-linked, sequence-specific DNA binding reagents can visualize sequence information on a large DNA molecule. In this paper, we synthesized newly designed TAMRA-linked polypyrrole to visualize adenine and thymine base pairs. A fluorescent image of the stained DNA molecule generates an intensity profile based on A/T frequency, revealing a characteristic sequence composition pattern. Computer-aided comparison of this intensity pattern with the genome sequence allowed us to determine the DNA sequence on a visualized DNA molecule from possible intensity profile pattern candidates for a given genome. Moreover, TAMRA-polypyrrole offers robust advantages for single DNA molecule detection: no fluorophore-mediated photocleavage and no structural deformation, since it exhibits a sequence-specific pattern alone without the use of intercalating dyes such as YOYO-1. Accordingly, we were able to identify genomic DNA fragments from Escherichia coli cells by aligning them to the genomic A/T frequency map based on TAMRA-polypyrrole-generated intensity profiles. Furthermore, we showed band and interband patterns of polytene chromosomal DNA stained with TAMRA-polypyrrole because it prefers to bind AT base pairs.

Bioorthogonal Metabolic DNA Labelling using Vinyl Thioether-Modified Thymidine and o-Quinolinone Quinone Methide.

Bioorthogonal metabolic DNA labeling with fluorochromes is a powerful strategy to visualize DNA molecules and their functions. Here, we report the development of a new DNA metabolic labeling 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 labeling 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 labeling 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.

Nanochannel-Confined TAMRA-Polypyrrole Stained DNA Stretching by Varying the Ionic Strength from Micromolar to Millimolar Concentrations.

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.

Electrostatic confinement and manipulation of DNA molecules for genome analysis.

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 Mesoplasmaflorum 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.

DNA Binding Peptide Directed Synthesis of Continuous DNA Nanowires for Analysis of Large DNA Molecules by Scanning Electron Microscope.

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.