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Topics in Current Chemistry (Cham) Apr 2020Cellular functions rely on a series of organized and regulated multienzyme cascade reactions. The catalytic efficiencies of these cascades depend on the precise spatial... (Review)
Review
Cellular functions rely on a series of organized and regulated multienzyme cascade reactions. The catalytic efficiencies of these cascades depend on the precise spatial organization of the constituent enzymes, which is optimized to facilitate substrate transport and regulate activities. Mimicry of this organization in a non-living, artificial system would be very useful in a broad range of applications-with impacts on both the scientific community and society at large. Self-assembled DNA nanostructures are promising applications to organize biomolecular components into prescribed, multidimensional patterns. In this review, we focus on recent progress in the field of DNA-scaffolded assembly and confinement of multienzyme reactions. DNA self-assembly is exploited to build spatially organized multienzyme cascades with control over their relative distance, substrate diffusion paths, compartmentalization and activity actuation. The combination of addressable DNA assembly and multienzyme cascades can deliver breakthroughs toward the engineering of novel synthetic and biomimetic reactors.
Topics: DNA; Enzymes; Protein Engineering
PubMed: 32248317
DOI: 10.1007/s41061-020-0299-3 -
Sensors (Basel, Switzerland) Nov 2020Contamination by pesticides in the food chain and the environment is a worldwide problem that needs to be actively monitored to ensure safety. Unfortunately, standard... (Review)
Review
Contamination by pesticides in the food chain and the environment is a worldwide problem that needs to be actively monitored to ensure safety. Unfortunately, standard pesticide analysis based on mass spectrometry takes a lot of time, money and effort. Thus, simple, reliable, cost-effective and field applicable methods for pesticide detection have been actively developed. One of the most promising technologies is an aptamer-based biosensor or so-called aptasensor. It utilizes aptamers, short single-stranded DNAs or RNAs, as pesticide recognition elements to integrate with various innovative biosensing technologies for specific and sensitive detection of pesticide residues. Several platforms for aptasensors have been dynamically established, such as colorimetry, fluorometry, electrochemistry, electrochemiluminescence (ECL) and so forth. Each platform has both advantages and disadvantages depending on the purpose of use and readiness of technology. For example, colorimetric-based aptasensors are more affordable than others because of the simplicity of fabrication and resource requirements. Electrochemical-based aptasensors have mainly shown better sensitivity than others with exceedingly low detection limits. This paper critically reviews the progression of pesticide aptasensors throughout the development process, including the selection, characterization and modification of aptamers, the conceptual frameworks of integrating aptamers and biosensors, the ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end users) criteria of different platforms and the future outlook.
Topics: Aptamers, Nucleotide; Biosensing Techniques; Colorimetry; DNA, Single-Stranded; Pesticides
PubMed: 33260648
DOI: 10.3390/s20236809 -
Nature Communications Feb 2020Genome-scale engineering holds great potential to impact science, industry, medicine, and society, and recent improvements in DNA synthesis have enabled the manipulation... (Review)
Review
Genome-scale engineering holds great potential to impact science, industry, medicine, and society, and recent improvements in DNA synthesis have enabled the manipulation of megabase genomes. However, coordinating and integrating the workflows and large teams necessary for gigabase genome engineering remains a considerable challenge. We examine this issue and recommend a path forward by: 1) adopting and extending existing representations for designs, assembly plans, samples, data, and workflows; 2) developing new technologies for data curation and quality control; 3) conducting fundamental research on genome-scale modeling and design; and 4) developing new legal and contractual infrastructure to facilitate collaboration.
Topics: Animals; DNA; DNA Replication; Databases, Genetic; Genetic Engineering; Genome; Humans
PubMed: 32019919
DOI: 10.1038/s41467-020-14314-z -
Trends in Biochemical Sciences Nov 2021Single-molecule localization microscopy (SMLM) is a potent tool to examine biological systems with unprecedented resolution, enabling the investigation of increasingly... (Review)
Review
Single-molecule localization microscopy (SMLM) is a potent tool to examine biological systems with unprecedented resolution, enabling the investigation of increasingly smaller structures. At the forefront of these developments is DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT), which exploits the stochastic and transient binding of fluorescently labeled DNA probes. In its early stages the implementation of DNA-PAINT was burdened by low-throughput, excessive acquisition time, and difficult integration with live-cell imaging. However, recent advances are addressing these challenges and expanding the range of applications of DNA-PAINT. We review the current state of the art of DNA-PAINT in light of these advances and contemplate what further developments remain indispensable to realize live-cell imaging.
Topics: DNA; Microscopy, Fluorescence; Single Molecule Imaging
PubMed: 34247944
DOI: 10.1016/j.tibs.2021.05.010 -
ACS Nano Jul 2021DNA origami has emerged as a powerful molecular breadboard with nanometer resolution that can integrate the world of bottom-up (bio)chemistry with large-scale,... (Review)
Review
DNA origami has emerged as a powerful molecular breadboard with nanometer resolution that can integrate the world of bottom-up (bio)chemistry with large-scale, macroscopic devices created by top-down lithography. Substituting the top-down patterning with self-assembled colloidal nanoparticles now takes the manufacturing complexity of top-down lithography out of the equation. As a result, the deterministic positioning of single molecules or nanoscale objects on macroscopic arrays is benchtop ready and easily accessible.
Topics: DNA; Nanotechnology; Printing
PubMed: 34255962
DOI: 10.1021/acsnano.1c04297 -
Physical Review Letters Jul 2021DNA torsional elastic properties play a crucial role in DNA structure, topology, and the regulation of motor protein progression. However, direct measurements of these...
DNA torsional elastic properties play a crucial role in DNA structure, topology, and the regulation of motor protein progression. However, direct measurements of these parameters are experimentally challenging. Here, we present a constant-extension method integrated into an angular optical trap to directly measure torque during DNA supercoiling. We measured the twist persistence length of extended DNA to be 22 nm under an extremely low force (∼0.02 pN) and the twist persistence length of plectonemic DNA to be 24 nm. In addition, we implemented a rigorous data analysis scheme that bridged our measurements with existing theoretical models of DNA torsional behavior. This comprehensive set of torsional parameters demonstrates that at least 20% of DNA supercoiling is partitioned into twist for both extended DNA and plectonemic DNA. This work provides a new experimental methodology, as well as an analytical and interpretational framework, which will enable, expand, and enhance future studies of DNA torsional properties.
Topics: DNA; DNA, Superhelical; Elasticity; Models, Chemical; Nucleic Acid Conformation; Thermodynamics
PubMed: 34296898
DOI: 10.1103/PhysRevLett.127.028101 -
Biophysical Journal May 2020Genomics is a sequence-based informatics science and a three-dimensional-structure-based material science. However, in practice, most genomics researchers utilize...
Genomics is a sequence-based informatics science and a three-dimensional-structure-based material science. However, in practice, most genomics researchers utilize sequence-based informatics approaches or three-dimensional-structure-based material science techniques, not both. This division is, at least in part, the result of historical developments rather than a fundamental necessity. The underlying computational tools, experimental techniques, and theoretical models were developed independently. The primary result presented here is a framework for the unification of informatics- and physics-based data associated with DNA, nucleosomes, and chromatin. The framework is based on the mathematical representation of geometrically exact rods and the generalization of DNA basepair step parameters. Data unification enables researchers to integrate computational, experimental, and theoretical approaches for the study of chromatin biology. The framework can be implemented using model-view-controller design principles, existing genome browsers, and existing molecular visualization tools. We developed a minimal, web-based genome dashboard, G-Dash-min, and applied it to two simple examples to demonstrate the usefulness of data unification and proof of concept. Genome dashboards developed using the framework and design principles presented here are extensible and customizable and are therefore more broadly applicable than the examples presented. We expect a number of purpose-specific genome dashboards to emerge as a novel means of investigating structure-function relationships for genomes that range from basepairs to entire chromosomes and for generating, validating, and testing mechanistic hypotheses.
Topics: Chromatin; DNA; Genomics; Nucleosomes; Software
PubMed: 32171420
DOI: 10.1016/j.bpj.2020.02.018 -
Viruses Jan 2021Human hepatitis B virus (HBV) can cause chronic, lifelong infection of the liver that may lead to persistent or episodic immune-mediated inflammation against... (Review)
Review
Human hepatitis B virus (HBV) can cause chronic, lifelong infection of the liver that may lead to persistent or episodic immune-mediated inflammation against virus-infected hepatocytes. This immune response results in elevated rates of killing of virus-infected hepatocytes, which may extend over many years or decades, lead to fibrosis and cirrhosis, and play a role in the high incidence of hepatocellular carcinoma (HCC) in HBV carriers. Immune-mediated inflammation appears to cause oxidative DNA damage to hepatocytes, which may also play a major role in hepatocarcinogenesis. An additional DNA damaging feature of chronic infections is random integration of HBV DNA into the chromosomal DNA of hepatocytes. While HBV DNA integration does not have a role in virus replication it may alter gene expression of the host cell. Indeed, most HCCs that arise in HBV carriers contain integrated HBV DNA and, in many, the integrant appears to have played a role in hepatocarcinogenesis. Clonal expansion of hepatocytes, which is a natural feature of liver biology, occurs because the hepatocyte population is self-renewing and therefore loses complexity due to random hepatocyte death and replacement by proliferation of surviving hepatocytes. This process may also represent a risk factor for the development of HCC. Interestingly, during chronic HBV infection, hepatocyte clones detected using integrated HBV DNA as lineage-specific markers, emerge that are larger than those expected to occur by random death and proliferation of hepatocytes. The emergence of these larger hepatocyte clones may reflect a survival advantage that could be explained by an ability to avoid the host immune response. While most of these larger hepatocyte clones are probably not preneoplastic, some may have already acquired preneoplastic changes. Thus, chronic inflammation in the HBV-infected liver may be responsible, at least in part, for both initiation of HCC via oxidative DNA damage and promotion of HCC via stimulation of hepatocyte proliferation through immune-mediated killing and compensatory division.
Topics: Animals; DNA, Viral; Hepatitis B virus; Hepatitis B, Chronic; Hepatocytes; Humans; Liver; Virus Integration
PubMed: 33573130
DOI: 10.3390/v13020210 -
International Journal of Molecular... Aug 2021The transcriptome of every cell is orchestrated by the complex network of interaction between transcription factors (TFs) and their binding sites on DNA. Disruption of... (Review)
Review
The transcriptome of every cell is orchestrated by the complex network of interaction between transcription factors (TFs) and their binding sites on DNA. Disruption of this network can result in many forms of organism malfunction but also can be the substrate of positive natural selection. However, understanding the specific determinants of each of these individual TF-DNA interactions is a challenging task as it requires integrating the multiple possible mechanisms by which a given TF ends up interacting with a specific genomic region. These mechanisms include DNA motif preferences, which can be determined by nucleotide sequence but also by DNA's shape; post-translational modifications of the TF, such as phosphorylation; and dimerization partners and co-factors, which can mediate multiple forms of direct or indirect cooperative binding. Binding can also be affected by epigenetic modifications of putative target regions, including DNA methylation and nucleosome occupancy. In this review, we describe how all these mechanisms have a role and crosstalk in one specific family of TFs, the basic helix-loop-helix (bHLH), with a very conserved DNA binding domain and a similar DNA preferred motif, the E-box. Here, we compile and discuss a rich catalog of strategies used by bHLH to acquire TF-specific genome-wide landscapes of binding sites.
Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Chromatin; DNA; Humans; Protein Binding; Transcriptional Activation
PubMed: 34502060
DOI: 10.3390/ijms22179150 -
Methods (San Diego, Calif.) Nov 2023The transcription, replication, packaging, and repair of genetic information ubiquitously involves DNA:protein interactions and other biological processes that require...
The transcription, replication, packaging, and repair of genetic information ubiquitously involves DNA:protein interactions and other biological processes that require local mechanical distortions of DNA. The energetics of such DNA-deforming processes are thus dependent on the local mechanical properties of DNA such as bendability or torsional rigidity. Such properties, in turn, depend on sequence, making it possible for sequence to regulate diverse biological processes by controlling the local mechanical properties of DNA. A deeper understanding of how such a "mechanical code" can encode broad regulatory information has historically been hampered by the absence of technology to measure in high throughput how local DNA mechanics varies with sequence along large regions of the genome. This was overcome in a recently developed technique called loop-seq. Here we describe a variant of the loop-seq protocol, that permits making rapid flexibility measurements in low-throughput, without the need for next-generation sequencing. We use our method to validate a previous prediction about how the binding site for the bacterial transcription factor Integration Host Factor (IHF) might serve as a rigid roadblock, preventing efficient enhancer-promoter contacts in IHF site containing promoters in E. coli, which can be relieved by IHF binding.
Topics: Bacterial Proteins; Escherichia coli; Base Sequence; Integration Host Factors; Promoter Regions, Genetic; DNA; DNA, Bacterial; Binding Sites
PubMed: 37769928
DOI: 10.1016/j.ymeth.2023.09.007