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Journal of Visualized Experiments : JoVE Feb 2022Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic marks in a...
Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic marks in a single cell and requiring data from other cells to distinguish signal from experimental background noise in a single cell. This paper describes a method to reuse the same single cell for iterative epigenomic analyses. In this experimental method, cellular proteins are first anchored to a polyacrylamide polymer instead of crosslinking them to protein and DNA, alleviating structural bias. This critical step allows repeated experiments with the same single cell. Next, a random primer with a scaffold sequence for proximity ligation is annealed to the genomic DNA, and the genomic sequence is added to the primer by extension using a DNA polymerase. Subsequently, an antibody against an epigenetic marker and control IgG, each labeled with different DNA probes, are bound to the respective targets in the same single cell. Proximity ligation is induced between the random primer and the antibody by adding a connector DNA with complementary sequences to the scaffold sequence of the random primer and the antibody-DNA probe. This approach integrates antibody information and nearby genome sequences in a single DNA product of proximity ligation. By enabling repeated experiments with the same single cell, this method allows an increase in data density from a rare cell and statistical analysis using only IgG and antibody data from the same cell. The reusable single cells prepared by this method can be stored for at least a few months and reused later to broaden epigenetic characterization and increase data density. This method provides flexibility to researchers and their projects.
Topics: DNA; DNA Probes; DNA-Directed DNA Polymerase; Epigenome; Epigenomics
PubMed: 35225292
DOI: 10.3791/63456 -
Journal of Virology Aug 2023Proliferating cell nuclear antigen (PCNA) belongs to the DNA sliding clamp family. Via interacting with various partner proteins, PCNA plays critical roles in DNA...
Proliferating cell nuclear antigen (PCNA) belongs to the DNA sliding clamp family. Via interacting with various partner proteins, PCNA plays critical roles in DNA replication, DNA repair, chromatin assembly, epigenetic inheritance, chromatin remodeling, and many other fundamental biological processes. Although PCNA and PCNA-interacting partner networks are conserved across species, PCNA of a given species is rarely functional in heterologous systems, emphasizing the importance of more representative PCNA studies. Here, we report two crystal structures of PCNA from African swine fever virus (ASFV), which is the only member of the family. Compared to the eukaryotic and archaeal PCNAs and the sliding clamp structural homologs from other viruses, PCNA possesses unique sequences and/or conformations at several regions, such as the J-loop, interdomain-connecting loop (IDCL), P-loop, and C-tail, which are involved in partner recognition or modification of sliding clamps. In addition to double-stranded DNA binding, we also demonstrate that PCNA can modestly enhance the ligation activity of the LIG protein. The unique structural features of PCNA can serve as a potential target for the development of ASFV-specific inhibitors and help combat the deadly virus. IMPORTANCE Two high-resolution crystal structures of African swine fever virus proliferating cell nuclear antigen (PCNA) are presented here. Structural comparison revealed that PCNA is unique at several regions, such as the J-loop, the interdomain-connecting loop linker, and the P-loop, which may play important roles in ASFV-specific partner selection of PCNA. Unlike eukaryotic and archaeal PCNAs, PCNA possesses high double-stranded DNA-binding affinity. Besides DNA binding, PCNA can also modestly enhance the ligation activity of the LIG protein, which is essential for the replication and repair of ASFV genome. The unique structural features make PCNA a potential target for drug development, which will help combat the deadly virus.
Topics: Animals; African Swine Fever; African Swine Fever Virus; DNA; Molecular Conformation; Proliferating Cell Nuclear Antigen; Swine; Viral Proteins
PubMed: 37534905
DOI: 10.1128/jvi.00748-23 -
Chembiochem : a European Journal of... Oct 2021The introduction of chemical modifications into long RNA molecules at specific positions for visualization, biophysical investigations, diagnostic and therapeutic... (Review)
Review
The introduction of chemical modifications into long RNA molecules at specific positions for visualization, biophysical investigations, diagnostic and therapeutic applications still remains challenging. In this review, we present recent approaches for covalent internal labeling of long RNAs. Topics included are the assembly of large modified RNAs via enzymatic ligation of short synthetic oligonucleotides and synthetic biology approaches preparing site-specifically modified RNAs via in vitro transcription using an expanded genetic alphabet. Moreover, recent approaches to employ deoxyribozymes (DNAzymes) and ribozymes for RNA labeling and RNA methyltransferase based labeling strategies are presented. We discuss the potentials and limits of the individual methods, their applicability for RNAs with several hundred to thousands of nucleotides in length and indicate future directions in the field.
Topics: DNA, Catalytic; Humans; RNA, Catalytic; RNA, Long Noncoding; Staining and Labeling
PubMed: 34043861
DOI: 10.1002/cbic.202100161 -
Nucleic Acids Research Aug 2021The low thermal stability of DNA nanostructures is the major drawback in their practical applications. Most of the DNA nanotubes/tiles and the DNA origami structures...
The low thermal stability of DNA nanostructures is the major drawback in their practical applications. Most of the DNA nanotubes/tiles and the DNA origami structures melt below 60°C due to the presence of discontinuities in the phosphate backbone (i.e., nicks) of the staple strands. In molecular biology, enzymatic ligation is commonly used to seal the nicks in the duplex DNA. However, in DNA nanotechnology, the ligation procedures are neither optimized for the DNA origami nor routinely applied to link the nicks in it. Here, we report a detailed analysis and optimization of the conditions for the enzymatic ligation of the staple strands in four types of 2D square lattice DNA origami. Our results indicated that the ligation takes overnight, efficient at 37°C rather than the usual 16°C or room temperature, and typically requires much higher concentration of T4 DNA ligase. Under the optimized conditions, up to 10 staples ligation with a maximum ligation efficiency of 55% was achieved. Also, the ligation is found to increase the thermal stability of the origami as low as 5°C to as high as 20°C, depending on the structure. Further, our studies indicated that the ligation of the staple strands influences the globular structure/planarity of the DNA origami, and the origami is more compact when the staples are ligated. The globular structure of the native and ligated origami was also found to be altered dynamically and progressively upon ethidium bromide intercalation in a concentration-dependent manner.
Topics: DNA; DNA Ligases; Electrophoresis, Agar Gel; Ethidium; Kinetics; Microscopy, Atomic Force; Nanostructures; Nucleic Acid Conformation; Nucleic Acid Denaturation; Phosphorylation; Temperature; Thermodynamics
PubMed: 34289063
DOI: 10.1093/nar/gkab611 -
PLoS Biology Mar 2021Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a...
Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3' ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3' ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.
Topics: 3' Untranslated Regions; Animals; DNA; DNA Breaks, Double-Stranded; DNA Repair; DNA Replication; DNA-Binding Proteins; Genome; Humans; Sequence Analysis, DNA
PubMed: 33760805
DOI: 10.1371/journal.pbio.3000886 -
Proceedings of the National Academy of... Dec 2022The 53BP1-RIF1 pathway restricts the resection of DNA double-strand breaks (DSBs) and promotes blunt end-ligation by non-homologous end joining (NHEJ) repair. The...
The 53BP1-RIF1 pathway restricts the resection of DNA double-strand breaks (DSBs) and promotes blunt end-ligation by non-homologous end joining (NHEJ) repair. The Shieldin complex is a downstream effector of the 53BP1-RIF1 pathway. Here, we identify a component of this pathway, CCAR2/DBC1, which is also required for restriction of DNA end-resection. CCAR2 co-immunoprecipitates with the Shieldin complex, and knockout of CCAR2 in a BRCA1-deficient cell line results in elevated DSB end-resection, RAD51 loading, and PARP inhibitor (PARPi) resistance. Knockout of CCAR2 is epistatic with knockout of other Shieldin proteins. The S1-like RNA-binding domain of CCAR2 is required for its interaction with the Shieldin complex and for suppression of DSB end-resection. CCAR2 functions downstream of the Shieldin complex, and CCAR2 knockout cells have delayed resolution of Shieldin complex foci. Forkhead-associated (FHA)-dependent targeting of CCAR2 to DSB sites re-sensitized BRCA1-/-SHLD2-/- cells to PARPi. Taken together, CCAR2 is a functional component of the 53BP1-RIF1 pathway, promotes the refill of resected DSBs, and suppresses homologous recombination.
Topics: Poly(ADP-ribose) Polymerase Inhibitors; DNA Breaks, Double-Stranded; DNA End-Joining Repair; Homologous Recombination; DNA
PubMed: 36442094
DOI: 10.1073/pnas.2214935119 -
Astrobiology Jul 2018Most strategies for life detection rely upon finding features known to be associated with terran life, such as particular classes of molecules. But life may be vastly...
Most strategies for life detection rely upon finding features known to be associated with terran life, such as particular classes of molecules. But life may be vastly different on other planets and moons, particularly as we expand our efforts to explore ocean worlds like Europa and Enceladus. We propose a new concept for life detection that harnesses the power of DNA sequencing to yield intricate informatics fingerprints, even for life that is not nucleic acid-based. The concept is based on the fact that folded nucleic acid structures (aptamers) have been shown to be capable of binding a wide variety of compounds, whether inorganic, organic, or polymeric, and irrespective of being from a biotic or abiotic source. Each nucleic acid sequence can be thought of as a code, and a combination of codes as a "fingerprint." Over multiple analytes, the "fingerprint" of a non-terran sample can be analyzed by chemometric protocols to provide a classifier of molecular patterns and complexity. Ultimately the chemometric fingerprints of living systems, which may differ significantly from nonliving systems, could provide an empirical, agnostic means of detecting life. Because nucleic acids are exponentially amplified by the polymerase chain reaction, even very small input signals could be translated into a robust readable output. The derived sequences could be identified by a small, portable sequencing device or by capture and optical imaging on a DNA microarray. Without presupposing any particular molecular framework, this agnostic approach to life detection could be used from Mars to the far reaches of the Solar System, all within the framework of an instrument drawing little heat and power. Key Words: Agnostic biosignatures-Astrobiology-Chemometrics-DNA sequencing-Life detection-Proximity ligation assay. Astrobiology 18, 915-922.
Topics: DNA; DNA Fingerprinting; Exobiology; Extraterrestrial Environment; Life; Planets; Sequence Analysis, DNA
PubMed: 29634318
DOI: 10.1089/ast.2017.1712 -
Biomolecules Jun 2023Programmable endonucleases, such as Cas (Clustered Regularly-Interspaced Short Repeats-associated proteins) and prokaryotic Argonaute (pAgo), depend on base pairing of... (Review)
Review
Programmable endonucleases, such as Cas (Clustered Regularly-Interspaced Short Repeats-associated proteins) and prokaryotic Argonaute (pAgo), depend on base pairing of the target DNA with the guide RNA or DNA to cleave DNA strands. Therefore, they are capable of recognizing and cleaving DNA sequences at virtually any arbitrary site. The present review focuses on the commonly used in vivo and in vitro recombination-based gene cloning methods and the application of programmable endonucleases in these sequence- and ligation-independent DNA assembly methods. The advantages and shortcomings of the programmable endonucleases utilized as tools for gene cloning are also discussed in this review.
Topics: Endonucleases; DNA; Cloning, Molecular; Prokaryotic Cells
PubMed: 37509059
DOI: 10.3390/biom13071022 -
DNA Repair Oct 2023DNA double strand breaks (DSBs) are common lesions whose misrepair are drivers of oncogenic transformations. The non-homologous end joining (NHEJ) pathway repairs the... (Review)
Review
DNA double strand breaks (DSBs) are common lesions whose misrepair are drivers of oncogenic transformations. The non-homologous end joining (NHEJ) pathway repairs the majority of these breaks in vertebrates by directly ligating DNA ends back together. Upon formation of a DSB, a multiprotein complex is assembled on DNA ends which tethers them together within a synaptic complex. Synapsis is a critical step of the NHEJ pathway as loss of synapsis can result in mispairing of DNA ends and chromosome translocations. As DNA ends are commonly incompatible for ligation, the NHEJ machinery must also process ends to enable rejoining. This review describes how recent progress in single-molecule approaches and cryo-EM have advanced our molecular understanding of DNA end synapsis during NHEJ and how synapsis is coordinated with end processing to determine the fidelity of repair.
Topics: Animals; DNA End-Joining Repair; DNA; DNA-Binding Proteins; DNA Breaks, Double-Stranded; Chromosome Pairing; DNA Repair
PubMed: 37572577
DOI: 10.1016/j.dnarep.2023.103553 -
Chemical Society Reviews Nov 2021The self-assembly of inorganic nanoparticles to larger structures is of great research interest as it allows the fabrication of novel materials with collective... (Review)
Review
The self-assembly of inorganic nanoparticles to larger structures is of great research interest as it allows the fabrication of novel materials with collective properties correlated to the nanoparticles' individual characteristics. Recently developed methods for controlling nanoparticle organisation have enabled the fabrication of a range of new materials. Amongst these, the assembly of nanoparticles using DNA has attracted significant attention due to the highly selective recognition between complementary DNA strands, DNA nanostructure versatility, and ease of DNA chemical modification. In this review we discuss the application of various chemical DNA modifications and molecular intercalators as tools for the manipulation of DNA-nanoparticle structures. In detail, we discuss how DNA modifications and small molecule intercalators have been employed in the chemical and photochemical DNA ligation in nanostructures; DNA rotaxanes and catenanes associated with reconfigurable nanoparticle assemblies; and DNA backbone modifications including locked nucleic acids, peptide nucleic acids and borane nucleic acids, which affect the stability of nanostructures in complex environments. We conclude by highlighting the importance of maximising the synergy between the communities of DNA chemistry and nanoparticle self-assembly with the aim to enrich the library of tools available for the manipulation of nanostructures.
Topics: DNA; Intercalating Agents; Nanoparticles; Nanostructures; Nucleic Acids
PubMed: 34792047
DOI: 10.1039/d1cs00632k