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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 -
Methods in Molecular Biology (Clifton,... 2022DNA-encoded library (DEL) links the powers of genetics and chemicals via high-efficient enzymatic ligation of DNA barcodes and the "split and pool" combinatorial...
DNA-encoded library (DEL) links the powers of genetics and chemicals via high-efficient enzymatic ligation of DNA barcodes and the "split and pool" combinatorial synthesis. Natural products (NPs) are evolutionary optimized compounds that have played a key role in the history of human drug discovery. Herein, we describe a method for functionality-independent annotation of complex natural products with amplifiable DNA barcodes to generate a DNA-encoded natural product library (nDEL). This method provides a simple and practical solution to leverage natural products by DNA barcoding.
Topics: Biological Products; Combinatorial Chemistry Techniques; DNA; Gene Library; Humans; Small Molecule Libraries
PubMed: 36083548
DOI: 10.1007/978-1-0716-2545-3_13 -
Genome Biology Oct 2023Genomic abnormalities are strongly associated with cancer and infertility. In this study, we develop a simple and efficient method - multiple genetic abnormality...
Genomic abnormalities are strongly associated with cancer and infertility. In this study, we develop a simple and efficient method - multiple genetic abnormality sequencing (MGA-Seq) - to simultaneously detect structural variation, copy number variation, single-nucleotide polymorphism, homogeneously staining regions, and extrachromosomal DNA (ecDNA) from a single tube. MGA-Seq directly sequences proximity-ligated genomic fragments, yielding a dataset with concurrent genome three-dimensional and whole-genome sequencing information, enabling approximate localization of genomic structural variations and facilitating breakpoint identification. Additionally, by utilizing MGA-Seq, we map focal amplification and oncogene coamplification, thus facilitating the exploration of ecDNA's transcriptional regulatory function.
Topics: DNA Copy Number Variations; Oncogenes; Genomics; Gene Expression Regulation; DNA
PubMed: 37904244
DOI: 10.1186/s13059-023-03081-x -
Genes & Development Jul 2023Punctuated bursts of structural genomic variations (SVs) have been described in various organisms, but their etiology remains incompletely understood. Homologous...
Punctuated bursts of structural genomic variations (SVs) have been described in various organisms, but their etiology remains incompletely understood. Homologous recombination (HR) is a template-guided mechanism of repair of DNA double-strand breaks and stalled or collapsed replication forks. We recently identified a DNA break amplification and genome rearrangement pathway originating from the endonucleolytic processing of a multi-invasion (MI) DNA joint molecule formed during HR. Genome-wide approaches confirmed that multi-invasion-induced rearrangement (MIR) frequently leads to several repeat-mediated SVs and aneuploidies. Using molecular and genetic analysis and a novel, highly sensitive proximity ligation-based assay for chromosomal rearrangement quantification, we further delineate two MIR subpathways. MIR1 is a universal pathway occurring in any sequence context, which generates secondary breaks and frequently leads to additional SVs. MIR2 occurs only if recombining donors exhibit substantial homology and results in sequence insertion without additional breaks or SVs. The most detrimental MIR1 pathway occurs late on a subset of persisting DNA joint molecules in a PCNA/Pol-independent manner, unlike recombinational DNA synthesis. This work provides a refined mechanistic understanding of these HR-based SV formation pathways and shows that complex repeat-mediated SVs can occur without displacement DNA synthesis. Sequence signatures for inferring MIR1 from long-read data are proposed.
Topics: Genomic Instability; Gene Rearrangement; Homologous Recombination; Selection, Genetic; DNA; Chromosomes, Fungal; Saccharomyces cerevisiae
PubMed: 37541760
DOI: 10.1101/gad.350618.123 -
Journal of Visualized Experiments : JoVE Jun 2022The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the hyperactive Tn5...
The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the hyperactive Tn5 transposase. Tn5 cuts and ligates adapters for high-throughput sequencing within accessible chromatin regions. In eukaryotic cells, genomic DNA is packaged into chromatin, a complex of DNA, histones, and other proteins, which acts as a physical barrier to the transcriptional machinery. In response to extrinsic signals, transcription factors recruit chromatin remodeling complexes to enable access to the transcriptional machinery for gene activation. Therefore, identifying open chromatin regions is useful when monitoring enhancer and gene promoter activities during biological events such as cancer progression. Since this protocol is easy to use and has a low cell input requirement, ATAC-seq has been widely adopted to define open chromatin regions in various cell types, including cancer cells. For successful data acquisition, several parameters need to be considered when preparing ATAC-seq libraries. Among them, the choice of cell lysis buffer, the titration of the Tn5 enzyme, and the starting volume of cells are crucial for ATAC-seq library preparation in cancer cells. Optimization is essential for generating high-quality data. Here, we provide a detailed description of the ATAC-seq optimization methods for epithelial cell types.
Topics: Chromatin; Chromatin Immunoprecipitation Sequencing; DNA; Epigenesis, Genetic; High-Throughput Nucleotide Sequencing; Neoplasms; Sequence Analysis, DNA; Transcription Factors
PubMed: 35848835
DOI: 10.3791/64242 -
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 -
Chembiochem : a European Journal of... Apr 2021Template-guided chemical reactions between nucleic acid strands are an important process in biomedical research. However, almost all of these reactions employ an...
Template-guided chemical reactions between nucleic acid strands are an important process in biomedical research. However, almost all of these reactions employ an oligonucleotide-templated approach that is based on the double-helix alignment. The moderate stability of the double helix makes this approach unsuitable for many chemical reactions, so alternative nucleic acid alignment mechanisms, demonstrating higher thermal and chemical stability, are desirable. Earlier, we described a noncovalent coupling mechanism between DNA strands through a quadruplex-and-Mg connection (QMC). QMC is based on G-quadruplexes and allows unusually stable and specific interactions. Herein, a novel catalytic nucleic acid reaction, based on QMC, is described. This approach uses G-tetrads as a structural and recognition element without employing Watson-Crick complementarity rules at any stage of substrate/catalyst formation or interaction between them. Quadruplex-templated ligation can be achieved through the self-ligation of two nucleic acid strands, or through a quadruplex catalyst, which forms a G-triplex and specifically connects the strands. The process is extraordinarily robust and efficient. For instance, the ligation of carbodiimide-activated substrates can proceed in boiling solutions, and complete ligation is demonstrated within a minute. The quadruplex-templated and catalyzed reactions will create new opportunities for chemical reactions requiring harsh experimental conditions.
Topics: Catalysis; DNA; G-Quadruplexes; Magnesium; Nucleic Acid Conformation; Phase Transition; Thermodynamics
PubMed: 33217115
DOI: 10.1002/cbic.202000754 -
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 -
Methods in Molecular Biology (Clifton,... 2022Phosphorothioate (PS) modification refers to replacing one of the nonbridging oxygen atoms in nucleic acids with sulfur. PS modifications can be easily introduced during...
Phosphorothioate (PS) modification refers to replacing one of the nonbridging oxygen atoms in nucleic acids with sulfur. PS modifications can be easily introduced during solid-phase DNA synthesis. It has been extensively used in ribozyme and DNAzyme research to achieve a bioinorganic understanding of metal binding, bioanalytical applications of metal detection, and chemical biology of DNA modification. It allows for the access of new chemistry, not available to natural DNA. Since each PS modification is accompanied by the production of a chiral phosphorus center, a key technical challenge is to separate the two diastereomers called R and S. In this chapter, we describe our methods of HPLC-based separation followed by ligation to generate a long and fluorescently modified DNAzyme substrate. Subsequently, the use of the modified substrate for activity assay to understand metal binding and for metal ion detection is also described.
Topics: DNA, Catalytic; Ions; Metals
PubMed: 35226327
DOI: 10.1007/978-1-0716-2047-2_17