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Molecules (Basel, Switzerland) Jan 2021The expanding scope of chemical reactions applied to nucleic acids has diversified the design of nucleic acid-based technologies that are essential to medicinal... (Review)
Review
The expanding scope of chemical reactions applied to nucleic acids has diversified the design of nucleic acid-based technologies that are essential to medicinal chemistry and chemical biology. Among chemical reactions, visible light photochemical reaction is considered a promising tool that can be used for the manipulations of nucleic acids owing to its advantages, such as mild reaction conditions and ease of the reaction process. Of late, inspired by the development of visible light-absorbing molecules and photocatalysts, visible light-driven photochemical reactions have been used to conduct various molecular manipulations, such as the cleavage or ligation of nucleic acids and other molecules as well as the synthesis of functional molecules. In this review, we describe the recent developments (from 2010) in visible light photochemical reactions involving nucleic acids and their applications in the design of nucleic acid-based technologies including DNA photocleaving, DNA photoligation, nucleic acid sensors, the release of functional molecules, and DNA-encoded libraries.
Topics: Catalysis; DNA; Fluorescent Dyes; Light; Photochemical Processes
PubMed: 33494512
DOI: 10.3390/molecules26030556 -
The Journal of Biological Chemistry Jul 2023Force and torque spectroscopy have provided unprecedented insights into the mechanical properties, conformational transitions, and dynamics of DNA and DNA-protein...
Force and torque spectroscopy have provided unprecedented insights into the mechanical properties, conformational transitions, and dynamics of DNA and DNA-protein complexes, notably nucleosomes. Reliable single-molecule manipulation measurements require, however, specific and stable attachment chemistries to tether the molecules of interest. Here, we present a functionalization strategy for DNA that enables high-yield production of constructs for torsionally constrained and very stable attachment. The method is based on two subsequent PCRs: first ∼380 bp long DNA strands are generated that contain multiple labels, which are used as "megaprimers" in a second PCR to generate ∼kbp long double-stranded DNA constructs with multiple labels at the respective ends. To achieve high-force stability, we use dibenzocyclooctyne-based click chemistry for covalent attachment to the surface and biotin-streptavidin coupling to the bead. The resulting tethers are torsionally constrained and extremely stable under load, with an average lifetime of 70 ± 3 h at 45 pN. The high yield of the approach enables nucleosome reconstitution by salt dialysis on the functionalized DNA, and we demonstrate proof-of-concept measurements on nucleosome assembly statistics and inner turn unwrapping under force. We anticipate that our approach will facilitate a range of studies of DNA interactions and nucleoprotein complexes under forces and torques.
Topics: Nucleosomes; DNA; Mechanical Phenomena; Biophysical Phenomena; Polymerase Chain Reaction
PubMed: 37257819
DOI: 10.1016/j.jbc.2023.104874 -
Heliyon May 2023A method to build sequence-restricted DNA dumbbells was developed. 5'-exonuclease converts end sequences of DNA targets into sticky ends. Self-looping oligonucleotides...
A method to build sequence-restricted DNA dumbbells was developed. 5'-exonuclease converts end sequences of DNA targets into sticky ends. Self-looping oligonucleotides with complementary 3'-overhangs are ligated to form dumbbells by DNA polymerase and ligase in a sequence-restricted manner. These reactions proceed in one pot at one temperature. We demonstrated one use of this method to 'tunnel' sequencing libraries into dumbbells for the Pacific Biosciences (PacBio) platform. Readouts of an Illumina P5/P7-ended 16S library from a standard microbial community confirmed successful tunneling. Twelve fecal samples additionally showed significant correlations between standard and tunneled 16S sequence variants on the PacBio platform. We further extended the method at a genomic scale to build a ∼0.45 Mbp giant dumbbell on chromosome 6. Sequences inside the dumbbells were successfully protected from a cocktail of exonucleases. Roughly 11-fold enrichment was achieved for the dumbbell-guarded region compared to the vicinity.
PubMed: 37215900
DOI: 10.1016/j.heliyon.2023.e16035 -
Cells Jul 2022Reperfusion injury after extended ischemia accounts for approximately 50% of myocardial infarct size, and there is no standard therapy. HDAC inhibition reduces infarct...
Reperfusion injury after extended ischemia accounts for approximately 50% of myocardial infarct size, and there is no standard therapy. HDAC inhibition reduces infarct size and enhances cardiomyocyte autophagy and PGC1α-mediated mitochondrial biogenesis when administered at the time of reperfusion. Furthermore, a specific autophagy-inducing peptide, Tat-Beclin 1 (TB), reduces infarct size when administered at the time of reperfusion. However, since SAHA affects multiple pathways in addition to inducing autophagy, whether autophagic flux induced by TB maintains mitochondrial homeostasis during ischemia/reperfusion (I/R) injury is unknown. We tested whether the augmentation of autophagic flux by TB has cardioprotection by preserving mitochondrial homeostasis both in vitro and in vivo. Wild-type mice were randomized into two groups: Tat-Scrambled (TS) peptide as the control and TB as the experimental group. Mice were subjected to I/R surgery (45 min coronary ligation, 24 h reperfusion). Autophagic flux, mitochondrial DNA (mtDNA), mitochondrial morphology, and mitochondrial dynamic genes were assayed. Cultured neonatal rat ventricular myocytes (NRVMs) were treated with a simulated I/R injury to verify cardiomyocyte specificity. The essential autophagy gene, ATG7, conditional cardiomyocyte-specific knockout (ATG7 cKO) mice, and isolated adult mouse ventricular myocytes (AMVMs) were used to evaluate the dependency of autophagy in adult cardiomyocytes. In NRVMs subjected to I/R, TB increased autophagic flux, mtDNA content, mitochondrial function, reduced reactive oxygen species (ROS), and mtDNA damage. Similarly, in the infarct border zone of the mouse heart, TB induced autophagy, increased mitochondrial size and mtDNA content, and promoted the expression of PGC1α and mitochondrial dynamic genes. Conversely, loss of ATG7 in AMVMs and in the myocardium of ATG7 cKO mice abolished the beneficial effects of TB on mitochondrial homeostasis. Thus, autophagic flux is a sufficient and essential process to mitigate myocardial reperfusion injury by maintaining mitochondrial homeostasis and partly by inducing PGC1α-mediated mitochondrial biogenesis.
Topics: Animals; Autophagy; Beclin-1; DNA, Mitochondrial; Homeostasis; Mice; Mitochondria; Myocardial Infarction; Myocardial Reperfusion Injury; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Rats; Rats, Sprague-Dawley
PubMed: 35805195
DOI: 10.3390/cells11132111 -
Proceedings of the National Academy of... Jun 2023The nonhomologous end-joining (NHEJ) pathway is a major DNA double-strand break repair pathway in mammals and is essential for lymphocyte development. Ku70 and Ku80...
The nonhomologous end-joining (NHEJ) pathway is a major DNA double-strand break repair pathway in mammals and is essential for lymphocyte development. Ku70 and Ku80 heterodimer (KU) initiates NHEJ, thereby recruiting and activating the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). While DNA-PKcs deletion only moderately impairs end-ligation, the expression of kinase-dead DNA-PKcs completely abrogates NHEJ. Active DNA-PK phosphorylates DNA-PKcs at two clusters-PQR around S2056 (S2053 in mouse) and ABCDE around T2609. Alanine substitution at the S2056 cluster moderately compromises end-ligation on plasmid-based assays. But, mice carrying alanine substitution at all five serine residues within the S2056 cluster () display no defect in lymphocyte development, leaving the physiological significance of S2056 cluster phosphorylation elusive. Xlf is a nonessential NHEJ factor. mice have substantial peripheral lymphocytes that are completely abolished by the loss of DNA-PKcs, the related ATM kinases, other chromatin-associated DNA damage response factors (e.g., 53BP1, MDC1, H2AX, and MRI), or RAG2-C-terminal regions, suggesting functional redundancy. While ATM inhibition does not further compromise end-ligation, here we show that in XLF-deficient background, DNA-PKcs S2056 cluster phosphorylation is critical for normal lymphocyte development. Chromosomal V(D)J recombination from B cells is efficient but often has large deletions that jeopardize lymphocyte development. Class-switch recombination junctions from mice are less efficient and the residual junctions display decreased fidelity and increased deletion. These findings establish a role for DNA-PKcs S2056 cluster phosphorylation in physiological chromosomal NHEJ, implying that S2056 cluster phosphorylation contributes to the synergy between XLF and DNA-PKcs in end-ligation.
Topics: Animals; Mice; Phosphorylation; Protein Kinases; Protein Processing, Post-Translational; Alanine; B-Lymphocytes; DNA-Activated Protein Kinase; Mammals; DNA-Binding Proteins
PubMed: 37307443
DOI: 10.1073/pnas.2221894120 -
Journal of the American Chemical Society Aug 2023Previously, nonenzymatic primer extension reaction of l-threoninol nucleic acid (L-TNA) was achieved in the presence of -cyanoimidazole (CNIm) and Mn; however, the...
Previously, nonenzymatic primer extension reaction of l-threoninol nucleic acid (L-TNA) was achieved in the presence of -cyanoimidazole (CNIm) and Mn; however, the reaction conditions were not optimized and a mechanistic insight was not sufficient. Herein, we report investigation of the kinetics and reaction mechanism of the chemical ligation of L-TNA to L-TNA and of DNA to DNA. We found that Cd, Ni, and Co accelerated ligation of both L-TNA and DNA and that the rate-determining step was activation of the phosphate group. The activation was enhanced by duplex formation between a phosphorylated L-TNA fragment and template, resulting in unexpectedly more effective L-TNA ligation than DNA ligation. Under optimized conditions, an 8-mer L-TNA primer could be elongated by ligation to L-TNA trimers to produce a 29-mer full-length oligomer with 60% yield within 2 h at 4 °C. This highly effective chemical ligation system will allow construction of artificial genomes, robust DNA nanostructures, and xeno nucleic acids for use in selection methods. Our findings also shed light on the possible pre-RNA world.
Topics: Nucleic Acids; DNA; Amino Alcohols; RNA; Nucleic Acid Conformation
PubMed: 37466125
DOI: 10.1021/jacs.3c04979 -
DNA Repair Oct 2022Chromosomal DNA double-strand breaks (DSBs) are the effective lesion of radiotherapy and other clastogenic cancer therapeutics, and are also the initiating event of many... (Review)
Review
Chromosomal DNA double-strand breaks (DSBs) are the effective lesion of radiotherapy and other clastogenic cancer therapeutics, and are also the initiating event of many approaches to gene editing. Ligation of the DSBs by end joining (EJ) pathways can restore the broken chromosome, but the repair junctions can have insertion/deletion (indel) mutations. The indel patterns resulting from DSB EJ are likely defined by the initial structure of the DNA ends, how the ends are processed and synapsed prior to ligation, and the factors that mediate the ligation step. In this review, we describe key factors that influence these steps of DSB EJ in mammalian cells, which is significant both for understanding mutagenesis resulting from clastogenic cancer therapeutics, and for developing approaches to manipulating gene editing outcomes.
Topics: Animals; Chromosome Breakage; DNA; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Repair; Humans; Mammals; Mutagenesis
PubMed: 35926296
DOI: 10.1016/j.dnarep.2022.103380 -
Chemical Research in Toxicology Apr 20211,2-Naphthoquinone, a secondary metabolite of naphthalene, is an environmental pollutant found in diesel exhaust particles that displays cytotoxic and genotoxic...
1,2-Naphthoquinone, a secondary metabolite of naphthalene, is an environmental pollutant found in diesel exhaust particles that displays cytotoxic and genotoxic properties. Because many quinones have been shown to act as topoisomerase II poisons, the effects of this compound on DNA cleavage mediated by human topoisomerase IIα and IIβ were examined. The compound increased the levels of double-stranded DNA breaks generated by both enzyme isoforms and did so better than a series of naphthoquinone derivatives. Furthermore, 1,2-naphthoquinone was a more efficacious poison against topoisomerase IIα than IIβ. Topoisomerase II poisons can be classified as interfacial (which interact noncovalently at the enzyme-DNA interface and increase DNA cleavage by blocking ligation) or covalent (which adduct the protein and increase DNA cleavage by closing the N-terminal gate of the enzyme). Therefore, experiments were performed to determine the mechanistic basis for the actions of 1,2-naphthoquinone. In contrast to results with etoposide (an interfacial poison), the activity of 1,2-naphthoquinone against topoisomerase IIα was abrogated in the presence of sulfhydryl and reducing agents. Moreover, the compound inhibited cleavage activity when incubated with the enzyme prior to the addition of DNA and induced virtually no cleavage with the catalytic core of the enzyme. It also induced stable covalent topoisomerase IIα-DNA cleavage complexes and was a partial inhibitor of DNA ligation. Findings were also consistent with 1,2-naphthoquinone acting as a covalent poison of topoisomerase IIβ; however, mechanistic studies with this isoform were less conclusive. Whereas the activity of 1,2-naphthoquinone was blocked in the presence of a sulfhydryl reagent, it was much less sensitive to the presence of a reducing agent. Furthermore, the reduced form of 1,2-naphthoquinone, 1,2-dihydroxynaphthalene, displayed high activity against the β isoform. Taken together, results suggest that 1,2-naphthoquinone increases topoisomerase II-mediated double-stranded DNA scission (at least in part) by acting as a covalent poison of the human type II enzymes.
Topics: DNA Cleavage; DNA Topoisomerases, Type II; Humans; Molecular Structure; Naphthoquinones; Recombinant Proteins; Topoisomerase II Inhibitors
PubMed: 33760604
DOI: 10.1021/acs.chemrestox.0c00492 -
Clinical Chemistry and Laboratory... Jan 2024Cancer morbidity and mortality can be reduced if the cancer is detected early. Cell-free DNA (cfDNA) fragmentomics emerged as a novel epigenetic biomarker for early...
OBJECTIVES
Cancer morbidity and mortality can be reduced if the cancer is detected early. Cell-free DNA (cfDNA) fragmentomics emerged as a novel epigenetic biomarker for early cancer detection, however, it is still at its infancy and requires technical improvement. We sought to apply a single-strand DNA sequencing technology, for measuring genetic and fragmentomic features of cfDNA and evaluate the performance in detecting multiple cancers.
METHODS
Blood samples of 364 patients from six cancer types (colorectal, esophageal, gastric, liver, lung, and ovarian cancers) and 675 healthy individuals were included in this study. Circulating tumor DNA mutations, cfDNA fragmentomic features and a set of protein biomarkers were assayed. Sensitivity and specificity were reported by cancer types and stages.
RESULTS
Circular Ligation Amplification and sequencing (CLAmp-seq), a single-strand DNA sequencing technology, yielded a population of ultra-short fragments (<100 bp) than double-strand DNA preparation protocols and reveals a more significant size difference between cancer and healthy cfDNA fragments (25.84 bp vs. 16.05 bp). Analysis of the subnucleosomal peaks in ultra-short cfDNA fragments indicates that these peaks are regulatory element "footprints" and correlates with gene expression and cancer stages. At 98 % specificity, a prediction model using ctDNA mutations alone showed an overall sensitivity of 46 %; sensitivity reaches 60 % when protein is added, sensitivity further increases to 66 % when fragmentomics is also integrated. More improvements observed for samples representing earlier cancer stages than later ones.
CONCLUSIONS
These results suggest synergistic properties of protein, genetic and fragmentomics features in the identification of early-stage cancers.
Topics: Humans; Cell-Free Nucleic Acids; Early Detection of Cancer; Mutation; Circulating Tumor DNA; Neoplasms; Biomarkers, Tumor
PubMed: 37678194
DOI: 10.1515/cclm-2023-0541 -
Annual Review of Biochemistry Jun 2021DNA double-strand breaks pose a serious threat to genome stability. In vertebrates, these breaks are predominantly repaired by nonhomologous end joining (NHEJ), which... (Review)
Review
DNA double-strand breaks pose a serious threat to genome stability. In vertebrates, these breaks are predominantly repaired by nonhomologous end joining (NHEJ), which pairs DNA ends in a multiprotein synaptic complex to promote their direct ligation. NHEJ is a highly versatile pathway that uses an array of processing enzymes to modify damaged DNA ends and enable their ligation. The mechanisms of end synapsis and end processing have important implications for genome stability. Rapid and stable synapsis is necessary to limit chromosome translocations that result from the mispairing of DNA ends. Furthermore, end processing must be tightly regulated to minimize mutations at the break site. Here, we review our current mechanistic understanding of vertebrate NHEJ, with a particular focus on end synapsis and processing.
Topics: Animals; DNA Breaks, Double-Stranded; DNA End-Joining Repair; Enzymes; Genomic Instability; Humans; Models, Biological; Multiprotein Complexes; V(D)J Recombination
PubMed: 33556282
DOI: 10.1146/annurev-biochem-080320-110356