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Nature Communications May 2020CRISPR-associated (Cas) DNA-endonucleases are remarkably effective tools for genome engineering, but have limited target ranges due to their protospacer adjacent motif...
CRISPR-associated (Cas) DNA-endonucleases are remarkably effective tools for genome engineering, but have limited target ranges due to their protospacer adjacent motif (PAM) requirements. We demonstrate a critical expansion of the targetable sequence space for a type II-A CRISPR-associated enzyme through identification of the natural 5[Formula: see text]-NAAN-3[Formula: see text] PAM preference of Streptococcus macacae Cas9 (SmacCas9). To achieve efficient editing activity, we graft the PAM-interacting domain of SmacCas9 to its well-established ortholog from Streptococcus pyogenes (SpyCas9), and further engineer an increased efficiency variant (iSpyMac) for robust genome editing activity. We establish that our hybrids can target all adenine dinucleotide PAM sequences and possess robust and accurate editing capabilities in human cells.
Topics: Adenine; Amino Acid Sequence; CRISPR-Associated Protein 9; Dinucleoside Phosphates; Gene Editing; HEK293 Cells; Humans; Nucleotide Motifs; Reproducibility of Results; Streptococcus
PubMed: 32424114
DOI: 10.1038/s41467-020-16117-8 -
International Journal of Molecular... Dec 2022Novel sulfur and selenium substituted 5',5'-linked dinucleoside pyrophate analogues were prepared in a vibration ball mill from the corresponding persilylated...
Novel sulfur and selenium substituted 5',5'-linked dinucleoside pyrophate analogues were prepared in a vibration ball mill from the corresponding persilylated monophosphate. The chemical hydrolysis of pyrophosphorochalcogenolate-linked dimers was studied over a wide pH-range. The effect of the chalcogeno-substitution on the reactivity of dinucleoside pyrophosphates was surprisingly modest, and the chemical stability is promising considering the potential therapeutic or diagnostic applications. The chemical stability of the precursor phosphorochalcogenolate monoesters was also investigated. Hydrolytic desilylation of these materials was effected in aqueous buffer at pH 3, 7 or 11 and resulted in phosphorus-chalcogen bond scission which was monitored using P NMR. The rate of dephosphorylation was dependent upon both the nature of the chalcogen and the pH. The integrity of the P-S bond in the corresponding phosphorothiolate was maintained at high pH but rapidly degraded at pH 3. In contrast, P-Se bond cleavage of the phosphoroselenolate monoester was rapid and the rate increased with alkalinity. The results obtained in kinetic experiments provide insight on the reactivity of the novel pyrophosphates studied as well as of other types of thiosubstituted biological phosphates. At the same time, these results also provide evidence for possible formation of unexpectedly reactive intermediates as the chalcogen-substituted analogues are metabolised.
Topics: Nucleosides; Phosphates; Hydrolysis; Diphosphates; Chalcogens
PubMed: 36555224
DOI: 10.3390/ijms232415582 -
Scientific Reports Jul 2022In this study, we probe the role of secondary messenger c-di-AMP in drug tolerance, which includes both persister and resistant mutant characterization of Mycobacterium...
In this study, we probe the role of secondary messenger c-di-AMP in drug tolerance, which includes both persister and resistant mutant characterization of Mycobacterium smegmatis. Specifically, with the use of c-di-AMP null and overproducing mutants, we showed how c-di-AMP plays a significant role in resistance mutagenesis against antibiotics with different mechanisms of action. We elucidated the specific molecular mechanism linking the elevated intracellular c-di-AMP level and high mutant generation and highlighted the significance of non-homology-based DNA repair. Further investigation enabled us to identify the unique mutational landscape of target and non-target mutation categories linked to intracellular c-di-AMP levels. Overall fitness cost of unique target mutations was estimated in different strain backgrounds, and then we showed the critical role of c-di-AMP in driving epistatic interactions between resistance genes, resulting in the evolution of multi-drug tolerance. Finally, we identified the role of c-di-AMP in persister cells regrowth and mutant enrichment upon cessation of antibiotic treatment.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Dinucleoside Phosphates; Drug Tolerance; Mycobacterium smegmatis; Phenotype
PubMed: 35907936
DOI: 10.1038/s41598-022-17051-z -
Chemistry (Weinheim An Der Bergstrasse,... May 2020Bacterial cyclic dinucleotides (CDNs) play important roles in regulating biofilm formation, motility and virulence. In eukaryotic cells, theses bacterial CDNs are...
Bacterial cyclic dinucleotides (CDNs) play important roles in regulating biofilm formation, motility and virulence. In eukaryotic cells, theses bacterial CDNs are recognized as pathogen-associated molecular patterns (PAMPs) and trigger an innate immune response. We report the photophysical analyses of a novel group of enzymatically synthesized emissive CDN analogues comprised of two families of isomorphic ribonucleotides. The highly favorable photophysical features of the CDN analogues, when compared to their non-emissive natural counterparts, are used to monitor in real time the dinucleotide cyclase-mediated synthesis and phosphodiesterase (PDE)-mediated hydrolysis of homodimeric and mixed CDNs, providing effective means to probe the activities of two classes of bacterial enzymes and insight into their biomolecular recognition and catalytic features.
Topics: Bacterial Proteins; Dinucleoside Phosphates; Immunity, Innate; Protein Biosynthesis; Vibrio cholerae
PubMed: 32157755
DOI: 10.1002/chem.202001194 -
Journal of Bacteriology Apr 2020Diadenosine tetraphosphate (ApA) is a dinucleotide found in both prokaryotes and eukaryotes. In bacteria, its cellular levels increase following exposure to various...
Diadenosine tetraphosphate (ApA) is a dinucleotide found in both prokaryotes and eukaryotes. In bacteria, its cellular levels increase following exposure to various stress signals and stimuli, and its accumulation is generally correlated with increased sensitivity to a stressor(s), decreased pathogenicity, and enhanced antibiotic susceptibility. ApA is produced as a by-product of tRNA aminoacylation, and is cleaved to ADP molecules by hydrolases of the ApaH and Nudix families and/or by specific phosphorylases. Here, considering evidence that the recombinant protein YqeK from copurified with ADP, and aided by thermal shift and kinetic analyses, we identified the YqeK family of proteins (COG1713) as an unprecedented class of symmetrically cleaving ApA hydrolases. We validated the functional assignment by confirming the ability of YqeK to affect levels of ApA in YqeK shows a catalytic efficiency toward ApA similar to that of the symmetrically cleaving ApA hydrolases of the known ApaH family, although it displays a distinct fold that is typical of proteins of the HD domain superfamily harboring a diiron cluster. Analysis of the available 3D structures of three members of the YqeK family provided hints to the mode of substrate binding. Phylogenetic analysis revealed the occurrence of YqeK proteins in a consistent group of Gram-positive bacteria that lack ApaH enzymes. Comparative genomics highlighted that and genes share a similar genomic context, where they are frequently found in operons involved in integrated responses to stress signals. Elevation of ApA level in bacteria is associated with increased sensitivity to heat and oxidative stress, reduced antibiotic tolerance, and decreased pathogenicity. ApaH is the major ApA hydrolase in gamma- and betaproteobacteria and has been recently proposed as a novel target to weaken the bacterial resistance to antibiotics. Here, we identified the orphan YqeK protein family (COG1713) as a highly efficient ApA hydrolase family, with members distributed in a consistent group of bacterial species that lack the ApaH enzyme. Among them are the pathogens , , and By identifying the player contributing to ApA homeostasis in these bacteria, we disclose a novel target to develop innovative antibacterial strategies.
Topics: Acid Anhydride Hydrolases; Adenosine Diphosphate; Amino Acid Sequence; Bacteria; Bacterial Proteins; Catalysis; Cloning, Molecular; Dinucleoside Phosphates; Kinetics; Multigene Family; Phylogeny; Sequence Alignment; Staphylococcus aureus
PubMed: 32152217
DOI: 10.1128/JB.00053-20 -
Open Heart Aug 2022To estimate the incidence and HRs for bleeding for different dual antiplatelet therapies (DAPT) in a real-world population with acute coronary syndrome (ACS) undergoing... (Randomized Controlled Trial)
Randomized Controlled Trial
Real-world bleeding in patients with acute coronary syndrome (ACS) undergoing percutaneous coronary intervention (PCI) and prescribed different combinations of dual antiplatelet therapy (DAPT) in England: a population-based cohort study emulating a 'target trial'.
OBJECTIVE
To estimate the incidence and HRs for bleeding for different dual antiplatelet therapies (DAPT) in a real-world population with acute coronary syndrome (ACS) undergoing percutaneous coronary intervention (PCI) in England.
DESIGN
A retrospective, population-based cohort study emulating a target randomised controlled trial (tRCT).
DATA SOURCES
Linked Clinical Practice Research Datalink (CPRD) and Hospital Episode Statistics (HES).
SETTING
Primary and secondary care.
PARTICIPANTS
Patients ≥18 years old with ACS undergoing emergency PCI.
INTERVENTIONS
Aspirin and clopidogrel (AC, reference) versus aspirin and prasugrel (AP) or aspirin and ticagrelor (AT); AP evaluated only in patients with ST-elevation myocardial infarction (STEMI).
MAIN OUTCOME MEASURES
Primary: any bleeding up to 12 months after the index event (HES- or CPRD- recorded). Secondary: HES-recorded bleeding, CPRD-recorded bleeding, all-cause and cardiovascular mortality, mortality from bleeding, myocardial infarction, stroke, additional coronary intervention and major adverse cardiovascular and cerebrovascular events (MACCE).
RESULTS
In ACS, the rates of any bleeding for AC and AT were 89 per 1000 person years and 134 per 1000 person years, respectively. In STEMI, rates for AC, AP and AT were 93 per 1000 person years, 138 per 1000 person years and 143 per 100 person years, respectively. In ACS, compared with AC, AT increased the hazard of any bleeding (HR: 1.47, 95% CI 1.19 to 1.82) but did not reduce MACCE (HR: 1.06, 95% CI 0.89 to 1.27). In STEMI, compared with AC, AP and AT increased the hazard of any bleeding (HR: 1.77, 95% CI 1.21 to 2.59 and HR: 1.50, 95% CI 1.10 to 2.05, respectively) but did not reduce MACCE (HR: 1.10, 95% CI 0.80 to 1.51 and HR: 1.21, 95% CI 0.94 to 1.51, respectively). Non-adherence to the prescribed DAPT regimen was 28% in AC (29% in STEMI only), 31% in AP (STEMI only) and 33% in AT (32% in STEMI only).
CONCLUSIONS
In a real-world population with ACS, DAPT with ticagrelor or prasugrel are associated with increased bleeding compared with DAPT with clopidogrel.
TRIAL REGISTRATION NUMBER
ISRCTN76607611.
Topics: Acute Coronary Syndrome; Adolescent; Aspirin; Clopidogrel; Cohort Studies; Dinucleoside Phosphates; Hemorrhage; Humans; Percutaneous Coronary Intervention; Platelet Aggregation Inhibitors; Prasugrel Hydrochloride; Retrospective Studies; ST Elevation Myocardial Infarction; Ticagrelor
PubMed: 35961692
DOI: 10.1136/openhrt-2022-001999 -
Scientific Reports Feb 2023Epithelial ovarian cancer is the most lethal gynecological malignancy, owing notably to its high rate of therapy-resistant recurrence in spite of good initial response...
Inhibition of nicotinamide dinucleotide salvage pathway counters acquired and intrinsic poly(ADP-ribose) polymerase inhibitor resistance in high-grade serous ovarian cancer.
Epithelial ovarian cancer is the most lethal gynecological malignancy, owing notably to its high rate of therapy-resistant recurrence in spite of good initial response to chemotherapy. Although poly(ADP-ribose) polymerase inhibitors (PARPi) have shown promise for ovarian cancer treatment, extended therapy usually leads to acquired PARPi resistance. Here we explored a novel therapeutic option to counter this phenomenon, combining PARPi and inhibitors of nicotinamide phosphoribosyltransferase (NAMPT). Cell-based models of acquired PARPi resistance were created through an in vitro selection procedure. Using resistant cells, xenograft tumors were grown in immunodeficient mice, while organoid models were generated from primary patient tumor samples. Intrinsically PARPi-resistant cell lines were also selected for analysis. Our results show that treatment with NAMPT inhibitors effectively sensitized all in vitro models to PARPi. Adding nicotinamide mononucleotide, the resulting NAMPT metabolite, abrogated the therapy-induced cell growth inhibition, demonstrating the specificity of the synergy. Treatment with olaparib (PARPi) and daporinad (NAMPT inhibitor) depleted intracellular NAD+ , induced double-strand DNA breaks, and promoted apoptosis as monitored by caspase-3 cleavage. The two drugs were also synergistic in mouse xenograft models and clinically relevant patient-derived organoids. Therefore, in the context of PARPi resistance, NAMPT inhibition could offer a promising new option for ovarian cancer patients.
Topics: Humans; Animals; Mice; Female; Poly(ADP-ribose) Polymerase Inhibitors; Niacinamide; Antineoplastic Agents; Ovarian Neoplasms; Dinucleoside Phosphates
PubMed: 36849518
DOI: 10.1038/s41598-023-30081-5 -
The Journal of Biological Chemistry Jul 2022The bacterial second messenger c-di-AMP controls essential cellular processes, including potassium and osmolyte homeostasis. This makes synthesizing enzymes and...
The bacterial second messenger c-di-AMP controls essential cellular processes, including potassium and osmolyte homeostasis. This makes synthesizing enzymes and components involved in c-di-AMP signal transduction intriguing as potential targets for drug development. The c-di-AMP receptor protein DarB of Bacillus subtilis binds the Rel protein and triggers the Rel-dependent stringent response to stress conditions; however, the structural basis for this trigger is unclear. Here, we report crystal structures of DarB in the ligand-free state and of DarB complexed with c-di-AMP, 3'3'-cGAMP, and AMP. We show that DarB forms a homodimer with a parallel, head-to-head assembly of the monomers. We also confirm the DarB dimer binds two cyclic dinucleotide molecules or two AMP molecules; only one adenine of bound c-di-AMP is specifically recognized by DarB, while the second protrudes out of the donut-shaped protein. This enables DarB to bind also 3'3'-cGAMP, as only the adenine fits in the active site. In absence of c-di-AMP, DarB binds to Rel and stimulates (p)ppGpp synthesis, whereas the presence of c-di-AMP abolishes this interaction. Furthermore, the DarB crystal structures reveal no conformational changes upon c-di-AMP binding, leading us to conclude the regulatory function of DarB on Rel must be controlled directly by the bound c-di-AMP. We thus derived a structural model of the DarB-Rel complex via in silico docking, which was validated with mass spectrometric analysis of the chemically crosslinked DarB-Rel complex and mutagenesis studies. We suggest, based on the predicted complex structure, a mechanism of stringent response regulation by c-di-AMP.
Topics: Adenine; Adenosine Monophosphate; Bacillus subtilis; Bacterial Proteins; Dinucleoside Phosphates
PubMed: 35714772
DOI: 10.1016/j.jbc.2022.102144 -
Cell Reports Apr 2023Programmed cell suicide of infected bacteria, known as abortive infection (Abi), serves as an immune defense strategy to prevent the propagation of bacteriophage...
Programmed cell suicide of infected bacteria, known as abortive infection (Abi), serves as an immune defense strategy to prevent the propagation of bacteriophage viruses. Many Abi systems utilize bespoke cyclic nucleotide immune messengers generated upon infection to mobilize cognate death effectors. Here, we identify a family of bacteriophage nucleotidyltransferases (NTases) that synthesize competitor cyclic dinucleotide (CDN) ligands and inhibit TIR NADase effectors activated via a linked STING CDN sensor domain (TIR-STING). Through a functional screen of NTase-adjacent phage genes, we uncover candidate inhibitors of cell suicide induced by heterologous expression of tonically active TIR-STING. Among these, we demonstrate that a virus MazG-like nucleotide pyrophosphohydrolase, Atd1, depletes the starvation alarmone (p)ppGpp, revealing a potential role for the alarmone-activated host toxin MazF as an executioner of TIR-driven Abi. Phage NTases and counterdefenses like Atd1 preserve host viability to ensure virus propagation and represent tools to modulate TIR and STING immune responses.
Topics: Bacteria; Bacteriophages; Dinucleoside Phosphates; Guanosine Pentaphosphate; Immunity; Nucleotides; Nucleotidyltransferases
PubMed: 36952342
DOI: 10.1016/j.celrep.2023.112305 -
Journal of the American Chemical Society Oct 2021Triazole linkages (TLs) are mimics of the phosphodiester bond in oligonucleotides with applications in synthetic biology and biotechnology. Here we report the...
Triazole linkages (TLs) are mimics of the phosphodiester bond in oligonucleotides with applications in synthetic biology and biotechnology. Here we report the RuAAC-catalyzed synthesis of a novel 1,5-disubstituted triazole (TL) dinucleoside phosphoramidite as well as its incorporation into oligonucleotides and compare its DNA polymerase replication competency with other TL analogues. We demonstrate that TL has superior replication kinetics to these analogues and is accurately replicated by polymerases. Derived structure-biocompatibility relationships show that linker length and the orientation of a hydrogen bond acceptor are critical and provide further guidance for the rational design of artificial biocompatible nucleic acid backbones.
Topics: Catalysis; DNA; DNA-Directed DNA Polymerase; Dinucleoside Phosphates; Molecular Mimicry; Triazoles
PubMed: 34546729
DOI: 10.1021/jacs.1c08057