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1.
Nucleic Acids Res ; 49(5): 2777-2789, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33590098

RESUMO

Cells and organisms have a wide range of mechanisms to defend against infection by viruses and other mobile genetic elements (MGE). Type III CRISPR systems detect foreign RNA and typically generate cyclic oligoadenylate (cOA) second messengers that bind to ancillary proteins with CARF (CRISPR associated Rossman fold) domains. This results in the activation of fused effector domains for antiviral defence. The best characterised CARF family effectors are the Csm6/Csx1 ribonucleases and DNA nickase Can1. Here we investigate a widely distributed CARF family effector with a nuclease domain, which we name Can2 (CRISPR ancillary nuclease 2). Can2 is activated by cyclic tetra-adenylate (cA4) and displays both DNase and RNase activity, providing effective immunity against plasmid transformation and bacteriophage infection in Escherichia coli. The structure of Can2 in complex with cA4 suggests a mechanism for the cA4-mediated activation of the enzyme, whereby an active site cleft is exposed on binding the activator. These findings extend our understanding of type III CRISPR cOA signalling and effector function.


Assuntos
Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Desoxirribonuclease I/química , Ribonucleases/química , Clostridiales/enzimologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/química , Desoxirribonuclease I/metabolismo , Ativação Enzimática , Escherichia coli/virologia , Sequências Repetitivas Dispersas , Metais/química , Modelos Moleculares , Domínios Proteicos , Ribonucleases/metabolismo
2.
Nucleic Acids Res ; 49(2): 1046-1064, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33410911

RESUMO

Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and for anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay, termed HUH-seq, to interrogate subtleties in Rep specificity and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed engineering of only four amino acids in a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.


Assuntos
DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Desoxirribonuclease I/metabolismo , Conformação de Ácido Nucleico , Conformação Proteica , Engenharia de Proteínas/métodos , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Transativadores/metabolismo , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Circoviridae/enzimologia , Sequência Conservada , Cristalografia por Raios X , DNA Helicases/química , DNA de Cadeia Simples/química , Desoxirribonuclease I/química , Biblioteca Gênica , Modelos Moleculares , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Vírus de Plantas/enzimologia , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Origem de Replicação , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Endonucleases Específicas para DNA e RNA de Cadeia Simples/química , Especificidade por Substrato , Transativadores/química , Proteínas Virais/química
3.
Int J Nanomedicine ; 16: 185-199, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33447034

RESUMO

Background: Therapy for glioblastoma (GBM) has always been very challenging, not only because of the presence of the blood-brain barrier (BBB) but also due to susceptibility to drug resistance. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) has revolutionized gene editing technology and is capable of treating a variety of genetic diseases, including human tumors, but there is a lack of safe and effective targeting delivery systems in vivo, especially in the central nervous system (CNS). Methods: Lipid-polymer hybrid nanoparticles (LPHNs-cRGD) were constructed for efficient and targeting delivery of CRISPR/Cas9 plasmids targeting O6-methylguanine-DNA methyltransferase (MGMT), a drug-resistance gene to temozolomide (TMZ). Focused ultrasound (FUS)-microbubbles (MBs) were used to non-invasively and locally open the BBB to further facilitate gene delivery into glioblastoma in vivo. The gene editing efficiency and drug sensitivity changes were evaluated both in vitro and in vivo. Results: The gene-loaded LPHNs-cRGD were successfully synthesized and could protect pCas9/MGMT from enzyme degradation. LPHNs-cRGD could target GBM cells and mediate the transfection of pCas9/MGMT to downregulate the expression of MGMT, resulting in an increased sensitivity of GBM cells to TMZ. MBs-LPHNs-cRGD complexes could safely and locally increase the permeability of the BBB with FUS irradiation in vivo and facilitated the accumulation of nanoparticles at the tumor region in orthotopic tumor-bearing mice. Furthermore, the FUS-assisted MBs-LPHNspCas9/MGMT-cRGD enhanced the therapeutic effects of TMZ in glioblastoma, inhibited tumor growth, and prolonged survival of tumor-bearing mice, with a high level of biosafety. Conclusion: In this work, we constructed LPHNs-cRGD for targeting delivery of the CRISPR/Cas9 system, in combination with FUS-MBs to open the BBB. The MBs-LPHNs-cRGD delivery system could be a potential alternative for efficient targeting gene delivery for the treatment of glioblastoma.


Assuntos
Neoplasias Encefálicas/terapia , Resistencia a Medicamentos Antineoplásicos , Terapia Genética , Glioblastoma/terapia , Ablação por Ultrassom Focalizado de Alta Intensidade , Lipídeos/química , Nanopartículas/química , Polímeros/química , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Desoxirribonuclease I/metabolismo , Glioblastoma/tratamento farmacológico , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Microbolhas , Nanopartículas/ultraestrutura , Peptídeos Cíclicos/química , Plasmídeos/genética , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Distribuição Tecidual , Transfecção
4.
Nucleic Acids Res ; 49(2): 1173-1198, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33398349

RESUMO

RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by 'tiptoeing' over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of DNA knockout and knock-in procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.


Assuntos
Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Desoxirribonuclease I/metabolismo , Edição de Genes/métodos , Proteínas de Bactérias/genética , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Células Clonais , Desoxirribonuclease I/genética , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Genes Reporter , Técnicas de Genotipagem , Células HEK293 , Células HeLa , Heterocromatina/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas , Polimorfismo Genético , RNA Guia/genética , Proteínas Recombinantes/metabolismo , Streptococcus pyogenes/enzimologia , Especificidade por Substrato , Transfecção
5.
Methods Mol Biol ; 2196: 27-37, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32889710

RESUMO

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has enabled efficient, markerless genome editing in a wide range of organisms. However, there is an off-target effect and a limit to the area of precise editing. Bases that can be precisely edited are limited to within the 20-base pair gRNA-targeting site and protospacer adjacent motif (PAM) sequence. We have developed a CRISPR nickase system that can perform a precise genome-wide base editing in Saccharomyces cerevisiae using a single Cas9 nickase. This system can precisely edit a broader genomic region by the avoidance of double-strand break (DSB) and subsequent non-homologous end joining (NHEJ). Furthermore, unintended mutations were not found at off-target sites in this system. In combination with yeast gap repair cloning, precise genome editing of yeast cells can be performed in 5 days. Here, we describe the methods for precise and convenient genome editing using this novel CRISPR nickase system.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desoxirribonuclease I/metabolismo , Edição de Genes , Leveduras/genética , Leveduras/metabolismo , Sistemas CRISPR-Cas , Reparo do DNA , Ordem dos Genes , Vetores Genéticos/genética , Mutagênese , RNA Guia , Reparo de DNA por Recombinação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
6.
Nucleic Acids Res ; 49(2): 1023-1032, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33367848

RESUMO

Pseudomonas putida MPE exemplifies a novel clade of manganese-dependent single-strand DNA endonuclease within the binuclear metallophosphoesterase superfamily. MPE is encoded within a widely conserved DNA repair operon. Via structure-guided mutagenesis, we identify His113 and His81 as essential for DNA nuclease activity, albeit inessential for hydrolysis of bis-p-nitrophenylphosphate. We propose that His113 contacts the scissile phosphodiester and serves as a general acid catalyst to expel the OH leaving group of the product strand. We find that MPE cleaves the 3' and 5' single-strands of tailed duplex DNAs and that MPE can sense and incise duplexes at sites of short mismatch bulges and opposite a nick. We show that MPE is an ambidextrous phosphodiesterase capable of hydrolyzing the ssDNA backbone in either orientation to generate a mixture of 3'-OH and 3'-PO4 cleavage products. The directionality of phosphodiester hydrolysis is dictated by the orientation of the water nucleophile vis-à-vis the OH leaving group, which must be near apical for the reaction to proceed. We propose that the MPE active site and metal-bound water nucleophile are invariant and the enzyme can bind the ssDNA productively in opposite orientations.


Assuntos
Proteínas de Bactérias/metabolismo , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Desoxirribonuclease I/metabolismo , Pseudomonas putida/enzimologia , Proteínas de Bactérias/química , Pareamento de Bases , Domínio Catalítico , Reparo de Erro de Pareamento de DNA , Reparo do DNA , Enzimas Reparadoras do DNA/química , Desoxirribonuclease I/química , Histidina/química , Hidrólise , Manganês/química , Modelos Moleculares , Nitrofenóis/metabolismo , Fosfatos/química , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Água
7.
Food Chem ; 339: 128059, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33152864

RESUMO

A colorimetric aptasensing strategy for detection of kanamycin was designed based on aptamer biorecognition and signal amplification assisted by nicking enzyme. The aptamer of kanamycin was designed to be contained in the metastable state hairpin DNA. The target DNA as recycling DNA was located in the loop of hairpin DNA. The presence of kanamycin stimulates the continuous actions, including specific recognition of the aptamer to kanamycin, the hybridization between target DNA and signal probe, the cleavage function of nicking enzyme. The actions induced accumulation of numerous free short sequences modified by platinum nanoparticles (PtNPs), which can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 to produce a colorimetric response. The aptasensor exhibited good selectivity and sensitivity for kanamycin in milk with a detection limit as low as 0.2 pg·mL-1. In addition, the proposed assay is potentially to be extended for other antibiotics detection in foods by adapting the corresponding aptamer sequence.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , Colorimetria/métodos , Desoxirribonuclease I/metabolismo , Sequências Repetidas Invertidas , Canamicina/análise , Leite/química , Animais , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Benzidinas/química , Sondas de DNA/química , Contaminação de Alimentos/análise , Peróxido de Hidrogênio/química , Canamicina/metabolismo , Limite de Detecção , Nanopartículas Metálicas/química , Hibridização de Ácido Nucleico , Platina/química
8.
Nat Commun ; 11(1): 5352, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097693

RESUMO

Prime editing is a recent genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. Here, we develop prime editing for primary adult stem cells grown in organoid culture models. First, we generate precise in-frame deletions in the gene encoding ß-catenin (CTNNB1) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers disease-causing mutations in intestinal organoids from patients with DGAT1-deficiency and liver organoids from a patient with Wilson disease (ATP7B). Prime editing is as efficient in 3D grown organoids as in 2D grown cell lines and offers greater precision than Cas9-mediated homology directed repair (HDR). Base editing remains more reliable than prime editing but is restricted to a subgroup of pathogenic mutations. Whole-genome sequencing of four prime-edited clonal organoid lines reveals absence of genome-wide off-target effects underscoring therapeutic potential of this versatile and precise gene editing strategy.


Assuntos
Edição de Genes/métodos , Organoides/metabolismo , beta Catenina/genética , Sistemas CRISPR-Cas , Linhagem Celular , Proliferação de Células , ATPases Transportadoras de Cobre/genética , Desoxirribonuclease I/metabolismo , Diacilglicerol O-Aciltransferase/genética , Células HEK293 , Degeneração Hepatolenticular/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Reparo de DNA por Recombinação , Células-Tronco , Reparo Gênico Alvo-Dirigido/métodos
9.
Nat Commun ; 11(1): 5241, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067443

RESUMO

To understand how the RuvC catalytic domain of Class 2 Cas proteins cleaves DNA, it will be necessary to elucidate the structures of RuvC-containing Cas complexes in their catalytically competent states. Cas12i2 is a Class 2 type V-I CRISPR-Cas endonuclease that cleaves target dsDNA by an unknown mechanism. Here, we report structures of Cas12i2-crRNA-DNA complexes and a Cas12i2-crRNA complex. We reveal the mechanism of DNA recognition and cleavage by Cas12i2, and activation of the RuvC catalytic pocket induced by a conformational change of the Helical-II domain. The seed region (nucleotides 1-8) is dispensable for RuvC activation, but the duplex of the central spacer (nucleotides 9-15) is required. We captured the catalytic state of Cas12i2, with both metal ions and the ssDNA substrate bound in the RuvC catalytic pocket. Together, our studies provide significant insights into the DNA cleavage mechanism by RuvC-containing Cas proteins.


Assuntos
DNA de Cadeia Simples/metabolismo , Desoxirribonuclease I/química , Desoxirribonuclease I/metabolismo , Metais/metabolismo , Catálise , Domínio Catalítico , Clivagem do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Desoxirribonuclease I/genética , Íons/química , Íons/metabolismo , Metais/química
10.
Proc Natl Acad Sci U S A ; 117(34): 20689-20695, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32788345

RESUMO

RNA abasic sites and the mechanisms involved in their regulation are mostly unknown; in contrast, DNA abasic sites are well-studied. We found surprisingly that, in yeast and human cells, RNA abasic sites are prevalent. When a base is lost from RNA, the remaining ribose is found as a closed-ring or an open-ring sugar with a reactive C1' aldehyde group. Using primary amine-based reagents that react with the aldehyde group, we uncovered evidence for abasic sites in nascent RNA, messenger RNA, and ribosomal RNA from yeast and human cells. Mass spectroscopic analysis confirmed the presence of RNA abasic sites. The RNA abasic sites were found to be coupled to R-loops. We show that human methylpurine DNA glycosylase cleaves N-glycosidic bonds on RNA and that human apurinic/apyrimidinic endonuclease 1 incises RNA abasic sites in RNA-DNA hybrids. Our results reveal that, in yeast and human cells, there are RNA abasic sites, and we identify a glycosylase that generates these sites and an AP endonuclease that processes them.


Assuntos
Sequência de Bases/genética , RNA/química , RNA/genética , Sítios de Ligação , DNA/química , Dano ao DNA/genética , DNA Glicosilases/metabolismo , Reparo do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Desoxirribonuclease I/metabolismo , Humanos , Nucleotídeos/genética , Estruturas R-Loop/genética , Saccharomyces cerevisiae/genética , Especificidade por Substrato , Leveduras/genética
11.
Nature ; 584(7820): 244-251, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32728217

RESUMO

DNase I hypersensitive sites (DHSs) are generic markers of regulatory DNA1-5 and contain genetic variations associated with diseases and phenotypic traits6-8. We created high-resolution maps of DHSs from 733 human biosamples encompassing 438 cell and tissue types and states, and integrated these to delineate and numerically index approximately 3.6 million DHSs within the human genome sequence, providing a common coordinate system for regulatory DNA. Here we show that these maps highly resolve the cis-regulatory compartment of the human genome, which encodes unexpectedly diverse cell- and tissue-selective regulatory programs at very high density. These programs can be captured comprehensively by a simple vocabulary that enables the assignment to each DHS of a regulatory barcode that encapsulates its tissue manifestations, and global annotation of protein-coding and non-coding RNA genes in a manner orthogonal to gene expression. Finally, we show that sharply resolved DHSs markedly enhance the genetic association and heritability signals of diseases and traits. Rather than being confined to a small number of distal elements or promoters, we find that genetic signals converge on congruently regulated sets of DHSs that decorate entire gene bodies. Together, our results create a universal, extensible coordinate system and vocabulary for human regulatory DNA marked by DHSs, and provide a new global perspective on the architecture of human gene regulation.


Assuntos
Cromatina/genética , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Anotação de Sequência Molecular , Cromatina/química , Cromatina/metabolismo , DNA/química , DNA/genética , Regulação da Expressão Gênica , Genes/genética , Genoma Humano/genética , Humanos , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genética
12.
Nature ; 583(7818): 729-736, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32728250

RESUMO

Combinatorial binding of transcription factors to regulatory DNA underpins gene regulation in all organisms. Genetic variation in regulatory regions has been connected with diseases and diverse phenotypic traits1, but it remains challenging to distinguish variants that affect regulatory function2. Genomic DNase I footprinting enables the quantitative, nucleotide-resolution delineation of sites of transcription factor occupancy within native chromatin3-6. However, only a small fraction of such sites have been precisely resolved on the human genome sequence6. Here, to enable comprehensive mapping of transcription factor footprints, we produced high-density DNase I cleavage maps from 243 human cell and tissue types and states and integrated these data to delineate about 4.5 million compact genomic elements that encode transcription factor occupancy at nucleotide resolution. We map the fine-scale structure within about 1.6 million DNase I-hypersensitive sites and show that the overwhelming majority are populated by well-spaced sites of single transcription factor-DNA interaction. Cell-context-dependent cis-regulation is chiefly executed by wholesale modulation of accessibility at regulatory DNA rather than by differential transcription factor occupancy within accessible elements. We also show that the enrichment of genetic variants associated with diseases or phenotypic traits in regulatory regions1,7 is almost entirely attributable to variants within footprints, and that functional variants that affect transcription factor occupancy are nearly evenly partitioned between loss- and gain-of-function alleles. Unexpectedly, we find increased density of human genetic variation within transcription factor footprints, revealing an unappreciated driver of cis-regulatory evolution. Our results provide a framework for both global and nucleotide-precision analyses of gene regulatory mechanisms and functional genetic variation.


Assuntos
Pegada de DNA/normas , Genoma Humano/genética , Fatores de Transcrição/metabolismo , Sequência Consenso , DNA/genética , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Genética Populacional , Estudo de Associação Genômica Ampla , Humanos , Modelos Moleculares , Polimorfismo de Nucleotídeo Único , Sequências Reguladoras de Ácido Nucleico/genética
13.
Nucleic Acids Res ; 48(14): 7991-8005, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32621607

RESUMO

DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5' to 3' ssDNA translocase and 5' to 3' helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Desoxirribonuclease I/metabolismo , Geobacillus stearothermophilus/enzimologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/isolamento & purificação , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , DNA , DNA Helicases/química , DNA Helicases/isolamento & purificação , Desoxirribonuclease I/química , Desoxirribonuclease I/isolamento & purificação
14.
Mol Genet Genomics ; 295(6): 1431-1442, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32685987

RESUMO

DNase I hypersensitive sites (DHSs) are highly sensitive active chromatin regions to DNase I enzymes, which provide the basis for the study of gene transcriptional regulation mechanism and play an important role in the analysis of gene expression regulatory elements. The identification of DHSs has contributed to biomedical research and genome analysis. There are already southern blotting technology and high-throughput sequencing technology to identify DHSs, but these experimental methods are often time-consuming and expensive, thus, novel and powerful computational methods are needed to predict DHSs. It is understood that researchers in related fields have proposed many feasible methods for the identification of DNase I hypersensitive sites. However, the accuracy of these methods is not satisfactory, so it is necessary to use more effective methods to predict DHSs. Therefore, on the basis of previous studies, we design a novel predictor called iDHS-DXG. First of all, we choose three sequence-derived feature representation methods to extract features, including kmer, mismatch and the dinucleotide property matrix based on Moran coefficient. Truncated singular value decomposition is selected for reducing the dimensionality of the benchmark dataset, and the optimal dimension is obtained through the test. Then, synthetic minority over-sampling technique is utilized to balance the positive and negative samples. After that, we introduce extreme gradient boosting ensemble classifier to predict DHSs. Compared with the previous research results, the main performance evaluation metrics of our method have been improved after five-fold cross-validation test. DHSs were identified on two human genome datasets with an accuracy of 90.84% and 91.27% respectively. This result shows that our method is a feasible, effective and competitive tool for the analysis of gene regulatory elements. Our research is helpful for biologists and geneticists to study genome analysis and gene regulation mechanism. Meanwhile, it is also of great significance to the development of human disease and drug design. Furthermore, the datasets and codes of iDHS-DXG can be obtained from the website: http://github.com/Xtian-696/iDHS-DXG/ .


Assuntos
Algoritmos , Cromatina/metabolismo , Desoxirribonuclease I/metabolismo , Repetições de Dinucleotídeos/genética , Genoma Humano , Cromatina/genética , Desoxirribonuclease I/química , Humanos , Sequências Reguladoras de Ácido Nucleico
15.
Arterioscler Thromb Vasc Biol ; 40(9): 2265-2278, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32673525

RESUMO

OBJECTIVE: Macrophages are immune cells, capable to remodel the extracellular matrix, which can harbor extracellular DNA incorporated into neutrophil extracellular traps (NETs). To study the breakdown of NETs we studied the capability of macrophage subsets to degrade these structures in vitro and in vivo in a murine thrombosis model. Furthermore, we analyzed human abdominal aortic aneurysm samples in support of our in vitro and in vivo results. Approach and Results: Macrophages were seeded onto blood clots or isolated NETs and polarized. All macrophages were capable to degrade NETs. For initial breakdown, macrophages relied on extracellular deoxyribonucleases. Proinflammatory polarization enhanced NET degradation. The boost in degradation was because of increased macropinocytosis, as inhibition by imipramine diminished their NET breakdown. Inhibition of macropinocytosis in a murine thrombosis model led to increased NET burden and reduced thrombus resolution in vivo. When analyzing abdominal aortic aneurysm samples, macrophage density furthermore corresponded negatively with the amount of local NETs in the intraluminal thrombi as well as in the vessel wall, as increased macrophage density was associated with a reduction in NET burden. CONCLUSIONS: We provide evidence that macrophages degrade NETs by extracellular predigestion and subsequent uptake. Furthermore, we show that proinflammatory macrophages increase NET degradation through enhanced macropinocytosis, priming them for NET engulfment. Based on our findings, that inhibition of macropinocytosis in mice corresponded to increased NET amounts in thrombi and that local macrophage density in human abdominal aortic aneurysm is negatively associated with surrounding NETs, we hypothesize, that macrophages are able to degrade NETs in vivo.


Assuntos
Endodesoxirribonucleases/metabolismo , Armadilhas Extracelulares/metabolismo , Ativação de Macrófagos , Macrófagos/enzimologia , Neutrófilos/metabolismo , Pinocitose , Animais , Aneurisma da Aorta Abdominal/metabolismo , Células Cultivadas , Desoxirribonuclease I/metabolismo , Desoxirribonucleases/metabolismo , Modelos Animais de Doenças , Exodesoxirribonucleases/metabolismo , Feminino , Humanos , Imipramina/farmacologia , Interferon gama/farmacologia , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Cinética , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares/metabolismo , Fagocitose , Fenótipo , Fosfoproteínas/metabolismo , Pinocitose/efeitos dos fármacos , Veia Cava Inferior/metabolismo , Trombose Venosa/metabolismo
16.
Nat Biotechnol ; 38(5): 582-585, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32393904

RESUMO

Prime editors, which are CRISPR-Cas9 nickase (H840A)-reverse transcriptase fusions programmed with prime editing guide RNAs (pegRNAs), can edit bases in mammalian cells without donor DNA or double-strand breaks. We adapted prime editors for use in plants through codon, promoter, and editing-condition optimization. The resulting suite of plant prime editors enable point mutations, insertions and deletions in rice and wheat protoplasts. Regenerated prime-edited rice plants were obtained at frequencies of up to 21.8%.


Assuntos
Edição de Genes/métodos , Oryza/crescimento & desenvolvimento , Triticum/crescimento & desenvolvimento , Sistemas CRISPR-Cas , Desoxirribonuclease I/metabolismo , Genoma de Planta , Oryza/genética , Triticum/genética
17.
Physiol Genomics ; 52(5): 217-221, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32275178
18.
PLoS One ; 15(3): e0229017, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32130239

RESUMO

We have earlier reported that cell-free chromatin (cfCh) particles that are released from dying cells, or those that circulate blood, can readily enter into healthy cells, illegitimately integrate into their genomes and induce dsDNA breaks, apoptosis and intense activation of inflammatory cytokines. We hypothesized that sepsis is caused by cfCh released from dying host cells following microbial infection leading to bystander host cell apoptosis and inflammation which are perpetuated in a vicious cycle with release of more cfCh from dying host cells. To test this hypothesis we used three cfCh inactivating agents namely 1) anti-histone antibody complexed nanoparticles which inactivate cfCh by binding to histones; 2) DNase I which inactivates cfCh by degrading its DNA component, and 3) a novel pro-oxidant combination of Resveratrol and Copper which, like DNase I, inactivates cfCh by degrading its DNA component. Female C57 BL/6 mice, 6-8 weeks old, were administered a single i.p. injection of LPS at a dose of 10 mg/Kg or 20 mg/Kg with or without concurrent treatment with the above cfCh inactivating agents. Administration of cfCh inactivating agents concurrently with LPS resulted in prevention of following pathological parameters: 1) release of cfCh in extra-cellular spaces of brain, lung and heart and in circulation; 2) release of inflammatory cytokines in circulation; 3) activation of DNA damage, apoptosis and inflammation in cells of thymus, spleen and in PBMCs; 4) DNA damage, apoptosis and inflammation in cells of lung, liver, heart, brain, kidney and small intestine; 5) liver and kidney dysfunction and elevation of serum lactate; 6) coagulopathy, fibrinolysis and thrombocytopenia; 7) lethality. We conclude that cfCh that are released from dying host cells in response to bacterial endotoxin represents a global instigator of sepsis. cfCh inactivation may provide a novel approach to management of sepsis in humans.


Assuntos
Morte Celular , Ácidos Nucleicos Livres/metabolismo , Cromatina/metabolismo , Endotoxinas , Sepse/metabolismo , Sepse/patologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Cromatina/patologia , Cromatina/fisiologia , Cobre/administração & dosagem , Citocinas/metabolismo , Dano ao DNA/efeitos dos fármacos , Desoxirribonuclease I/metabolismo , Desoxirribonuclease I/uso terapêutico , Feminino , Histonas/metabolismo , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/uso terapêutico , Resveratrol/administração & dosagem , Sepse/induzido quimicamente , Sepse/prevenção & controle
19.
PLoS Biol ; 18(3): e3000651, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32191696

RESUMO

Rapid antibiotic susceptibility testing (AST) for Neisseria gonorrhoeae (Ng) is critically needed to counter widespread antibiotic resistance. Detection of nucleic acids in genotypic AST can be rapid, but it has not been successful for ß-lactams (the largest antibiotic class used to treat Ng). Rapid phenotypic AST for Ng is challenged by the pathogen's slow doubling time and the lack of methods to quickly quantify the pathogen's response to ß-lactams. Here, we asked two questions: (1) Is it possible to use nucleic acid quantification to measure the ß-lactam susceptibility phenotype of Ng very rapidly, using antibiotic-exposure times much shorter than the 1- to 2-h doubling time of Ng? (2) Would such short-term antibiotic exposures predict the antibiotic resistance profile of Ng measured by plate growth assays over multiple days? To answer these questions, we devised an innovative approach for performing a rapid phenotypic AST that measures DNA accessibility to exogenous nucleases after exposure to ß-lactams (termed nuclease-accessibility AST [nuc-aAST]). We showed that DNA in antibiotic-susceptible cells has increased accessibility upon exposure to ß-lactams and that a judiciously chosen surfactant permeabilized the outer membrane and enhanced this effect. We tested penicillin, cefixime, and ceftriaxone and found good agreement between the results of the nuc-aAST after 15-30 min of antibiotic exposure and the results of the gold-standard culture-based AST measured over days. These results provide a new pathway toward developing a critically needed phenotypic AST for Ng and additional global-health threats.


Assuntos
Antibacterianos/farmacologia , DNA Bacteriano/metabolismo , Desoxirribonuclease I/metabolismo , Neisseria gonorrhoeae/efeitos dos fármacos , Tensoativos/farmacologia , beta-Lactamas/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Gonorreia/microbiologia , Gonorreia/urina , Humanos , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/crescimento & desenvolvimento , Neisseria gonorrhoeae/isolamento & purificação , Fenótipo , Reprodutibilidade dos Testes , Fatores de Tempo , Fluxo de Trabalho
20.
J Am Chem Soc ; 142(7): 3311-3315, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32011869

RESUMO

DNA nanostructures (DNs) have garnered a large amount of interest as a potential therapeutic modality. However, DNs are prone to nuclease-mediated degradation and are unstable in low Mg2+ conditions; this greatly limits their utility in physiological settings. Previously, PEGylated oligolysines were found to protect DNs against low-salt denaturation and to increase nuclease resistance by up to ∼400-fold. Here we demonstrate that glutaraldehyde cross-linking of PEGylated oligolysine-coated DNs extends survival by up to another ∼250-fold to >48 h during incubation with 2600 times the physiological concentration of DNase I. DNA origami with cross-linked oligolysine coats are non-toxic and are internalized into cells more readily than non-cross-linked origami. Our strategy provides an off-the-shelf and generalizable method for protecting DNs in vivo.


Assuntos
Reagentes para Ligações Cruzadas/metabolismo , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Glutaral/metabolismo , Polilisina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Reagentes para Ligações Cruzadas/química , Reagentes para Ligações Cruzadas/toxicidade , DNA/química , DNA/toxicidade , Glutaral/química , Glutaral/toxicidade , Células HEK293 , Humanos , Hidrólise , Nanoestruturas/química , Nanoestruturas/toxicidade , Conformação de Ácido Nucleico , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polietilenoglicóis/toxicidade , Polilisina/química , Polilisina/toxicidade
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