RESUMO
In DNA, methylation at the fifth position of cytosine (5mC) by DNA methyltransferases is essential for eukaryotic gene regulation. Methylation patterns are dynamically controlled by epigenetic machinery. Erasure of 5mC by Fe2+ and 2-ketoglutarate (2KG) dependent dioxygenases in the ten-eleven translocation family (TET1-3), plays a key role in nuclear processes. Through the event of active demethylation, TET proteins iteratively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), each of which has been implicated in numerous diseases when aberrantly generated. A wide range of biochemical assays have been developed to characterize TET activity, many of which require multi-step processing to detect and quantify the 5mC oxidized products. Herein, we describe the development and optimization of a sensitive MALDI mass spectrometry-based technique that directly measures TET activity and eliminates tedious processing steps. Employing optimized assay conditions, we report the steady-state activity of wild type TET2 enzymes to furnish 5hmC, 5fC and 5caC. We next determine IC50 values of several small-molecule inhibitors of TETs. The utility of this assay is further demonstrated by analyzing the activity of V1395A which is an activating mutant of TET2 that primarily generates 5caC. Lastly, we describe the development of a secondary assay that utilizes bisulfite chemistry to further examine the activity of wildtype TET2 and V1395A in a base-resolution manner. The combined results demonstrate that the activity of TET proteins can be gauged, and their products accurately quantified using our methods.
Assuntos
5-Metilcitosina , Proteínas de Ligação a DNA , Dioxigenases , Proteínas Proto-Oncogênicas , Dioxigenases/metabolismo , Dioxigenases/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análise , 5-Metilcitosina/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Ensaios Enzimáticos/métodos , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/química , Metilação de DNA , Citosina/análogos & derivados , Citosina/análise , Citosina/metabolismo , Citosina/química , OxirreduçãoRESUMO
RNA modifications play an important role in actively controlling recently created formation in cellular regulation mechanisms, which link them to gene expression and protein. The RNA modifications have numerous alterations, presenting broad glimpses of RNA's operations and character. The modification process by the TET enzyme oxidation is the crucial change associated with cytosine hydroxymethylation. The effect of CR is an alteration in specific biochemical ways of the organism, such as gene expression and epigenetic alterations. Traditional laboratory systems that identify 5-hydroxymethylcytosine (5hmC) samples are expensive and time-consuming compared to other methods. To address this challenge, the paper proposed XGB5hmC, a machine learning algorithm based on a robust gradient boosting algorithm (XGBoost), with different residue based formulation methods to identify 5hmC samples. Their results were amalgamated, and six different frequency residue based encoding features were fused to form a hybrid vector in order to enhance model discrimination capabilities. In addition, the proposed model incorporates SHAP (Shapley Additive Explanations) based feature selection to demonstrate model interpretability by highlighting the high contributory features. Among the applied machine learning algorithms, the XGBoost ensemble model using the tenfold cross-validation test achieved improved results than existing state-of-the-art models. Our model reported an accuracy of 89.97%, sensitivity of 87.78%, specificity of 94.45%, F1-score of 0.8934%, and MCC of 0.8764%. This study highlights the potential to provide valuable insights for enhancing medical assessment and treatment protocols, representing a significant advancement in RNA modification analysis.
Assuntos
5-Metilcitosina , Algoritmos , Aprendizado de Máquina , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Humanos , Citosina/análogos & derivados , Citosina/metabolismoRESUMO
The engineered TadA variants used in cytosine base editors (CBEs) present distinctive advantages, including a smaller size and fewer off-target effects compared to cytosine base editors that rely on natural deaminases. However, the current TadA variants demonstrate a preference for base editing in DNA with specific motif sequences and possess dual deaminase activity, acting on both cytosine and adenosine in adjacent positions, limiting their application scope. To address these issues, we employ TadA orthologs screening and multi sequence alignment (MSA)-guided protein engineering techniques to create a highly effective cytosine base editor (aTdCBE) without motif and adenosine deaminase activity limitations. Notably, the delivery of aTdCBE to a humanized mouse model of Duchenne muscular dystrophy (DMD) mice achieves robust exon 55 skipping and restoration of dystrophin expression. Our advancement in engineering TadA ortholog for cytosine editing enriches the base editing toolkits for gene-editing therapy and other potential applications.
Assuntos
Adenosina , Citosina , Edição de Genes , Distrofia Muscular de Duchenne , Citosina/metabolismo , Animais , Edição de Genes/métodos , Adenosina/metabolismo , Camundongos , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Engenharia de Proteínas , Distrofina/genética , Distrofina/metabolismo , Adenosina Desaminase/metabolismo , Adenosina Desaminase/genética , Modelos Animais de Doenças , Éxons/genética , Células HEK293 , Sistemas CRISPR-Cas , Proteínas de Escherichia coliAssuntos
Cidofovir , Citosina , Organofosfonatos , Cidofovir/administração & dosagem , Cidofovir/uso terapêutico , Humanos , Citosina/análogos & derivados , Citosina/administração & dosagem , Citosina/uso terapêutico , Organofosfonatos/administração & dosagem , Organofosfonatos/uso terapêutico , Injeções Intralesionais , Papiloma/tratamento farmacológico , Administração Tópica , Neoplasias dos Seios Paranasais/tratamento farmacológico , Neoplasias dos Seios Paranasais/diagnóstico por imagem , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêuticoRESUMO
Human adenovirus infection (HAdV) may be fatal in patients undergoing allogeneic hematopoietic cell transplantation (HCT). Cidofovir is effective in only a part of the post-HCT HAdV infection. Therefore, posttransplant immune reconstitution is important for HAdV clearance. We describe the detailed immune reconstitution and response of adenovirus-specific T cells in a patient with inborn errors of immunity who had disseminated HAdV infection with hepatitis post-HCT and was treated with cidofovir. Though the patient received cidofovir for only 19 days starting from Day 72 after HCT because of renal dysfunction, we observed T-cell reconstitution, a decrease in HAdV copy number, and amelioration of the symptoms of HAdV infection after Day 90. We initially observed expanded NK and CD8+CD45RO+ memory subsets and later gradual increase of naïve T cells eveloped after cessation of cidofovir treatment. An increase in adenovirus-specific IFN-γ secretion from 2 to 4 months after HCT was confirmed by ELISpot assay. The progression of immune reconstitution and cidofovir treatment are considered to have contributed to survival in this patient. Optimization of transplantation methods, prompt appropriate antiviral medication, and virus-specific T-cell therapy would be necessary as the better strategy for systemic HAdV infection.
Assuntos
Infecções por Adenovirus Humanos , Antivirais , Cidofovir , Citosina , Transplante de Células-Tronco Hematopoéticas , Reconstituição Imune , Organofosfonatos , Humanos , Cidofovir/uso terapêutico , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Organofosfonatos/uso terapêutico , Citosina/análogos & derivados , Citosina/uso terapêutico , Infecções por Adenovirus Humanos/tratamento farmacológico , Infecções por Adenovirus Humanos/imunologia , Infecções por Adenovirus Humanos/terapia , Antivirais/uso terapêutico , Transplante Homólogo , Adenovírus Humanos/imunologia , Masculino , Hepatite Viral Humana/tratamento farmacológico , Hepatite Viral Humana/imunologiaRESUMO
The conversion of DNA 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by TET enzymes represents a significant epigenetic modification, yet its role in early human embryos remains largely unknown. Here we showed that the early human embryo inherited a significant amount of 5hmCs from an oocyte, which unexpectedly underwent de novo hydroxymethylation during its growth. Furthermore, the generation of 5hmC in the paternal genome after fertilization roughly followed the maternal pattern, which was linked to DNA methylation dynamics and regions of sustained methylation. The 5hmCs persisted until the eight-cell stage and exhibited high enrichment at OTX2 binding sites, whereas knockdown of OTX2 in human embryos compromised the expression of early lineage genes. Specifically, the depletion of 5hmC affected the activation of embryonic genes, which was further evaluated by ectopically expressing mouse Tet3 in human early embryos. These findings revealed distinct dynamics of 5hmC and unravelled its multifaceted functions in early human embryonic development.
Assuntos
5-Metilcitosina , Citosina , Metilação de DNA , Proteínas de Ligação a DNA , Dioxigenases , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição Otx , Proteínas Proto-Oncogênicas , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Humanos , Animais , Fatores de Transcrição Otx/metabolismo , Fatores de Transcrição Otx/genética , Camundongos , Dioxigenases/metabolismo , Dioxigenases/genética , Citosina/análogos & derivados , Citosina/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Masculino , Blastocisto/metabolismo , Linhagem da Célula/genética , Oócitos/metabolismo , Epigênese Genética , Sítios de LigaçãoRESUMO
Epigenetic cytosine methylation covers most of genomic CpG dinucleotides in human cells. In addition to common deamination-mediated mutagenesis at CpG sites, an alternative deamination-independent pathway associated with DNA polymerase activity was previously described. This mutagenesis is characterized by the TCGâTTG mutational signature and is believed to arise from dAMP misincorporation opposite 5-methylcytosine (mC) or its oxidized derivative 5-hydroxymethylcytosine (hmC) by B-family replicative DNA polymerases with disrupted proofreading 3â5'-exonuclease activity. In addition to being less stable and pro-mutagenic themselves, cytosine modifications also increase the risk of adjacent nucleotides damage, including the formation of 8-oxo-2'-deoxyguanosine (8-oxoG), a well-known mutagenic lesion. The effect of cytosine methylation on error-prone DNA polymerases lacking proofreading activity and involved in repair and DNA translesion synthesis remains unexplored. Here we analyze the efficiency and fidelity of translesion Y-family polymerases (Pol κ, Pol η, Pol ι and REV1) and primase-polymerase PrimPol opposite mC and hmC as well as opposite 8-oxoG adjacent to mC in the TCG context. We demonstrate that epigenetic cytosine modifications suppress Pol ι and REV1 activities and lead to increasing dAMP misincorporation by PrimPol, Pol κ and Pol ι in vitro. Cytosine methylation also increases misincorporation of dAMP opposite the adjacent 8-oxoG by PrimPol, decreases the TLS activity of Pol η opposite the lesion but increases dCMP incorporation opposite 8-oxoG by REV1. Altogether, these data suggest that methylation and hydroxymethylation of cytosine alter activity and fidelity of translesion DNA polymerases.
Assuntos
5-Metilcitosina , Citosina , Metilação de DNA , DNA Polimerase Dirigida por DNA , Humanos , DNA Polimerase Dirigida por DNA/metabolismo , Citosina/metabolismo , Citosina/análogos & derivados , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análogos & derivados , Reparo do DNA , Dano ao DNA , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , DNA Polimerase iota , DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Replicação do DNA , 8-Hidroxi-2'-Desoxiguanosina/metabolismoRESUMO
DNA methyltransferases are drug targets for myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), acute myelogenous leukemia (AML) and possibly ß-hemoglobinopathies. We characterize the interaction of nucleoside analogues in DNA with a prokaryotic CpG-specific DNA methyltransferase (M.MpeI) as a model for mammalian DNMT1 methyltransferases. We tested DNA containing 5-hydroxymethylcytosine (5hmC), 5-hydroxycytosine (5OHC), 5-methyl-2-pyrimidinone (in the ribosylated form known as 5-methylzebularine, 5mZ), 5,6-dihydro-5-azacytosine (dhaC), 5-fluorocytosine (5FC), 5-chlorocytosine (5ClC), 5-bromocytosine (5BrC) and 5-iodocytosine (5IC). Covalent complex formation was by far most efficient for 5FC. Non-covalent complexes were most abundant for dhaC and 5mZ. Surprisingly, we observed methylation of 5IC and 5BrC, and to a lesser extent 5ClC and 5FC, in the presence, but not the absence of small molecule thiol nucleophiles. For 5IC and 5BrC, we demonstrated by mass spectrometry that the reactions were due to methyltransferase driven dehalogenation, followed by methylation. Crystal structures of M.MpeI-DNA complexes capture the 'in' conformation of the active site loop for analogues with small or rotatable (5mZ) 5-substituents and its 'out' form for bulky 5-substituents. Since very similar 'in' and 'out' loop conformations were also observed for DNMT1, it is likely that our conclusions generalize to other DNA methyltransferases.
Assuntos
Citosina , DNA , Citosina/análogos & derivados , Citosina/química , Citosina/metabolismo , DNA/metabolismo , DNA/química , Especificidade por Substrato , Metilação de DNA , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA (Citosina-5-)-Metiltransferases/química , Humanos , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/química , 5-Metilcitosina/metabolismo , 5-Metilcitosina/química , 5-Metilcitosina/análogos & derivados , Modelos MolecularesRESUMO
Polyomaviruses are species-specific DNA viruses that can cause disease in immunocompromised individuals. Despite their role as the causative agents for several diseases, there are no currently approved antivirals for treating polyomavirus infection. Brincidofovir (BCV) is an antiviral approved for the treatment of poxvirus infections and has shown activity against other double-stranded DNA viruses. In this study, we tested the efficacy of BCV against polyomavirus infection in vitro and in vivo using mouse polyomavirus (MuPyV). BCV inhibited virus production in primary mouse kidney cells and brain cortical cells. BCV treatment of cells transfected with MuPyV genomic DNA resulted in a reduction in virus levels, indicating that viral inhibition occurs post-entry. Although in vitro BCV treatment had a limited effect on viral DNA and RNA levels, drug treatment was associated with a reduction in viral protein, raising the possibility that BCV acts post-transcriptionally to inhibit MuPyV infection. In mice, BCV treatment was well tolerated, and prophylactic treatment resulted in a reduction in viral DNA levels and a potent suppression of infectious virus production in the kidney and brain. In mice with chronic polyomavirus infection, therapeutic administration of BCV decreased viremia and reduced infection in the kidney. These data demonstrate that BCV exerts antiviral activity against polyomavirus infection in vivo, supporting further investigation into the use of BCV to treat clinical polyomavirus infections. IMPORTANCE: Widespread in the human population and able to persist asymptomatically for the life of an individual, polyomavirus infections cause a significant disease burden in the immunocompromised. Individuals undergoing immune suppression, such as kidney transplant patients or those treated for autoimmune diseases, are particularly at high risk for polyomavirus-associated diseases. Because no antiviral agent exists for treating polyomavirus infections, management of polyomavirus-associated diseases typically involves reducing or discontinuing immunomodulatory therapy. This can be perilous due to the risk of transplant rejection and the potential development of adverse immune reactions. Thus, there is a pressing need for the development of antivirals targeting polyomaviruses. Here, we investigate the effects of brincidofovir, an FDA-approved antiviral, on polyomavirus infection in vivo using mouse polyomavirus. We show that the drug is well-tolerated in mice, reduces infectious viral titers, and limits viral pathology, indicating the potential of brincidofovir as an anti-polyomavirus therapeutic.
Assuntos
Antivirais , Citosina , Organofosfonatos , Infecções por Polyomavirus , Polyomavirus , Animais , Citosina/análogos & derivados , Citosina/farmacologia , Citosina/uso terapêutico , Infecções por Polyomavirus/tratamento farmacológico , Infecções por Polyomavirus/virologia , Polyomavirus/efeitos dos fármacos , Camundongos , Antivirais/farmacologia , Antivirais/uso terapêutico , Organofosfonatos/farmacologia , Organofosfonatos/uso terapêutico , Replicação Viral/efeitos dos fármacos , Rim/virologia , Rim/efeitos dos fármacos , Feminino , DNA Viral/genética , Células Cultivadas , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Encéfalo/virologiaRESUMO
Nucleobases control the assembly of DNA, RNA, etc. due to hydrogen bond complementarity. By combining these unique molecules with state-of-the-art synthetic polymers, it is possible to form nanoparticles whose self-assembly behavior could be altered under orthogonal stimuli (pH and temperature). Herein, we report the synthesis of cytosine-containing nanoparticles via aqueous reversible addition-fragmentation chain transfer polymerization-induced self-assembly. A poly(N-acryloylmorpholine) macromolecular chain transfer agent (mCTA) was chain-extended with cytosine acrylamide, and a morphological phase diagram was constructed. By exploiting the ability of cytosine to form dimers via hydrogen bonding, the self-assembly behavior of cytosine-containing polymers was altered when performed under acidic conditions. Under these conditions, stable nanoparticles could be formed at longer polymer chain lengths. Furthermore, the resulting nanoparticles displayed different morphologies compared to those at pH 7. Additionally, particle stability post-assembly could be controlled by varying pH and temperature. Finally, small-angle X-ray scattering was performed to probe their dynamic behavior under thermal cycling.
Assuntos
Citosina , Ligação de Hidrogênio , Nanopartículas , Citosina/química , Concentração de Íons de Hidrogênio , Nanopartículas/química , Temperatura , Polimerização , Polímeros/químicaRESUMO
Non-CpG methylation is associated with several cellular processes, especially neuronal development and cancer, while its effect on DNA structure remains unclear. We have determined the crystal structures of DNA duplexes containing -CGCCG- regions as CCG repeat motifs that comprise a non-CpG site with or without cytosine methylation. Crystal structure analyses have revealed that the mC:G base-pair can simultaneously form two alternative conformations arising from non-CpG methylation, including a unique water-mediated cis Watson-Crick/Hoogsteen, (w)cWH, and Watson-Crick (WC) geometries, with partial occupancies of 0.1 and 0.9, respectively. NMR studies showed that an alternative conformation of methylated mC:G base-pair at non-CpG step exhibits characteristics of cWH with a syn-guanosine conformation in solution. DNA duplexes complexed with the DNA binding drug echinomycin result in increased occupancy of the (w)cWH geometry in the methylated base-pair (from 0.1 to 0.3). Our structural results demonstrated that cytosine methylation at a non-CpG step leads to an antiâsyntransition of its complementary guanosine residue toward the (w)cWH geometry as a partial population of WC, in both drug-bound and naked mC:G base pairs. This particular geometry is specific to non-CpG methylated dinucleotide sites in B-form DNA. Overall, the current study provides new insights into DNA conformation during epigenetic regulation.
Assuntos
Pareamento de Bases , Citosina , Metilação de DNA , DNA , Conformação de Ácido Nucleico , Água , DNA/química , Citosina/química , Água/química , Cristalografia por Raios X , Modelos MolecularesRESUMO
The BisI family of restriction endonucleases is unique in requiring multiple methylated or hydroxymethylated cytosine residues within a short recognition sequence (GCNGC), and in cleaving directly within this sequence, rather than at a distance. Here, we report that the number of modified cytosines that are required for cleavage can be tuned by the salt concentration. We present crystal structures of two members of the BisI family, NhoI and Eco15I_Ntd (N-terminal domain of Eco15I), in the absence of DNA and in specific complexes with tetra-methylated GCNGC target DNA. The structures show that NhoI and Eco15I_Ntd sense modified cytosine bases in the context of double-stranded DNA (dsDNA) without base flipping. In the co-crystal structures of NhoI and Eco15I_Ntd with DNA, the internal methyl groups (G5mCNGC) interact with the side chains of an (H/R)(V/I/T/M) di-amino acid motif near the C-terminus of the distal enzyme subunit and arginine residue from the proximal subunit. The external methyl groups (GCNG5mC) interact with the proximal enzyme subunit, mostly through main chain contacts. Surface plasmon resonance analysis for Eco15I_Ntd shows that the internal and external methyl binding pockets contribute about equally to sensing of cytosine methyl groups.
Assuntos
DNA , Modelos Moleculares , DNA/química , DNA/metabolismo , Cristalografia por Raios X , Citosina/química , Citosina/metabolismo , Metilação de DNA , Enzimas de Restrição do DNA/química , Enzimas de Restrição do DNA/metabolismo , Enzimas de Restrição do DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Especificidade por Substrato , Domínio CatalíticoRESUMO
The i-motif, a secondary structure of a four-helix formed by cytosine-rich DNA (i-DNA) through C-C+ base pairing, is prevalent in human telomeres and promoters. This structure creates steric hindrance, thereby inhibiting both gene expression and protein coding. The conformation of i-DNA is intricately linked to the intracellular ionic environment. Hence, investigating its conformation under various ion conditions holds significant importance. In this study, we explored the impact of cations on the i-motif structure at the single-molecule level using the α-hemolysin (α-HL) nanochannel. Our findings reveal that the ability of i-DNA to fold into the i-motif structure follows the order Cs+ > Na+ > K+ > Li+ for monovalent cations. Furthermore, we observed the interconversion of single-stranded DNA (ss-DNA) and the i-motif structure at high and low concentrations of Mg2+ and Ba2+ electrolyte solutions. This study not only has the potential to extend the application of i-motif-based sensors in complex solution environments but also provides a new idea for the detection of metal ions.
Assuntos
Cátions , DNA , Proteínas Hemolisinas , Nanoporos , DNA/química , Cátions/química , Proteínas Hemolisinas/química , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Citosina/química , DNA de Cadeia Simples/químicaRESUMO
Despite the widely accepted involvement of DNA methylation in the regulation of rDNA transcription, the relative participation of different cytosine methylation pathways is currently described only for a few model plants. Using PacBio, Bisulfite, and RNA sequencing; PCR; Southern hybridizations; and FISH, the epigenetic consequences of rDNA copy number variation were estimated in two T. porrifolius lineages, por1 and por2, the latter with more than twice the rDNA copy numbers distributed approximately equally between NORs on chromosomes A and D. The lower rDNA content in por1 correlated with significantly reduced (>90%) sizes of both D-NORs. Moreover, two (L and S) prominent rDNA variants, differing in the repetitive organization of intergenic spacers, were detected in por2, while only the S-rDNA variant was detected in por1. Transcriptional activity of S-rDNA in por1 was associated with secondary constriction of both A-NORs. In contrast, silencing of S-rDNA in por2 was accompanied by condensation of A-NORs, secondary constriction on D-NORs, and L-rDNA transcriptional activity, suggesting (i) bidirectional nucleolar dominance and (ii) association of S-rDNAs with A-NORs and L-rDNAs with D-NORs in T. porrifolius. Each S- and L-rDNA array was formed of several sub-variants differentiating both genetically (specific SNPs) and epigenetically (transcriptional efficiency and cytosine methylation). The most significant correlations between rDNA silencing and methylation were detected for symmetric CWG motifs followed by CG motifs. No correlations were detected for external cytosine in CCGs or asymmetric CHHs, where methylation was rather position-dependent, particularly for AT-rich variants. We conclude that variations in rDNA copy numbers in plant diploids can be accompanied by prompt epigenetic responses to maintain an appropriate number of active rDNAs. The methylation dynamics of CWGs are likely to be the most responsible for regulating silent and active rDNA states.
Assuntos
Citosina , Metilação de DNA , DNA Ribossômico , Inativação Gênica , Citosina/metabolismo , DNA Ribossômico/genética , Variações do Número de Cópias de DNA , Transcrição Gênica , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Cromossomos de Plantas/genéticaRESUMO
Base editing (BE) faces protospacer adjacent motif (PAM) constraints and off-target effects in both eukaryotes and prokaryotes. For Streptomyces, renowned as one of the most prolific bacterial producers of antibiotics, the challenges are more pronounced due to its diverse genomic content and high GC content. Here, we develop a base editor named eSCBE3-NG-Hypa, tailored with both high efficiency and -fidelity for Streptomyces. Of note, eSCBE3-NG-Hypa recognizes NG PAM and exhibits high activity at challenging sites with high GC content or GC motifs, while displaying minimal off-target effects. To illustrate its practicability, we employ eSCBE3-NG-Hypa to achieve precise key amino acid conversion of the dehydratase (DH) domains within the modular polyketide synthase (PKS) responsible for the insecticide avermectins biosynthesis, achieving domains inactivation. The resulting DH-inactivated mutants, while ceasing avermectins production, produce a high yield of oligomycin, indicating competitive relationships among multiple biosynthetic gene clusters (BGCs) in Streptomyces avermitilis. Leveraging this insight, we use eSCBE3-NG-Hypa to introduce premature stop codons into competitor gene cluster of ave in an industrial S. avermitilis, with the mutant Δolm exhibiting the highest 4.45-fold increase in avermectin B1a compared to the control. This work provides a potent tool for modifying biosynthetic pathways and advancing metabolic engineering in Streptomyces.
Assuntos
Sistemas CRISPR-Cas , Citosina , Edição de Genes , Policetídeo Sintases , Streptomyces , Streptomyces/genética , Streptomyces/metabolismo , Edição de Genes/métodos , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Citosina/metabolismo , Ivermectina/análogos & derivados , Ivermectina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , OligomicinasRESUMO
Laccases are the most commonly used agents for the treatment of phenolic pollutants. To address the instability and high cost of natural laccases, we investigated nucleobase-modulated copper nanomaterial with laccase-like activity. Various nucleobases, including adenine, guanine, cytosine, and thymine, were investigated as templates for Cu2+ reduction and copper nanomaterials formation due to their coordination capacity. By comparing structure and catalytic activity, the cytosine-mediated copper nanomaterial (C-Cu) had the best laccase-like activity and other nucleobase-templated copper nanomaterials exhibited low catalytic activity under the same conditions. The mechanism of nucleobase regulation of the catalytic activity of copper nanomaterials was further analyzed using X-ray photoelectron spectroscopy and density functional theory. The possible catalytic mechanisms of C-Cu, including substrate adsorption, substrate oxidation, oxygen binding, and oxygen reduction, were proposed. Remarkably, nucleobase-modulated copper nanozymes showed high stability and catalytic oxidation performance at various pH values, temperatures, long-term storage, and high salinity. In combination with electrochemical techniques, a portable electrochemical sensor for measuring phenolic pollutants was developed. This novel sensor exhibited a good linear response to catechol (10-1000 µM) with a limit of detection of 1.8 µM and excellent selectivity and anti-interference ability. This study provides not only a new strategy for the regulation of the laccase-like activity of copper nanomaterials but also a novel tool for the effective removal and low-cost detection of phenolic pollutants.
Assuntos
Cobre , Lacase , Nanoestruturas , Poluentes Químicos da Água , Cobre/química , Lacase/química , Lacase/metabolismo , Nanoestruturas/química , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/química , Oxirredução , Fenóis/química , Fenóis/análise , Catálise , Técnicas Eletroquímicas , Citosina/química , Catecóis/química , Adenina/química , Adenina/análise , Guanina/química , Guanina/análiseRESUMO
5-formylcytosine (5 fC) and 5-carboxylcytosine (5caC) serve as key intermediates in DNA demethylation process with significant implications for gene regulation and disease progression. In this study, we introduce a novel electrochemical sensing platform specifically designed for the sensitive and selective detection of 5 fC and 5caC in DNA. Protein A-modified magnetic beads (ProtA-MBs) coupled with specific antibodies facilitate the immunorecognition and enrichment of these modified bases. Signal amplification is achieved through several chemical reactions involving the interaction between N3-kethonaxl and guanine, copper-free click chemistry for the attachment of dibenzocyclooctyne (DBCO)-Biotin, and the subsequent recognition by streptavidin-conjugated horseradish peroxidase (SA-HRP). The assay's readout is performed on a disposable laser-induced graphene (LIG) electrode, modified with the bead-antibody-DNA complex in a magnetic field, and analyzed using differential pulse voltammetry in a system employing hydroquinone (HQ) as the redox mediator and H2O2 as the substrate. This immunosensor displayed excellent sensitivity, with detection limits of 14.8 fM for 5 fC across a 0.1-1000 pM linear range and 87.4 fM for 5caC across a 0.5-5000 pM linear range, and maintained high selectivity even in the presence of interferences from other DNA modifications. Successful application in quantifying 5 fC and 5caC in genomic DNA from cell extracts, with recovery rates between 97.7% to 102.9%, underscores its potential for clinical diagnostics. N3-kethoxal was used for the first time in an electrochemical sensor. This work not only broadens the toolkit for detecting DNA modifications but also provides a fresh impetus for the development of point-of-care testing (POCT) technologies.
Assuntos
Técnicas Biossensoriais , Citosina , DNA , Técnicas Eletroquímicas , Limite de Detecção , DNA/química , Técnicas Eletroquímicas/métodos , Citosina/química , Citosina/análogos & derivados , Humanos , Imunoensaio/métodos , Imunoensaio/instrumentação , Grafite/químicaRESUMO
The present study aimed to evaluate whether epigenetic markers are expressed in the dental follicles surrounding ectopically erupting teeth. Twenty-one dental follicles were collected in 20 adolescent children through surgical exposure of ectopic teeth. The epigenetic modifications of DNA methylation and histone acetylation were evaluated by immunohistochemistry. The results showed cells positive for DNA-methyltransferase 1 (DNMT1), DNA methyltransferase 3 beta (DNMT3B), ten-eleven translocation-2 (TET2), acetyl-histone H3 (AcH3), acetyl-histone H4 (AcH4), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC) were present in all the samples. The levels of epigenetic markers representing active chromatin (5hmC, AcH3, AcH4, and TET2) were statistically significantly higher than those of markers representing inactive chromatin (5mC, DNMT3B, DNMT1). In conclusion, follicles in ectopic teeth display major epigenetic modifications. In the follicles, epigenetic markers associated with the activation of bone-related genes are more abundant than markers associated with the inactivation of bone-related genes.
Assuntos
Metilação de DNA , Saco Dentário , Epigênese Genética , Histonas , Erupção Dentária , Humanos , Histonas/metabolismo , Adolescente , Acetilação , Criança , Feminino , Masculino , Erupção Dentária/genética , Saco Dentário/metabolismo , DNA Metiltransferase 3B , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Citosina/metabolismoRESUMO
The inherent chiral structures of DNA serve as attractive scaffolds to construct DNA hybrid catalysts for valuable enantioselective transformations. Duplex and G-quadruplex DNA-based enantioselective catalysis has made great progress, yet novel design strategies of DNA hybrid catalysts are highly demanding and atomistic analysis of active centers is still challenging. DNA i-motif structures could be finely tuned by different cytosine-cytosine base pairs, providing a new platform to design DNA catalysts. Herein, we found that a human telomeric i-motif DNA containing cytosine-silver(I)-cytosine (C-Ag+-C) base pairs interacting with Cu(II) ions (i-motif DNA(Ag+)/Cu2+) could catalyze Diels-Alder reactions with full conversions and up to 95 % enantiomeric excess. As characterized by various physicochemical techniques, the presence of Ag+ is proved to replace the protons in hemiprotonated cytosine-cytosine (C : C+) base pairs and stabilize the DNA i-motif to allow the acceptance of Cu(II) ions. The i-motif DNA(Ag+)/Cu2+ catalyst shows about 8-fold rate acceleration compared with DNA and Cu2+. Based on DNA mutation experiments, thermodynamic studies and density function theory calculations, the catalytic center of Cu(II) ion is proposed to be located in a specific loop region via binding to one nitrogen-7 atom of an unpaired adenine and two phosphate-oxygen atoms from nearby deoxythymidine monophosphate and deoxyadenosine monophosphate, respectively.
Assuntos
Cobre , Reação de Cicloadição , DNA , Prata , Prata/química , Catálise , DNA/química , Estereoisomerismo , Cobre/química , Citosina/química , Humanos , Pareamento de BasesRESUMO
i-Motifs (iMs) are non-canonical, four-stranded secondary structures formed by stacking of hemi-protonated CH+·C base pairs in cytosine-rich DNA sequences, predominantly at pH < 7. The presence of iM structures in cells was a matter of debate until the recent development of iM-specific antibody, iMab, which was instrumental for several studies that suggested the existence of iMs in live cells and their putative biological roles. We assessed the interaction of iMab with cytosine-rich oligonucleotides by biolayer interferometry (BLI), pull-down assay and bulk-FRET experiments. Our results suggest that binding of iMab to DNA oligonucleotides is governed by the presence of runs of at least two consecutive cytosines and is generally increased in acidic conditions, irrespectively of the capacity of the sequence to adopt, or not, an iM structure. Moreover, the results of the bulk-FRET assay indicate that interaction with iMab results in unfolding of iM structures even in acidic conditions, similarly to what has been observed with hnRNP K, well-studied single-stranded DNA binding protein. Taken together, our results strongly suggest that iMab actually binds to blocks of 2-3 cytosines in single-stranded DNA, and call for more careful interpretation of results obtained with this antibody.