DNA sensing
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DNA methylation plays an important role in physiological and pathological processes. Several genetic diseases and most malignancies tend to be associated with aberrant DNA methylation. Among other analytical methods, electrochemical approaches have been successfully employed for characterisation of DNA methylation patterns that are essential for the diagnosis and treatment of particular diseases. This article discusses current trends in the electrochemical sensing and biosensing of DNA methylation. Particularly, it provides an overview of applied electrode materials, electrode modifications and biorecognition elements applications with an emphasis on strategies that form the core DNA methylation detection approaches. The three main strategies as (i) bisulfite treatment, (ii) cleavage by restriction endonucleases, and (iii) immuno/affinity reaction were described in greater detail. Additionally, the availability of the reviewed platforms for early cancer diagnosis and the approval of methylation inhibitors for anticancer therapy were discussed.
- MeSH
- biosenzitivní techniky přístrojové vybavení metody MeSH
- DNA analýza genetika MeSH
- elektrochemické techniky přístrojové vybavení metody MeSH
- elektrody MeSH
- lidé MeSH
- metylace DNA * MeSH
- nádory diagnóza genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The mechanism by which DNA viruses interact with different DNA sensors and their connection with the activation of interferon (IFN) type I pathway are poorly understood. We investigated the roles of protein 204 (p204) and cyclic guanosine-adenosine synthetase (cGAS) sensors during infection with mouse polyomavirus (MPyV). The phosphorylation of IFN regulatory factor 3 (IRF3) and the stimulator of IFN genes (STING) proteins and the upregulation of IFN beta (IFN-β) and MX Dynamin Like GTPase 1 (MX-1) genes were detected at the time of replication of MPyV genomes in the nucleus. STING knockout abolished the IFN response. Infection with a mutant virus that exhibits defective nuclear entry via nucleopores and that accumulates in the cytoplasm confirmed that replication of viral genomes in the nucleus is required for IFN induction. The importance of both DNA sensors, p204 and cGAS, in MPyV-induced IFN response was demonstrated by downregulation of the IFN pathway observed in p204-knockdown and cGAS-knockout cells. Confocal microscopy revealed the colocalization of p204 with MPyV genomes in the nucleus. cGAS was found in the cytoplasm, colocalizing with viral DNA leaked from the nucleus and with DNA within micronucleus-like bodies, but also with the MPyV genomes in the nucleus. However, 2'3'-Cyclic guanosine monophosphate-adenosine monophosphate synthesized by cGAS was detected exclusively in the cytoplasm. Biochemical assays revealed no evidence of functional interaction between cGAS and p204 in the nucleus. Our results provide evidence for the complex interactions of MPyV and DNA sensors including the sensing of viral genomes in the nucleus by p204 and of leaked viral DNA and micronucleus-like bodies in the cytoplasm by cGAS.
- MeSH
- DNA virů genetika imunologie MeSH
- fosfoproteiny antagonisté a inhibitory genetika metabolismus MeSH
- fosforylace MeSH
- infekce onkogenními viry imunologie virologie MeSH
- interakce hostitele a patogenu MeSH
- interferon beta metabolismus MeSH
- jaderné proteiny antagonisté a inhibitory genetika metabolismus MeSH
- membránové proteiny antagonisté a inhibitory genetika metabolismus MeSH
- myši MeSH
- nukleotidyltransferasy antagonisté a inhibitory genetika metabolismus MeSH
- polyomavirové infekce imunologie virologie MeSH
- Polyomavirus genetika imunologie MeSH
- přirozená imunita imunologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Modern trends in electrochemical sensing deoxyribonucleic acids (DNA), particularly the use of electrochemical sensors for detection of DNA damage or hybridization, are discussed. Applications of electrochemical methods such as AC voltammetry, square-wave voltammetry and constant current chronopotentiometry as well as use of mercury and carbon electrodes are presented. Special attention is paid to application of Hg amalgams and carbon (pyrolytic graphite, C paste or glassy C) electrodes for monitoring reduction and oxidation processes (label-free detection). Techniques and procedures used for DNA labeling with electroactive tags such as transition-metal (Os, Ru, Cu) complexes or redox mediators are described. DNA interactions with heavy metal ions, drugs, and proteins are also mentioned. The review does not intend to give a complete overview of the topics considered but, rather, to present some historic consequences and modern electrochemical methods used in DNA research.
Specific DNA-protein interactions are vital for cellular life maintenance processes, such as transcriptional regulation, chromosome maintenance, replication and DNA repair, and their monitoring gives valuable information on molecular-level organization of those processes. Here, we propose a new method of label-free electrochemical sensing of sequence specific binding between the lysozyme protein and a single stranded DNA aptamer specific for lysozyme (DNAapta) that exploits the constant current chronopotentiometric stripping (CPS) analysis at modified mercury electrodes. Specific lysozyme-DNAapta binding was distinguished from nonspecific lysozyme-DNA interactions at thioglycolic acid-modified mercury electrodes, but not at the dithiothreitol-modified or bare mercury electrodes. Stability of the surface-attached lysozyme-DNAapta layer depended on the stripping current (Istr) intensity, suggesting that the integrity of the layer critically depends on the time of its exposure to negative potentials. Stabilities of different lysozyme-DNA complexes at the negatively polarized electrode surface were tested, and it was shown that structural transitions of the specific lysozyme-DNAapta complexes occur in the Istr ranges different from those observed for assemblies of lysozyme with DNA sequences capable of only nonspecific lysozyme-DNA interactions. Thus, the CPS allows distinct discrimination between specific and non-specific protein-DNA binding and provides valuable information on stability of the nucleic acid-protein interactions at the polarized interfaces.
BACKGROUND: The aberrant recognition of self-nucleic acids by the innate immune system contributes to the pathology of several autoimmune diseases. Although microbial DNA and, in certain instances, self-DNA that is released from damaged cells are primarily recognized by Toll-like receptor 9 (TLR9), recent evidence suggests that other cytosolic sequence-nonspecific DNA sensors contribute to DNA recognition. In this study, we focused on the sensing of microbial and host DNA in type 1 diabetes (T1D) patients. METHODS: Peripheral blood mononuclear cells (PBMCs) and monocytes from pediatric patients with T1D and from healthy donors were stimulated with microbial DNA (CpG) or with self-DNA (DNA contained within neutrophil extracellular traps, NETs). The production of cytokines was measured by flow cytometry and multiplex bead assays. The internalization of microbial DNA and its colocalization with STING was detected by image cytometry. Furthermore, the involvement of the TBK1 kinase was investigated by detecting its phosphorylation with phospho-flow cytometry or by using a TBK1 inhibition assay. RESULTS: We observed a prominent proinflammatory response in T1D PBMCs, especially pDCs and monocytes, to microbial DNA in comparison to that in controls. We further confirmed that monocytes could bind and internalize DNA and respond by releasing proinflammatory cytokines in a more pronounced manner in T1D patients than those in controls. Surprisingly, this cytokine production was not affected by TLR9 blockade, suggesting the involvement of intracellular receptors in DNA recognition. We further identified TBK1 and STING as two crucial molecules in the DNA-sensing pathway that were involved in CpG-DNA sensing by T1D cells. A similar DNA-sensing pathway that was dependent on intracellular DNA sensors and the STING-TBK1 interaction was employed in response to NETs, which were used to model self-DNA. CONCLUSIONS: Here, we show that there were significant differences in DNA sensing in T1D patients compared to that in controls. We demonstrate that monocytes from T1D patients are able to sense microbial- and self-DNA, leading to proinflammatory cytokine secretion through the adaptor protein STING and the TBK1 kinase.
- MeSH
- CpG ostrůvky genetika MeSH
- cytokiny metabolismus MeSH
- diabetes mellitus 1. typu genetika metabolismus MeSH
- dítě MeSH
- DNA metabolismus MeSH
- leukocyty mononukleární metabolismus MeSH
- lidé MeSH
- membránové proteiny metabolismus MeSH
- mladiství MeSH
- monocyty metabolismus MeSH
- protein-serin-threoninkinasy metabolismus MeSH
- signální transdukce fyziologie MeSH
- studie případů a kontrol MeSH
- toll-like receptor 9 metabolismus MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Guanine-rich sequences of DNA are able to create tetrastranded structures known as G-quadruplexes; they are formed by the stacking of planar G-quartets composed of four guanines paired by Hoogsteen hydrogen bonding. G-quadruplexes act as ligands for metal ions and aptamers for various molecules. Interestingly, the G-quadruplexes form a complex with anionic porphyrin hemin and exhibit peroxidase-like activity. This review focuses on overview of sensing techniques based on G-quadruplex complexes with anionic porphyrins for detection of various analytes, including metal ions such as K+, Ca2+, Ag+, Hg2+, Cu2+, Pb2+, Sr2+, organic molecules, nucleic acids, and proteins. Principles of G-quadruplex-based detection methods involve DNA conformational change caused by the presence of analyte which leads to a decrease or an increase in peroxidase activity, fluorescence, or electrochemical signal of the used probe. The advantages of various detection techniques are also discussed.
- MeSH
- biosenzitivní techniky * MeSH
- delece genu MeSH
- DNA katalytická chemie MeSH
- DNA chemie MeSH
- G-kvadruplexy * MeSH
- ionty analýza chemie MeSH
- kovy analýza chemie MeSH
- lidé MeSH
- nádorový supresorový protein p53 chemie genetika MeSH
- nanočástice chemie MeSH
- nukleové kyseliny analýza chemie MeSH
- organické látky analýza chemie MeSH
- proteiny analýza chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Stripping voltammetric determination of purine bases in the presence of copper ions at mercury, amalgam, or carbon-based electrodes has recently been utilized in analysis of DNA or synthetic oligodeoxynucleotides (ODNs). Here we report on copper-enhanced label-free anodic stripping detection of guanine and adenine bases in acid-hydrolyzed DNA at anodically oxidized boron-doped diamond electrode (AO-BDDE). The AO-BDDE was successfully applied in a three-electrode microcell in which an approximately 50 microL drop of the analyte solution can be efficiently stirred during the accumulation step by streaming of an inert gas. Accelerated mass transport due to the solution motion in the presence of copper resulted in enhancement of the guanine oxidation signal by about 2 orders of magnitude (compared to accumulation of the analyte from still solution not containing copper), allowing an easy detection of approximately 25 fmol of the ODNs. The proposed technique is shown to be suitable for a determination of purine (particularly guanine) content in DNA samples. Applications of the technique in magnetic bead-based DNA assays (such as hybridization with DNA sequences exhibiting asymmetrical distribution of purine/pyrimidine nucleotides between the complementary strands or monitoring of amplification of specific DNA fragments in a duplex polymerase chain reaction) are demonstrated.
- MeSH
- bor chemie MeSH
- diamant chemie MeSH
- DNA-dependentní DNA-polymerasy metabolismus MeSH
- DNA MeSH
- elektrochemie MeSH
- elektrody MeSH
- financování organizované MeSH
- hydrolýza MeSH
- kationty chemie MeSH
- kyseliny chemie MeSH
- měď chemie MeSH
- oligonukleotidy chemie MeSH
- oxidace-redukce MeSH
- puriny chemie MeSH
- sekvence nukleotidů MeSH
OBJECTIVE: Autosomal dominant hypocalcemia (ADH) is a rare disorder caused by activating mutations of the calcium-sensing receptor (CASR). The treatment of ADH patients with 1α-hydroxylated vitamin D derivatives can cause hypercalciuria leading to nephrocalcinosis. DESIGN AND METHODS: We studied a girl who presented with hypoparathyroidism and asymptomatic hypocalcemia at age 2.5 years. Mutations of CASR were investigated by DNA sequencing. Functional analyses of mutant and WT CASRs were done in transiently transfected human embryonic kidney (HEK293) cells. RESULTS: The proband and her father are heterozygous for an eight-nucleotide deletion c.2703_2710delCCTTGGAG in the CASR encoding the intracellular domain of the protein. Transient expression of CASR constructs in kidney cells in vitro suggested greater cell surface expression of the mutant receptor with a left-shifted extracellular calcium dose-response curve relative to that of the WT receptor consistent with gain of function. Initial treatment of the patient with calcitriol led to increased urinary calcium excretion. Evaluation for mosaicism in the paternal grandparents of the proband was negative. CONCLUSIONS: We describe a novel naturally occurring deletion mutation within the CASR that apparently arose de novo in the father of the ADH proband. Functional analysis suggests that the cytoplasmic tail of the CASR contains determinants that regulate the attenuation of signal transduction. Early molecular analysis of the CASR gene in patients with isolated idiopathic hypoparathyroidism is recommended because of its relevance to clinical outcome and treatment choice. In ADH patients, calcium supplementation and low-dose cholecalciferol avoids hypocalcemic symptoms without compromising renal function.
- MeSH
- cytoplazma MeSH
- dominantní geny * MeSH
- dospělí MeSH
- HEK293 buňky MeSH
- heterozygot MeSH
- hyperkalciurie genetika patologie MeSH
- hypokalcemie genetika patologie MeSH
- hypoparatyreóza vrozené genetika patologie MeSH
- lidé MeSH
- nesmyslný kodon genetika MeSH
- předškolní dítě MeSH
- receptory "calcium-sensing" chemie genetika MeSH
- rodina MeSH
- rodokmen MeSH
- sekvence nukleotidů MeSH
- sekvenční delece * MeSH
- terciární struktura proteinů genetika MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
A complex OsO(4), 2,2'-bipyridine (Os,bipy), has been used for electroactive labeling of biopolymers as well as for probing of nucleic acids and protein structure and interactions. In DNA, Os,bipy forms electrochemically active adducts with pyrimidine nucleobases, exhibiting highly selective modification of thymine residues in single-stranded DNA. Here, we show that modification of rare thymine residues (one thymine among several tens of unreactive purine bases) can easily be detected by means of a simple ex situ voltammetric analysis using carbon electrodes. Based on this remarkable sensitivity of detection, Os,bipy has been used as an electroactive probe for unpaired and/or mismatched thymine residues within DNA heteroduplexes. Site-specific chemical modification of the DNA with the Os,bipy has allowed a clear distinction between perfectly base-paired DNA homoduplexes and mismatched heteroduplexes, as well as discrimination among heteroduplexes containing one or two mispaired thymines, a single thymine insertion, or combination of a mispair and an insertion.
Wiley series in probability and statistics
1st ed. xiv, 246 s.
- MeSH
- DNA MeSH
- konformace proteinů MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- biochemie
- genetika, lékařská genetika
