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1.
Methods Mol Biol ; 2564: 223-246, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36107345

RESUMO

DNA binding fluorescent proteins are a powerful tool for single-molecule visualization. In this chapter, we discuss a protocol for the synthesis of DNA binding fluorescent proteins and visualization of single DNA molecules. This chapter includes stepwise methods for molecular cloning, reversible staining, two-color staining, sequence-specific staining, and microscopic visualization of single DNA molecules in a microfluidic device. This content will be useful for DNA characterization using DNA binding fluorescent proteins and its visualization at the single-molecule level.


Assuntos
Proteínas de Ligação a DNA , DNA , DNA/química , Proteínas de Ligação a DNA/metabolismo , Corantes Fluorescentes , Microscopia de Fluorescência/métodos , Coloração e Rotulagem
2.
Methods Mol Biol ; 2519: 65-72, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36066710

RESUMO

The comet assay is an effective method for identifying DNA breaks and alkali-labile sites induced by genotoxins. Performed as a single-cell electrophoresis, this assay is especially simplistic, and the results are easily reproducible. DNA breakage can be quantitatively assessed by the induced comet tail regions, which can be measured using a variety of comet software. This protocol will finish within approximately two hours with adequate preparation, and digitized images can be taken using a confocal or standard fluorescence microscopes after staining the cell nucleus with a DNA dye.


Assuntos
Dano ao DNA , Mutagênicos , Ensaio Cometa/métodos , DNA , Coloração e Rotulagem
3.
Methods Mol Biol ; 2519: 93-98, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36066713

RESUMO

After DNAs are damaged, DNA repair proteins accumulate and are activated at the DNA damaged site. These accumulated proteins are visualized as foci by fluorescent immunocytochemistry technique. This allows the DNA damage responses in interphase nuclei to be detected; it was earlier times difficult to analyze DNA damage in situ. In order to analyze DNA damage in interphase cells, either DNA is extracted to assay breaks biochemically, or premature chromosome condensation is conducted to observe as chromatin breaks. Although DNA damage-induced foci are typically analyzed in interphase cells, these foci can be also visualized on mitotic chromosomes. The foci where the repair proteins accumulate at the damage site is observed as mitotic chromosome break site. Since mitotic cells attach loosely or not attached to cell culture vessels, it is difficult to analyze foci on chromosomes in culture vessels under a microscope, so metaphase chromosome spread must be prepared for accurate analysis. The cytocentrifuge system is an ideal method to adhere mitotic cells to microscope slides for the fluorescent immunocytochemistry. This chapter introduces cytocentrifuge method to prepare metaphase spread for DNA damage foci analysis.


Assuntos
Cromossomos , Dano ao DNA , DNA , Interfase , Metáfase
4.
Methods Mol Biol ; 2519: 117-126, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36066717

RESUMO

A basic question of cell biology is how DNA folds to chromosome. A number of recently accumulated evidences have suggested that folding of chromosome proceeds tightly coupled with DNA replication progresses. Drug-induced PCC is a useful tool for visualization of the interphase nuclei, in particular, S-phase, as S-phase prematurely condensed chromosomes (S-phase PCC). Active replicating DNA is labeled directly with Cy3-dUTP by bead loading method, and then S-phase nuclei is immediately condensed prematurely by calyculin A to obtain S-phase PCC. Active replicating regions on S-PCC are observed under a scanning confocal microscope. Cy3-dUTP-labeled S-phase PCCs clearly reveal the drastic transitional change of chromosome formation through S-phase, starting from a "cloudy nebula" to numerous numbers of "beads on a string" and finally to "striped arrays of banding structured chromosome" known as G- or R-banding pattern. The number, distribution, and shape of replication foci were also measured in individual subphase of S-phase; maximally ~1400 foci of 0.35 µm average radius size were scored at the beginning of S-phase, and the number is reduced to ~100 at the end of S-phase. Drug-induced PCC clearly provided the new insight that eukaryote DNA replication is tightly coupled with the chromosome condensation/compaction for construction of eukaryote higher-ordered chromosome structure.


Assuntos
Cromossomos , Replicação do DNA , Núcleo Celular , Cromossomos/genética , DNA , Interfase/genética , Fase S
5.
Small ; 18(12): e2106196, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35322558

RESUMO

Cell mechanical forces play fundamental roles in regulating cellular responses to environmental stimulations. The shortcomings of conventional methods, including force resolution and cellular throughput, make them less accessible to mechanical heterogeneity at the single-cell level. Here, a DNA tensioner platform is introduced with high throughput (>10 000 cells per chip) and pN-level resolution. A microfluidic-based cell array is trapped on "hairpin-structured" DNA tensioners that enable transformation of the mechanical information of living cells into fluorescence signals. By using the platform, one can identify enhanced mechanical forces of drug-resistant cells as compared to their drug-sensitive counterparts, and mechanical differences between metastatic tumor cells in pleural effusion and nonmetastatic histiocytes. Further genetic analysis traces two genes, VEGFA and MINK1, that may play deterministic roles in regulating mechanical heterogeneities. In view of the ubiquity of cells' mechanical forces in the extracellular microenvironment (ECM), this platform shows wide potential to establish links of cellular mechanical heterogeneity to genetic heterogeneity.


Assuntos
DNA , Microfluídica
6.
Immunol Rev ; 305(1): 137-151, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34935162

RESUMO

Epigenetic regulation of gene transcription in the immune system is important for proper control of protective and pathogenic inflammation. Aberrant epigenetic modifications are often associated with dysregulation of the immune cells, including lymphocytes and macrophages, leading to pathogenic inflammation and autoimmune diseases. Two classical epigenetic markers-histone modifications and DNA cytosine methylation, the latter is the 5 position of the cytosine base in the context of CpG dinucleotides-play multiple roles in the immune system. CxxC domain-containing proteins, which basically bind to the non-methylated CpG (i.e., epigenetic "readers"), often function as "writers" of the epigenetic markers via their catalytic domain within the proteins or by interacting with other epigenetic modifiers. We herein report the most recent advances in our understanding of the functions of CxxC domain-containing proteins in the immune system and inflammation, mainly focusing on T cells and macrophages.


Assuntos
Metilação de DNA , Epigênese Genética , Ilhas de CpG , DNA , Humanos , Inflamação/genética
7.
Forensic Sci Int Genet ; 57: 102661, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35063923

RESUMO

Non-self DNA is normally present on skin due to DNA transfer occurring during daily activities. The understanding of persistence and accumulation of foreign DNA on the neck can assist in the interpretation of DNA evidence collected from an assaulted victim. Establishing the composition and level of non-self DNA present is relevant, especially in cases where the victim cohabits with other individuals, such as partner and children. This study investigated the persistence and accumulation of non-self DNA on the neck, over the course of 24 h. DNA samples were collected from the neck of 20 adult volunteers at three time-points, on two days. The detection of a partner's DNA and DNA from unknown sources was studied in relation to the living arrangement and to the activities performed by each individual. An increased number of non-self alleles were detected over time. Partner's DNA was observed to accumulate during the day and to persist when an individual was absent from the shared home environment. DNA from unknown contributors was found on the neck of individuals that used public transport, attended public spaces and had social interactions. The data acquired from this study will help to increase knowledge on the composition of DNA present on an individual's neck in a daily situation.


Assuntos
Vítimas de Crime , DNA , Adulto , Alelos , Criança , DNA/genética , Humanos , Fatores de Tempo
8.
Clin. transl. oncol. (Print) ; 24(10): 1856–1864, octubre 2022.
Artigo em Inglês | IBECS | ID: ibc-207942

RESUMO

Due to the bottlenecks encountered in traditional treatment for tumor, more effective drug targets need to be developed. Cell division cycle 7 kinase plays an important role in DNA replication, DNA repair and recombination signaling pathways. In this review, we first describe recent studies on the role of CDC7 in DNA replication in normal human tissues, and then we integrate new evidence focusing on the important role of CDC7 in replication stress tolerance of tumor cells and its impact on the prognosis of clinical oncology patients. Finally, we comb through the CDC7 inhibitors identified in recent studies as a reference for further research in clinical practice. (AU)


Assuntos
Humanos , Biomarcadores , Proteínas de Ciclo Celular , DNA , Proteínas Serina-Treonina Quinases , Neoplasias
9.
PLoS One ; 17(9): e0273717, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36067197

RESUMO

BACKGROUND: Low socioeconomic status neighborhood exposure to stress and violence may be sources of negative stimuli that poses significant health risks for children, adolescents and throughout the life course of an individual. The study aims to investigate if aberrant epigenetic DNA methylation changes may be a potential mechanism for regulating neighborhood exposures and health outcomes. METHODS: Exposure to environmental stressors identified in 98 young African American (AA) adults aged 18-25 years old from the Washington D.C., area were used in the study. We correlated the association between stress markers; cortisol, CRP, IgG, IGA, IgM, and self-reported exposure to violence and stress, with quantitative DNA methylation changes in a panel of gene-specific loci using saliva DNA. RESULTS: In all participants studied, the exposure to violence was significant and negatively correlated with DNA methylation of MST1R loci (p = 0.032; r = -0.971) and nominally significant with NR3C1 loci (p = 0.053; r = -0.948). In addition, we observed significant and negative correlation of DNA methylation changes of LINE1 (p = 0.044; r = -0.248); NR3C1 (p = 0.017; r = -0.186); MSTR1 (p = 0.022; r = -0.192); and DRD2 (p = 0.056; r = -0.184; albeit nominal significant correlation) with IgA expression. On the other hand, we observed a significant and position correlation of DNA methylation changes in DRD2 (p = 0.037; r = 0.184) with IgG expression. When participants were stratified by sex, we observed in AA young male adults, significant DNA methylation changes of MST1R (p< 0.05) and association with exposure to violence and IgG level. We also observed significant DNA methylation levels of DRD2 (p< 0.05) and association with IgA, IgG, and cortisol level. Furthermore, we observed significant DNA methylation changes of NR3C1 (p< 0.05) with stress, IgA, and IgG in the male participants only. On the other hand, we only observed significant and a positive association of IgG with DNA methylation levels of ESR1 (p = 0.041) in the young AA female participants. CONCLUSION: Our preliminary observation of significant DNA methylation changes in neuronal and immune genes in saliva samples supports our recently published genome-wide DNA methylations changes in blood samples from young AA male adults indicating that saliva offers a non-invasive means for DNA methylation prediction of exposure to environmental stressors in a gender-specific manner.


Assuntos
Metilação de DNA , Hidrocortisona , Adolescente , Adulto , Afro-Americanos/genética , Criança , DNA/metabolismo , Feminino , Humanos , Hidrocortisona/metabolismo , Imunoglobulina A/metabolismo , Imunoglobulina G/metabolismo , Masculino , Saliva/metabolismo , Caracteres Sexuais , Adulto Jovem
10.
PLoS One ; 17(9): e0273670, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36070298

RESUMO

Environmental DNA (eDNA) is increasingly used to noninvasively monitor aquatic animals in freshwater and coastal areas. However, the use of eDNA in the open ocean (hereafter referred to OceanDNA) is still limited because of the sparse distribution of eDNA in the open ocean. Small pelagic fish have a large biomass and are widely distributed in the open ocean. We tested the performance of two OceanDNA analysis methods-species-specific qPCR (quantitative polymerase chain reaction) and MiFish metabarcoding using universal primers-to determine the distribution of small pelagic fish in the open ocean. We focused on six small pelagic fish species (Sardinops melanostictus, Engraulis japonicus, Scomber japonicus, Scomber australasicus, Trachurus japonicus, and Cololabis saira) and selected the Kuroshio Extension area as a testbed, because distribution of the selected species is known to be influenced by the strong frontal structure. The results from OceanDNA methods were compared to those of net sampling to test for consistency. Then, we compared the detection performance in each target fish between the using of qPCR and MiFish methods. A positive correlation was evident between the qPCR and MiFish detection results. In the ranking of the species detection rates and spatial distribution estimations, comparable similarity was observed between results derived from the qPCR and MiFish methods. In contrast, the detection rate using the qPCR method was always higher than that of the MiFish method. Amplification bias on non-target DNA and low sample DNA quantity seemed to partially result in a lower detection rate for the MiFish method; the reason is still unclear. Considering the ability of MiFish to detect large numbers of species and the quantitative nature of qPCR, the combined usage of the two methods to monitor quantitative distribution of small pelagic fish species with information of fish community structures was recommended.


Assuntos
DNA Ambiental , Perciformes , Animais , Biodiversidade , DNA/análise , DNA/genética , DNA Ambiental/genética , Peixes/genética , Oceanos e Mares , Perciformes/genética
11.
Cell Rep ; 40(10): 111310, 2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-36070696

RESUMO

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that plays a critical role in regulating antiviral signaling. cGAS binds to DNA and catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which is essential for downstream signal transduction. The antiviral response is a rapid biological process; however, cGAS itself has relatively low DNA binding affinity, implying that formation of the cGAS-DNA complex requires an additional factor(s) that promotes cGAS-DNA binding, allowing efficient antiviral signal transduction. Here, we report that the Ku proteins (Ku80 and Ku70) directly interact with cGAS and positively regulate cGAS-mediated antiviral signaling. Mechanistically, we find that the interaction of the Ku proteins with cGAS significantly increases the DNA-binding affinity of cGAS and promotes cGAS condensation in the cytosol, thereby enhancing cGAS catalytic activity. Our results show that the Ku proteins are critical partners of cGAS in sensing DNA virus infection and ensuring efficient innate immune signal transduction.


Assuntos
Nucleotídeos Cíclicos , Nucleotidiltransferases , Antivirais , DNA/metabolismo , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/metabolismo
12.
Sci Rep ; 12(1): 15183, 2022 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-36071071

RESUMO

Enhancers regulate gene expression, by playing a crucial role in the synthesis of RNAs and proteins. They do not directly encode proteins or RNA molecules. In order to control gene expression, it is important to predict enhancers and their potency. Given their distance from the target gene, lack of common motifs, and tissue/cell specificity, enhancer regions are thought to be difficult to predict in DNA sequences. Recently, a number of bioinformatics tools were created to distinguish enhancers from other regulatory components and to pinpoint their advantages. However, because the quality of its prediction method needs to be improved, its practical application value must also be improved. Based on nucleotide composition and statistical moment-based features, the current study suggests a novel method for identifying enhancers and non-enhancers and evaluating their strength. The proposed study outperformed state-of-the-art techniques using fivefold and tenfold cross-validation in terms of accuracy. The accuracy from the current study results in 86.5% and 72.3% in enhancer site and its strength prediction respectively. The results of the suggested methodology point to the potential for more efficient and successful outcomes when statistical moment-based features are used. The current study's source code is available to the research community at https://github.com/csbioinfopk/enpred .


Assuntos
Elementos Facilitadores Genéticos , Aprendizado de Máquina , Biologia Computacional/métodos , DNA/metabolismo , Software
13.
J Law Med ; 29(3): 725-739, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36056663

RESUMO

Rapid developments in biotechnology have brought questions regarding ownership of human genetic material to the forefront of the public conscience. This article aims to determine the current approach of Australian and United Kingdom courts to property disputes regarding human biological material and adjudicate its relevance in the context of human deoxyribonucleic acid (DNA) ownership. Following initial exploration of the question of whether DNA ought to be considered an object of property, it argues that the dominant approach established by the landmark decision of Doodeward v Spence (1908) 6 CLR 406 is weaker than the newer "guided discretion" basis in the DNA context. It concludes this latter approach is far better equipped to respond to key policy concerns associated with recognising property rights in DNA.


Assuntos
DNA , Propriedade , Austrália , Humanos , Reino Unido
14.
Zhonghua Yi Xue Za Zhi ; 102(33): 2607-2613, 2022 Sep 06.
Artigo em Chinês | MEDLINE | ID: mdl-36058686

RESUMO

Objective: To investigate the clinical diagnostic value of multi-target stool fecal immunochemical test-DNA (FIT-DNA) test in colorectal cancer (CRC) and advanced adenoma (AA). Methods: A total of 235 patients who were undergoing colonoscopy or colorectal cancer surgery in the Cancer Hospital, Chinese Academy of Medical Sciences from April 2021 to January 2022 were prospectively enrolled. There were 141 males and 94 females, with an average age of (55±13) years (22-86). The patients were divided into two groups, including 215 patients who were first diagnosed but not treated (86 cases of CRC, 12 cases of AA, 25 cases of non-advanced adenoma, 8 cases of hyperplastic or other polyps and 84 apparently healthy cases) and 20 patients in the intervention group (2 cases with a history of CRC surgery, 6 cases with a history of endoscopic surgery, 4 non-CRC patients with special diseases and 8 cases with a history of neoadjuvant chemoradiotherapy). Fresh stool samples were collected before intestinal preparation or surgery for FIT-DNA test using the matching kit for sample processing and nucleic acid purification. KRAS mutation and methylation of BMP3 and NDRG4 genes were detected by fluorescence probe method, and FIT method was employed to detect fecal occult blood. Colonoscopy or pathological biopsy results were used as the gold standard. And the screening and diagnostic efficacy of FIT-DNA test for colorectal cancer and advanced adenoma were evaluated by receiver operating curve (ROC). Results: The sensitivity of FIT-DNA test for early colorectal cancer and advanced adenoma was 7/7 and 8/12, respectively. And the negative predictive value was 98.1% (104/106) and 93.7% (104/111), respectively. The overall screening sensitivity for both early colorectal cancer and advanced adenoma was 15/19, and the negative predictive value was 96.3% (104/108). Besides, the area under the curves (AUCs) were 0.982 (95%CI: 0.960-1.000, P<0.05), 0.758 (95%CI: 0.592-0.924, P<0.05) and 0.841 (95%CI: 0.724-0.957, P<0.05), respectively. Moreover, the diagnostic sensitivity of FIT-DNA test was 98.8% (85/86) for colorectal cancer, 8/12 for advanced adenoma, and 94.9% (93/98) for both colorectal cancer and advanced adenoma, with a specificity of 88.9% (104/117). The AUCs were 0.968 (95%CI: 0.937-0.997, P<0.05), 0.758 (95%CI: 0.592-0.924, P<0.05) and 0.942 (95%CI: 0.905-0.979, P<0.05), respectively. After the inclusion of intervention group, the overall diagnostic sensitivity and specificity of FIT-DNA test was 91.6% (98/107) and 89.1% (114/128), respectively. Conclusion: FIT-DNA test has a high early screening and diagnostic efficacy for colorectal cancer.


Assuntos
Adenoma , Neoplasias Colorretais , Adenoma/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/patologia , DNA , Detecção Precoce de Câncer/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sangue Oculto , Adulto Jovem
15.
Int J Nanomedicine ; 17: 3723-3733, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36061124

RESUMO

Introduction: Urinary tract infections (UTI) are one of the most serious human bacterial infections affecting millions of people every year. Therefore, simple and reliable identification of the urinary tract pathogenic bacteria within a few minutes would be of great significance for diagnosis and treatment of clinical patients with UTIs. In this study, the fluorescence sensor was reported to guide the detection of urinary tract bacterial infections rapidly. Methods: The Ami-AuNPs-DNAs sensor was fabricated by the amino-modified Au nanoparticles (Ami-AuNPs) and six DNAs signal molecules, which bound to the urinary tract pathogenic bacteria and generated corresponding response signals. Further, based on the collected response signals, identification was performed by principal component analysis (PCA) and linear discriminant analysis (LDA). The Ami-AuNPs and Ami-AuNPs-DNAs were characterized by transmission electron microscopy, UV-vis absorption spectrum, Fourier transform infrared spectrum, dynamic light scattering and zeta potentials. Thereafter, the Ami-AuNPs-DNAs sensor was used to discriminate and identify five kinds of urinary tract pathogenic bacteria. Moreover, the quantitative analysis performance towards individual bacteria at different concentrations were also evaluated. Results: The Ami-AuNPs-DNAs sensor were synthesized successfully in terms of spherical, well-dispersed and uniform in size, which could well discriminate five main urinary tract pathogenic bacteria with unique fingerprint-like patterns and was sufficiently sensitive to determine individual bacteria with a detection limit to 1×107 cfu/mL. Furthermore, the sensor had also been successfully applied to identify bacteria in urine samples collected from clinical UTIs. Conclusion: The developed fluorescence sensor could be applied to rapid and accurate discrimination of urinary tract pathogenic bacteria and holds great promise for the diagnosis of the disease caused by bacterial infection.


Assuntos
Infecções Bacterianas , Nanopartículas Metálicas , Infecções Urinárias , Sistema Urinário , Bactérias , Infecções Bacterianas/diagnóstico , DNA , Fluorescência , Ouro , Humanos , Infecções Urinárias/diagnóstico , Infecções Urinárias/microbiologia
16.
J Mol Model ; 28(10): 291, 2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36063245

RESUMO

Ruthenium (Ru)-based anticancer drugs are considered to be novel alternatives of platinum-based drugs. They exhibit potent cytotoxicity against the cancer cells and hence are useful for the treatment of cancer. Herein, the density functional theory calculations in the gas phase and aqueous media are carried out to study the reactions of two Ru(III)-based drugs such as KP1019 and KP418 with the N7 site of guanine (G), 2'-deoxyguanosine (dGua), and guanosine (Gua) to understand their reactivity against the DNA and RNA. All the reactions are found to be exothermic. The activation free energies and rate constants of these reactions indicate that KP1019 and KP418 would react with the dGua more readily than Gua. Hence, the binding of these drugs with the DNA would be more preferred as compared to RNA. It is further found that among these drugs, KP1019 would be more reactive than KP418 in agreement with the experimental observation. Thus, this study is expected to aid in the future development of potent anticancer drugs.


Assuntos
Antineoplásicos , Rutênio , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , DNA , Desoxiguanosina , Guanina/farmacologia , Guanosina/farmacologia , Indazóis , Compostos Organometálicos , RNA , Rutênio/farmacologia , Compostos de Rutênio
17.
Methods Cell Biol ; 172: 115-134, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36064219

RESUMO

When employed according to specific doses and fractionation schedules, radiation therapy (RT) elicits potent tumor-targeting immune responses that rely on the secretion of type I interferon (IFN) by irradiated cancer cells. Most often, this is initiated by the ability of RT to promote the cytosolic accumulation of double-stranded DNA (dsDNA) molecules, which are detected by cyclic GMP-AMP synthase (CGAS) to engage the stimulator of interferon response cGAMP interactor 1 (STING1)-dependent transactivation of type I IFN-coding genes via interferon regulatory factor 3 (IRF3). Here, we describe a simple protocol for the quantification of cytosolic dsDNA species by immunofluorescence microscopy coupled to automated image analysis, as enabled by precise sample processing conditions that permeabilize plasma-but not nuclear or inner mitochondrial-membranes. As compared to subcellular fractionation-based techniques, this approach is compatible with assessments in individual cells aimed at gauging inter-cellular heterogeneity, as well as subcellular tests including co-localization studies.


Assuntos
Interferon Tipo I , Núcleo Celular , Citosol , DNA , Microscopia de Fluorescência
18.
Int Rev Cell Mol Biol ; 372: 175-205, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36064264

RESUMO

RNA editing allows correction of pathological point mutations without permanently altering genomic DNA. Theoretically targetable to any RNA type and site, its flexibility and reversibility makes it a potentially powerful gene editing tool. RNA editing offers a host of potential advantages in specific niches when compared to currently available alternative gene manipulation techniques. Unlike DNA editors, which are currently too large to be delivered in vivo using a viral vector, smaller RNA editors fit easily within the capabilities of an adeno-associated virus (AAV). Unlike gene augmentation, which is limited by gene size and viral packaging constraints, RNA editing may correct transcripts too long to fit within a viral vector. In this article we examine the development of RNA editing and discuss potential applications and pitfalls. We argue that, although in its infancy, an RNA editing approach can offer unique advantages for selected retinal diseases.


Assuntos
Edição de Genes , Edição de RNA , Sistemas CRISPR-Cas , DNA , Edição de Genes/métodos , RNA , RNA Guia/genética , RNA Guia/metabolismo
19.
J Mol Model ; 28(10): 296, 2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-36066769

RESUMO

Genome methylation is a key epigenetic mechanism in various biological events such as development, cellular differentiation, cancer progression, aging, and iPSC reprogramming. Crosstalk between DNA methylation and gene expression is mediated by MBD2, known as the reader of DNA methylation and suggested as a drug target. Despite its magnitude of significance, a scarcely limited number of small molecules to be used as inhibitors have been detected so far. Therefore, we screened a comprehensive compound library to elicit MBD2 inhibitor candidates. Promising molecules were subjected to computational docking analysis by targeting the methylated DNA-binding domain of human MBD2. We could detect reasonable binding energies and docking residues, presumably located in druggable pockets. Docking results were also validated via MD simulation and per-residue energy decomposition calculation. Drug-likeness of these small molecules was assessed through ADMET prediction to foresee off-target side effects for future studies. All computational approaches notably highlighted two compounds named CID3100583 and 8,8-ethylenebistheophylline. These compounds have become prominent as novel candidates, possibly disrupting MBD2MBD-DNA interaction. Consequently, these compounds have been considered prospective inhibitors with the usage potential in a wide range of applications from cancer treatment to somatic cell reprogramming protocols.


Assuntos
Metilação de DNA , Proteínas de Ligação a DNA , Simulação por Computador , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Humanos
20.
Anal Chim Acta ; 1226: 340265, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36068063

RESUMO

Herein, a high-sensitivity photoelectrochemical (PEC) biosensor was developed based on CdS quantum dots (QDs) sensitized porous hexagonal boron nitride (h-BN) nanosheets (NSs) and the multiple sites tripodal DNA walker (TDW) formed by catalytic hairpin assembly (CHA). Noticeably, the porous structure of h-BN NSs gives it a lasting gift of large specific surface areas and extensive active reaction sites, which makes it possible to be employed as photoelectric substrate material. The h-BN/CdS QDs composite material promotes the transmission of photogenerated electrons and holes, the outstanding photoelectric conversion efficiency. Meanwhile, the CHA-formed TDWs triggered by miRNA-141 moved on track strand-functionalized electrode, so that a large number of alkaline phosphatase (ALP) was immobilized on the electrode surface and further in situ catalyzed ascorbic acid 2-phosphate (AAP) to produce ascorbic acid (AA) as the electron donor. As the result of the decisive influence of electron donor on PEC biosensor, the sensitive detection of miRNA-141 was realized. The proposed PEC biosensor displayed an excellent linear relationship ranging from 1 fM to 100 nM with a detection limit of 0.73 fM, providing a powerful strategy for early clinical diagnosis and biomedical research.


Assuntos
Técnicas Biossensoriais , Compostos de Cádmio , MicroRNAs , Pontos Quânticos , Compostos de Boro , Compostos de Cádmio/química , DNA/genética , Técnicas Eletroquímicas , Limite de Detecção , Pontos Quânticos/química
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