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1.
Org Biomol Chem ; 18(10): 1892-1899, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-31960874

RESUMO

Branched oligonucleotides containing a biologically relevant DNA lesion, dCyd341, which involves an interstrand crosslink between a cytosine base on one strand and a ribose moiety on the opposite strand, were prepared in a single automated solid-phase synthesis. For this, we first prepared the phosphoramidite analogue of dCyd341 bearing an orthogonal levulinyl protecting group. Then, following the synthesis of the first DNA strand containing dCyd341, the levulinic group was removed and the synthesis was then continued from the free base hydroxyl group at the branching point, using traditional phosphoramidites. The synthesized oligonucleotides were fully characterized by MALDI-TOF/MS and were enzymatically digested, and the presence of the lesion was confirmed by HPLC-MS/MS and the sequence was finally controlled upon exonuclease digestion followed by MALDI-TOF/MS analysis. The developed strategy was successfully employed for the preparation of several short linear and branched oligonucleotides containing the aforementioned lesion.


Assuntos
Dano ao DNA , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/genética , Técnicas de Síntese em Fase Sólida
2.
Int J Mol Sci ; 21(2)2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31952262

RESUMO

Hyperlipidemia is a chronic disorder that plays an important role in the development of cardiovascular diseases, type II diabetes, atherosclerosis, hypertension, and non-alcoholic fatty liver disease. Hyperlipidemias have created a worldwide health crisis and impose a substantial burden not only on personal health but also on societies and economies. Transcription factors in the sterol regulatory element binding protein (SREBP) family are key regulators of the lipogenic genes in the liver. SREBPs regulate lipid homeostasis by controlling the expression of a range of enzymes required for the synthesis of endogenous cholesterol, fatty acids, triacylglycerol, and phospholipids. Thereby, SREBPs have been considered as targets for the treatment of metabolic diseases. The aim of this study was to investigate the beneficial functions and the possible underlying molecular mechanisms of SREBP decoy ODN, which is a novel inhibitor of SREBPs, in high-fat diet (HFD)-fed hyperlipidemic mice. Our studies using HFD-induced hyperlipidemia animal model revealed that SREBB decoy ODN inhibited the increased expression of fatty acid synthetic pathway, such as SREBP-1c, FAS, SCD-1, ACC1, and HMGCR. In addition, SREBP decoy ODN decreased pro-inflammatory cytokines, including TNF-α, IL-1ß, IL-8, and IL-6 expression. These results suggest that SREBP decoy ODN exerts its anti-hyperlipidemia effects in HFD-induced hyperlipidemia mice by regulating their lipid metabolism and inhibiting lipogenesis through inactivation of the SREPB pathway.


Assuntos
Modelos Animais de Doenças , Hiperlipidemias/prevenção & controle , Oligodesoxirribonucleotídeos/farmacologia , Proteína de Ligação a Elemento Regulador de Esterol 1/antagonistas & inibidores , Animais , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Citocinas/genética , Citocinas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperlipidemias/etiologia , Hiperlipidemias/genética , Mediadores da Inflamação/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Masculino , Camundongos Endogâmicos C57BL , Oligodesoxirribonucleotídeos/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
3.
Cell Prolif ; 53(1): e12722, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31737959

RESUMO

OBJECTIVES: The mechanisms underlying the effects of Toll-like receptor 9 (TLR9) and autophagy on rheumatoid arthritis (RA)-aggravated periodontitis are unclear. We aimed to explore a novel target, cathepsin K (Ctsk)-mediated TLR9-related autophagy, during the progress of periodontitis with RA. MATERIALS AND METHODS: DBA/J1 mouse model of periodontitis with RA was created by local colonization of Porphyromonas gingivalis (Pg) and injection of collagen. The expression of Ctsk was inhibited by adeno-associated virus (AAV). Micro-CT, immunohistochemistry (IHC), Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of TLR9-related autophagy in periodontitis with RA. Small interfering RNA (siRNA) and CpG oligodeoxynucleotides (CpG ODN) were applied in macrophages. Western blot, immunofluorescence (IF) and qRT-PCR were used to verify the in vivo results. RESULTS: RA can promote periodontitis bone destruction in the lesion area, while inhibiting Ctsk could effectively alleviate this effect. The infiltration of macrophages, TLR9, autophagy proteins (TFEB and LC3) and inflammatory cytokines increased in the periodontitis-with-RA group and was reduced by the inhibition of Ctsk in the periodontal region. Macrophage stimulation confirmed the in vivo results. With the activation of TLR9 by CpG ODN, inhibition of Ctsk could suppress both TLR9 downstream signalling proteins and autophagy-related proteins. CONCLUSIONS: This study advanced a novel role for Ctsk in TLR9 and autophagy to explain the interaction between periodontitis and RA.


Assuntos
Artrite Reumatoide/tratamento farmacológico , Catepsina K/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Periodontite/tratamento farmacológico , Receptor Toll-Like 9/imunologia , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/imunologia , Catepsina K/genética , Catepsina K/imunologia , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos DBA , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/imunologia , Oligodesoxirribonucleotídeos/genética , Periodontite/genética , Periodontite/imunologia , Periodontite/patologia , Receptor Toll-Like 9/genética
4.
Anal Chim Acta ; 1094: 130-135, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31761039

RESUMO

Quantification of plasma membrane proteins (PMPs) is crucial for understanding the fundamentals of cellular signaling systems and their related diseases. In this work, a super-quadruplex scaffold was designed to regulate assembly of oligonucleotide-grafted AIEgens for detection of PMPs. The nonfluorescence oligonucleotide-grafted AIEgen (Oligo-AIEgen) was firstly synthesized by attaching the AIEgen to 3'-terminus of the oligonucleotide through click chemistry. Meanwhile, the tetramolecular hairpin-conjugated super-quadruplex (THP-G4) as cleavage element and signal enhancement scaffold composited of three elements: a substrate sequence of DNAzyme in the loop region, partial hybridization region in the stem, and six guanine nucleotides to form G-quadruplex. Once the DNAzyme was anchored on the specific PMPs through aptamer-protein recognition, the substrate sequence on the loop of THP-G4 was cleaved by DNAzyme with the aid of cofactor MnII, resulting in the conformation switch of THP-G4 to the activated G-quadruplex scaffold. The latter could assemble Oligo-AIEgens to generate aggregation-induced emission (AIE) enhancement, resulting in a simple and sensitive strategy for detection of membrane proteins. Moreover, the DNAzyme continuously cut the next THP-G4 to achieve recycling amplification. Under the optimized conditions, this AIE-based strategy exhibited good linear relationship with the logarithm of MUC1 concentration from 0.01 to 10 µg mL-1 with the limit of detection down to 4.3 ng mL-1. The G4-assembled AIEgens provides a universal platform for detecting various biomolecules and a proof-of concept for AIE biosensing.


Assuntos
Acrilonitrila/análogos & derivados , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Quadruplex G , Mucina-1/análise , Estilbenos/química , Acrilonitrila/síntese química , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Linhagem Celular Tumoral , DNA Catalítico/química , DNA Catalítico/genética , Corantes Fluorescentes/síntese química , Humanos , Limite de Detecção , Mucina-1/química , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Estudo de Prova de Conceito , Estilbenos/síntese química
5.
Nucleosides Nucleotides Nucleic Acids ; 39(1-3): 119-130, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31645189

RESUMO

Point mutations are well characterized activators of oncogenes but are often indistinguishable using common gene technologies. In general, the precise sites of point-mutated oncogenes are difficult to distinguish under physiological conditions primarily because single nucleotide mismatch do not affect the annealing temperatures of DNA probes sufficiently. To address this limitation, we developed photo-responsive oligodeoxyribonucleotides containing 2'-O-[N-(4,5',8-trimethylpsoralen-4'-ylmethylcarbamoyl)]adenosine (Ps-amd-Oligo), which can be used to selectively manipulate and identify genes with point mutations. Here we present time course analyses of the photo-cross-linking efficiency of Ps-amd-Oligo with DNA and RNA and show promising selectivity for the oncogene H-ras.


Assuntos
Adenosina/análogos & derivados , Adenosina/química , DNA , Mutação , Oligodesoxirribonucleotídeos/química , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenosina/síntese química , Reagentes para Ligações Cruzadas , DNA/química , DNA/genética , Estrutura Molecular , Oligodesoxirribonucleotídeos/genética , Mutação Puntual , Raios Ultravioleta
6.
J Vasc Surg ; 71(1): 229-241, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31204215

RESUMO

OBJECTIVE: Intimal hyperplasia (IH) is the main cause of therapeutic failure after vascular and endovascular surgery. However, there is currently no targeted therapy for the treatment of IH. We recently reported that the inhibition of cyclic adenosine monophosphate response element (CRE) binding protein (CREB) activation is important in vein graft IH. We focused on a decoy oligodeoxynucleotide (ODN) therapeutic strategy for suppressing IH as a clinical application. The objective of this study was to confirm the therapeutic effect of a CRE decoy ODN in an animal model as a novel therapy for preventing intimal hyperplasia as the first step of the preclinical study of our strategy. METHODS: We designed two phosphorothioate CREs and two scramble decoy ODNs and screened them using a CREB transcription assay to check their ability to bind to a CRE sequence. We chose a CRE decoy ODN with high first-binding ability and transfected it into vascular smooth muscle cells (VSMCs) in vitro. Proliferation and migration were assessed using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays and modified Boyden chamber assays. We examined CRE activity using a luciferase reporter gene assay. We assessed the expression of messenger RNAs by quantitative real-time polymerase chain reaction. In a wire-injury mouse model (C57BL6, n = 6), CRE decoy ODN was transfected into the injured vessel wall using an ultrasound-sonoporation method in vivo. Mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) and four and a half LIM domains 5 (FHL5) expression of pregrafting vein remnants were assessed by immunohistologic analyses. RESULTS: Compared with scramble decoy ODN, the selected CRE decoy ODN could significantly decrease CRE activity (mean ± standard error of the mean: 0.20 ± 0.03 vs 1.00 ± 0.16, n = 6; P < .05) as shown by a luciferase reporter gene assay, VSMC proliferation (0.73 ± 0.04 vs 0.89 ± 0.02, n = 6; P < .05) and migration (96.4 ± 6.1 vs 311.4 ± 19.1 migrated VSMCs/well, n = 6; P < .05) after 24-hour transfection. The CRE decoy ODN significantly suppressed the formation of IH at injured vessel walls in an animal model, as analyzed by pathologic staining (0.20 ± 0.02 vs 0.56 ± 0.08, area of the intima/area of the artery vs the control after 21 days' transfection, n = 6; P < .05). Furthermore, MAPKAPK3 and FHL5, which are CREB activators, were significantly expressed in pregrafting vein remnants in diabetes mellitus patients. CONCLUSIONS: CREB-CRE signaling is an important mechanism of IH formation, and CRE decoy therapy can help preventing IH. This study is the first part of the preclinical study of our strategy.


Assuntos
AMP Cíclico/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima , Oligodesoxirribonucleotídeos/genética , Elementos de Resposta/genética , Lesões do Sistema Vascular/prevenção & controle , Animais , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Oligodesoxirribonucleotídeos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/metabolismo , Lesões do Sistema Vascular/patologia
7.
J Microbiol ; 58(2): 153-162, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31872374

RESUMO

Oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG-ODN) can be specifically recognized by Toll-like receptor 9 (TLR9), provoking innate immune responses. Designed according to this structural feature, many synthetic phosphorothioate CpG-ODNs successfully activate macrophages. However, it is difficult to find potent stimulatory CpG-DNA fragments in microbial genomes. Therefore, whether microbial CpG-DNA substantially contributes to infectious and immune diseases remains controversial. In this study, high-throughput scanning was carried out for thousands of bacterial genomes with bioinformatics tools to comprehensively evaluate the distribution of CpG-DNA fragments. A random sampling test was then performed to verify their immunostimulatory properties by experiments in vitro and in vivo. Natural TLR9-dependent and potent stimulatory CpG-DNA fragments were found in microbial genomes. Interestingly, highly conserved stimulatory CpG-DNA fragments were found in 16S and 23S rDNA sequences with multiple copies, while others were species-specific. Additionally, we found that the reported active motifs were mostly non-stimulatory in natural CpG fragments. This evidence indicates that the previous structural descriptions of functional CpG-ODNs are incomplete. Our study has assessed the distribution of microbial CpG-DNA fragments, and identified natural stimulatory CpG-DNA fragments. These findings provide a deeper understanding of CpG-ODN structures and new evidence for microbial DNA inflammatory function and pathogenicity.


Assuntos
Adjuvantes Imunológicos/genética , Genoma Bacteriano/imunologia , Oligodesoxirribonucleotídeos/genética , Animais , Biologia Computacional , Escherichia coli/genética , Imunidade Inata , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/microbiologia , Camundongos , Streptococcus/genética , Receptor Toll-Like 9/imunologia
8.
Life Sci ; 239: 117067, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31738882

RESUMO

AIMS: Both CpG oligodeoxynucleotide (CpG-ODN) and melatonin have been reported to induce Th1 response and contribute to allergic asthma resistance. Here, we aimed to reveal how they confer such effect as well as whether they crosstalk with each other. MAIN METHODS: Six-week-old Female C57BL/6 mice were challenged by OVA to induce allergic airway inflammation, and were treated with CpG-ODN, CpG-ODN plus Luzindole or melatonin respectively. Bronchoalveolar lavage fluid (BALF) cellularity was classified and counted by Wright's-Giemsa staining. HE and PAS staining were used to analyze airway inflammation. The levels of IL-4, IL-5, IL-13,GM-CSF and IFN-γ, as well as IL-1ß and IL-18 were analyzed by ELISA. Protein expressions of ASMT, AANAT, NLRP3, IL-1ß and caspase-1 in lung tissue were detected by Western blotting, expression of ASMT and AANAT were further observed by immunohistochemistry. KEY FINDINGS: We found that CpG-ODN considerably suppressed OVA-induced airway leukocytes infiltration, goblet cell hyperplasia and Th2 cytokines production. Furthermore, the resolution effect of CpG-ODN on OVA-induced allergic airway inflammation occurred in parallel with decreased-activation of NLRP3 inflammasome and increased biosynthesis of melatonin. Blocking the effect of endogenous melatonin by Luzindole abolished the suppressive effect of CpG-ODN on OVA-induced airway inflammation and activation of NLRP3 inflammasome, suggesting such effect was mediated by endogenous melatonin. Moreover, exogenous melatonin pronouncedly ameliorated airway inflammation and decreased the activation of NLRP3 inflammasome. SIGNIFICANCE: These results proven that CpG-ODN protects against allergic airway inflammation via suppressing the activation of NLRP3 inflammasome, and such effect may be resulted from the restored-production of melatonin.


Assuntos
Inflamassomos/efeitos dos fármacos , Melatonina/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Animais , Asma , Líquido da Lavagem Broncoalveolar , Citocinas/metabolismo , Feminino , Hipersensibilidade/metabolismo , Inflamassomos/metabolismo , Inflamassomos/fisiologia , Inflamação/metabolismo , Inflamação/prevenção & controle , Pulmão/metabolismo , Melatonina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Células Th2 , Triptaminas/farmacologia
9.
Mikrochim Acta ; 186(12): 843, 2019 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-31768709

RESUMO

Voltammetric detection of the K-ras gene fragment was accomplished through the combined application of (a) a switchable DNA nanostructure, (b) the use of hairpin probe and exonuclease III (Exo III)-assisted signal amplification, (c) a split G-quadruplex, and (d) by exploiting the redox activity of DNAzyme. Three assistant oligonucleotides were designed to construct a DNA tweezer on a gold electrode. It is in "open state" in the absence of K-ras DNA. Then, a hairpin probe was introduced, whose stem-loop structure can be opened through hybridization with the K-ras DNA. Exo III is added which hydrolyzes the complementary region of the hairpin sequence to release a single-stranded rest fragment. The ssDNA hybridizes with the DNA tweezer on the electrode which thereby is switched to the "closed state". This leads to the formation of G-quadruplex due to the shortened distance of the split G-quadruplex-forming sequences in the tweezer. The voltammetric signal of the G-quadruplex-hemin complex, with a peak near -0.3 V vs. Ag/AgCl, is used as the signal output. Under the optimal conditions, the current response in differential pulse voltammetry (DPV) increases linearly with the concentration of K-ras DNA in the range of 0.01-1000 pM, and the detection limit is 2.4 fM. The assay can clearly discriminate K-ras DNA from a single-base mutation. The method has excellent selectivity and was applied to the determination of K-ras DNA in (spiked) serum samples. Graphical abstractSchematic representation of a method for the determination of the K-ras gene fragment through a combination of switchable DNA tweezer, split G-quadruplex, and exonuclease III (ExoIII)-assisted target recycling signal amplification.


Assuntos
Técnicas Biossensoriais/métodos , DNA/sangue , Técnicas Eletroquímicas/métodos , Genes ras , Nanoestruturas/química , Oligodesoxirribonucleotídeos/química , Sequência de Bases , DNA/genética , Técnicas Eletroquímicas/instrumentação , Eletrodos , Quadruplex G , Ouro/química , Hemina/química , Humanos , Sequências Repetidas Invertidas , Limite de Detecção , Mutação , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/genética
10.
Analyst ; 145(1): 172-176, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31724655

RESUMO

Single nucleotide polymorphisms (SNPs) have been proven to be important biomarkers for disease diagnosis, prognosis and disease pathogenesis. Here, taking the advantages of a self-assembled oligonucleotide sandwich structure and robust chemical reactions, we have developed a simple, high-throughput and effective colorimetric analytical technique termed CuAAC-based ligation-assisted assays (CuAAC-LA) for SNP detection using a DNA-BIND 96-well plate. With the 5'-azide and 3'-alkyne groups labelled on two oligonucleotide probes, the target DNA can direct a Cu(i)-catalyzed alkyne-azide cycloaddition (CuAAC) click reaction. Since the small difference in duplex stability caused by a single-nucleotide mismatch was amplified by the steric effects of these reactive groups for the ligation reaction of an unstable duplex, CuAAC-LA exhibited an ultra-sensitive discrimination ability for a mutant type target in the presence of large amounts of wild type targets. As low as 0.05% SNP could be clearly detected, which was better than most previously reported methods by various DNA ligases, indicating that a simple and rapid synthetic method i.e., the DNA template-directed click reaction held the potential to replace the ligase for SNP detection.


Assuntos
Colorimetria/métodos , DNA/análise , Polimorfismo de Nucleotídeo Único , Alcinos/química , Armoracia/enzimologia , Azidas/química , Benzidinas/química , Química Click , Corantes/química , Reação de Cicloadição , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Peroxidase do Rábano Silvestre/química , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/genética , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Oxirredução
11.
Analyst ; 145(1): 286-294, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31750449

RESUMO

Different from the classical antiparallel DNA double-stranded structure, parallel DNA duplexes possess unique structures and potential biological functions. In this work, we found that the parallel DNA homoduplex from the [TG(GA)3]n sequence ([TG(GA)3]n-dsDNA) can dramatically enhance the fluorescence of thioflavin T (ThT), and the fluorescence enhancement is proportional to the number (n) of TG(GA)3 units in [TG(GA)3]n. Compared with the traditional G-quadruplex/ThT system, [TG(GA)3]n/ThT showed more stable and stronger fluorescence emission. In addition, coupled with an isothermal exponential amplification reaction, [TG(GA)3]3/ThT was used as a label-free fluorescent probe to detect microRNA, and the [TG(GA)3]3/ThT probe exhibited higher sensitivity than the G-quadruplex/ThT probe. This work provides a new paradigm to design label-free fluorescent biosensing/imaging systems.


Assuntos
Benzotiazóis/química , DNA/química , Corantes Fluorescentes/química , MicroRNAs/análise , Oligodesoxirribonucleotídeos/química , Técnicas Biossensoriais/métodos , DNA/genética , MicroRNAs/genética , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/genética , Estudo de Prova de Conceito
12.
J Am Chem Soc ; 141(45): 18013-18020, 2019 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-31626550

RESUMO

Cell-cell interactions are mediated through compositions expressed on the membrane. Engineering the cell surface to display functional modules with high biocompatibility, high controllability, and high stability would offer great opportunities for studying and manipulating these intercellular reactions. However, it remains a technical challenge because of the complex and dynamic nature of the cell membrane. Herein, by using three-dimensional (3D) amphiphilic pyramidal DNA as the scaffold, we develop a biocompatible, effective, and versatile strategy for engineering the cell surface with DNA probes. Compared with linear DNA constructs, these pyramidal probes show higher (nearly 100-fold) membrane-anchoring stability and higher (about 2.5-fold) target accessibility. They enable specific, effective, and tunable connections between cells. Meanwhile, our results indicate that connecting cells in close proximity are critical to initiate intercellular communication. By combining high programmability and high diversity of DNA probes, this strategy is expected to provide a powerful and designable membrane-anchored nanoplatform for studying multicellular communication networks.


Assuntos
Comunicação Celular/efeitos dos fármacos , Membrana Celular/química , Sondas de DNA/química , DNA/química , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Engenharia Celular/métodos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , DNA/genética , DNA/metabolismo , Sondas de DNA/genética , Sondas de DNA/metabolismo , Fluoresceínas/química , Corantes Fluorescentes/química , Humanos , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo
13.
J Cell Mol Med ; 23(12): 8046-8057, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31631510

RESUMO

ß-thalassaemia is a prevalent hereditary haematological disease caused by mutations in the human haemoglobin ß (HBB) gene. Among them, the HBB IVS2-654 (C > T) mutation, which is in the intron, creates an aberrant splicing site. Bone marrow transplantation for curing ß-thalassaemia is limited due to the lack of matched donors. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), as a widely used tool for gene editing, is able to target specific sequence and create double-strand break (DSB), which can be combined with the single-stranded oligodeoxynucleotide (ssODN) to correct mutations. In this study, according to two different strategies, the HBB IVS2-654 mutation was seamlessly corrected in iPSCs by CRISPR/Cas9 system and ssODN. To reduce the occurrence of secondary cleavage, a more efficient strategy was adopted. The corrected iPSCs kept pluripotency and genome stability. Moreover, they could differentiate normally. Through CRISPR/Cas9 system and ssODN, our study provides improved strategies for gene correction of ß-Thalassaemia, and the expression of the HBB gene can be restored, which can be used for gene therapy in the future.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Oligodesoxirribonucleotídeos/genética , Splicing de RNA/genética , Globinas beta/genética , Talassemia beta/genética , DNA de Cadeia Simples/genética , Terapia Genética/métodos , Hematopoese , Humanos , Células-Tronco Pluripotentes Induzidas/química , Mutação , Clivagem do RNA/genética , Sítios de Splice de RNA , Sequenciamento Completo do Exoma , Globinas beta/metabolismo , Talassemia beta/metabolismo
14.
Analyst ; 144(23): 6936-6943, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31617512

RESUMO

MicroRNAs (miRNAs) are small non-coding RNA molecules that serve as important biomarkers for a variety of diseases such as cancer and vascular disease. However, sensitive and accurate detection of miR-21 is very challenging in that up-regulation of miR-21 is highly associated with several types of malignant tumors. Here, quartz crystal microbalance (QCM) biosensors were developed for sensitive and specific detection of miR-21 through formation of miR-21-DNA hybrid duplexes and non-specific intercalation of surface-modified pyrene molecules. High selectivity for miR-21 over other miRNAs came from the specific hybridization between miR-21 and gold nanoparticle (AuNP)-conjugated complementary oligonucleotides of miR-21. High sensitivity was obtained through formation of intercalated complexes on the surface with subsequent gold staining signal amplification. Under optimum condition using this strategic approach, our novel QCM biosensors could detect miR-21 concentration as low as 3.6 pM in the entire linear range from 2.5 pM to 2.5 µM with a correlation coefficient of 0.989. In addition, these sensors did not work at all for other miRNAs based on their high selectivity. miR-21 in human brain total RNA and total RNA extracted from A549 cell line could also be successfully detected. Therefore, miRNA detection technology using QCM biosensors and their detection mechanisms have potential as alternatives in biological studies and clinical diagnosis.


Assuntos
DNA/química , Substâncias Intercalantes/química , MicroRNAs/análise , Pirenos/química , Células A549 , Técnicas Biossensoriais/métodos , Química Encefálica , DNA/genética , Ouro/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , MicroRNAs/genética , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Técnicas de Microbalança de Cristal de Quartzo/métodos
15.
Cold Spring Harb Protoc ; 2019(10)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575797

RESUMO

This is a general protocol for the isolation of mRNA from total RNA using oligo(dT) coupled to magnetic beads. First, total RNA is dissolved in a high-salt buffer and heated briefly to 65°C-70°C, followed by immediate cooling on ice to disrupt secondary structures. The RNA is subsequently annealed to the oligo(dT)-magnetic beads at room temperature; the high-salt binding buffer stabilizes the poly(A)-oligo(dT) complexes. A high-salt washing buffer is then used to wash away unbound RNAs while retaining oligo(dT)-bound poly(A)+ mRNAs. To elute the poly(A)+ mRNAs from the beads, a low-salt buffer (or water) is used to destabilize the poly(A)-oligo(dT) complexes. Alternatively, poly(A)+ mRNAs can be retained on the beads for downstream applications (e.g., solid-phase cDNA synthesis).


Assuntos
Cromatografia de Afinidade/métodos , Magnetismo , Microesferas , Oligodesoxirribonucleotídeos/genética , Poli A/genética , RNA Mensageiro/genética , Celulose/análogos & derivados , DNA Complementar/genética , Fenômenos Magnéticos , Hibridização de Ácido Nucleico/métodos , Oligodesoxirribonucleotídeos/metabolismo , Poli A/isolamento & purificação , Poli A/metabolismo , RNA/genética , RNA/metabolismo , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo
16.
Int J Mol Sci ; 20(17)2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470511

RESUMO

Approximately 30% of pancreatic cancer patients harbor targetable mutations. However, there has been no therapy targeting these molecules clinically. Nucleic acid medicines show high specificity and can target RNAs. Nucleic acid medicine is expected to be the next-generation treatment next to small molecules and antibodies. There are several kinds of nucleic acid drugs, including antisense oligonucleotides, small interfering RNAs, microRNAs, aptamers, decoys, and CpG oligodeoxynucleotides. In this review, we provide an update on current research of nucleic acid-based therapies. Despite the challenging obstacles, we hope that nucleic acid drugs will have a significant impact on the treatment of pancreatic cancer. The combination of genetic diagnosis using next generation sequencing and targeted therapy may provide effective precision medicine for pancreatic cancer patients.


Assuntos
Aptâmeros de Nucleotídeos/uso terapêutico , Ensaios Clínicos como Assunto/métodos , Oligodesoxirribonucleotídeos/uso terapêutico , Oligonucleotídeos Antissenso/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , RNA Interferente Pequeno/uso terapêutico , Animais , Aptâmeros de Nucleotídeos/genética , Humanos , Oligodesoxirribonucleotídeos/genética , Oligonucleotídeos Antissenso/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , RNA Neoplásico/antagonistas & inibidores , RNA Neoplásico/genética , RNA Interferente Pequeno/genética
17.
Curr Protein Pept Sci ; 20(11): 1060-1068, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31470785

RESUMO

Single stranded microbial DNA fragments with unmethylated deoxycytidylyldeoxyguanosine dinucleotide (CpG) motifs are interpreted as danger signals by the innate immune system via recognition by the Toll-like Receptor 9 (TLR9). Their synthetic analogues, Oligodeoxynucleotides (ODN) comprise a promising class of immune modulators with potential applications in the treatment of multiple diseases, such as cancer, autoimmune diseases or allergy. ODN molecules contain a core hexamer sequence, which is species specific consisting of GACGTT and AACGT for mouse and GTCGTT in humans. Assessment of structural features of different type of ODNs is highly challenging. NMR spectroscopic insights were gained for a short, single CpG motif containing ODN 1668. The structural basis of ODN recognition by TLR9 recently started to unravel as crystal structures of TLR9 orthologues in complex with ODN 1668 were solved. Systematic investigations of ODN sequences revealed that ODNs with a single CpG motif are capable of activating mouse TLR9, but two closely positioned CpG motifs are necessary for activation of human TLR9. Furthermore, longer ODNs with TCC and TCG sequences at the 5' end were shown to activate TLR9 with higher efficiency. It was revealed that 5'-xCx motif containing short ODNs (sODN) are able to augment the immune response of short, single CpG containing ODNs, which are incapable of activating of TLR9 alone. All these observations pointed to the existence of a second binding site on TLR9, which was characterized in crystal structures that delivered further insights of the nucleic acid recognition of the innate immune system by TLR9.


Assuntos
DNA de Cadeia Simples/química , DNA de Cadeia Simples/farmacologia , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Oligodesoxirribonucleotídeos/metabolismo , Receptor Toll-Like 9/metabolismo , Sequência de Bases , DNA de Cadeia Simples/metabolismo , Humanos , Fatores Imunológicos/metabolismo , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética
18.
J Org Chem ; 84(21): 13374-13383, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31536351

RESUMO

In traditional oligodeoxynucleotide (ODN) synthesis, phosphate groups are protected with the 2-cyanoethyl group, and amino groups are protected with acyl groups. At the end of ODN synthesis, deprotection is achieved with strong bases and nucleophiles. Therefore, traditional technologies are not suitable for the synthesis of ODNs containing sensitive functionalities. To address the problem, we report the use of Dim and Dmoc groups, which are based on the 1,3-dithian-2-yl-methyl function, for phosphate and amine protection for the solid phase ODN synthesis. Using the new Dim-Dmoc protection, deprotection was achieved under mild oxidative conditions without using any strong bases and nucleophiles. As a result, the new technology is suitable for the synthesis of ODNs containing sensitive functions. To demonstrate feasibility, seven 20-mer ODNs including four that contain sensitive ester and alkyl chloride groups were synthesized, purified with RP HPLC, and characterized with MALDI-TOF MS and enzyme digestion essays. High purity ODNs were obtained.


Assuntos
Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/síntese química , Amidas/química , Sequência de Bases , Técnicas de Química Sintética , Oligodesoxirribonucleotídeos/genética , Ácidos Fosfóricos/química
19.
Anal Chim Acta ; 1081: 176-183, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31446955

RESUMO

Precise description of temperature at the microscale level is essential in many biological applications. In this study, we prepared a DNA-based thermometer that reports low and high temperatures by providing two distinct optical signals. The system is a molecular beacon that carries a loop and a stem, whose conformation is subject to change from a hairpin to a random coil when the temperature changes from low to high. A fluorophore, Cy5, and a quencher, BHQ3, are terminally labeled at the stem ends. Moreover, perylene is included in the middle of the 3'-end stem. The signaling state of Cy5 relies on the relative distance to BHQ3. However, the perylene emission is regulated by its microenvironment (i.e., the oligonucleotide or duplex state). With a temperature variation, the designed thermometer undergoes a change in conformation that leads to two signal patterns with Cy5/off and perylene/on at low temperature and Cy5/on and perylene/off at high temperature. The reversibility and biocompatibility of the thermometer design were examined for potential applications in biological systems.


Assuntos
Temperatura Baixa , DNA/química , Temperatura Alta , Termômetros , Carbocianinas/química , DNA/genética , Fluorescência , Corantes Fluorescentes/química , Células Hep G2 , Humanos , Sequências Repetidas Invertidas , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Concentração Osmolar , Perileno/química
20.
Int J Mol Sci ; 20(16)2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31426274

RESUMO

Over two decades ago, short oligodeoxynucleotides (ODNs) were proven to be an effective and rapid technique for analysis of gene function without interference in the plant genome. Our previous research has shown the successful regulation of chalcone synthase (CHS) gene expression in flax by ODN technology. The CHS gene encodes a pivotal enzyme in flavonoid biosynthesis. The manipulation of its transcript level was the result of the specific methylation status developed after treatment with ODNs. In further analysis of the application of oligodeoxynucleotides in plants, we will focus on maintaining the methylation status induced originally by ODNs homologous to the regulatory regions of the CHS gene in flax. This article reports the latest investigation applied to stabilization and inheritance of the epigenetic marks induced by plants' treatment with ODNs. The methylation status was analyzed in the particular CCGG motifs located in the CHS gene sequence. Individual plants were able to maintain alterations induced by ODNs. In order to confirm the impact of methylation marks on the nucleosome rearrangement, chromatin accessibility assay was performed. The perpetuation of targeted plant modulation induced by ODNs exhibits strong potential for improving crops and intensified application for medicine, nutrition and industry.


Assuntos
Aciltransferases/genética , Metilação de DNA , Linho/genética , Engenharia Genética/métodos , Proteínas de Plantas/genética , Sequência de Bases , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Oligodesoxirribonucleotídeos/genética , Plantas Geneticamente Modificadas/genética
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