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1.
Anal Chem ; 2020 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-32403919

RESUMO

Synthetic DNAzyme motors or machines hold great potential in the detection of intracellular microRNA (miRNA) and mRNA. However, to make intracellular DNAzyme motors or machines operate efficiently, adding exogenous metal ion cofactors as fuel is imperative, which limits their applications. Here, we reported a Na+-specific DNAzyme-based DNAzyme motor differentiating cell subtypes of nonsmall cell lung cancer by simultaneously sensing intracellular miRNA-21 and miRNA-205. The DNAzyme motor could be fueled by intracellular Na+, which avoids the necessity of adding exogenous cofactors. It could be also designed to detect other miRNAs or mRNAs by changing 12-nt DNA domain. Meanwhile, our DNAzyme motor had high sensitivity, excellent specificity, high biostability, and little cytotoxicity. Therefore, the miRNA-initiated and intracellular Na+-fueled DNAzyme motor can expand the application of DNAzyme motors or machines in sensing miRNA and has potential value in cancer clinical diagnosis and prognosis.

2.
Nat Commun ; 11(1): 1518, 2020 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251279

RESUMO

Size selectivity is an important mechanism for molecular recognition based on the size difference between targets and non-targets. However, rational design of an artificial size-selective molecular recognition system for biological targets in living cells remains challenging. Herein, we construct a DNA molecular sieve for size-selective molecular recognition to improve the biosensing selectivity in living cells. The system consists of functional nucleic acid probes (e.g., DNAzymes, aptamers and molecular beacons) encapsulated into the inner cavity of framework nucleic acid. Thus, small target molecules are able to enter the cavity for efficient molecular recognition, while large molecules are prohibited. The system not only effectively protect probes from nuclease degradation and nonspecific proteins binding, but also successfully realize size-selective discrimination between mature microRNA and precursor microRNA in living cells. Therefore, the DNA molecular sieve provides a simple, general, efficient and controllable approach for size-selective molecular recognition in biomedical studies and clinical diagnoses.

3.
Chem Commun (Camb) ; 56(19): 2901-2904, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-32037435

RESUMO

The enzymatic-assisted signal amplification of DNA sensors is rarely applied in living cells due to the difficulties in protein delivery. In this study, we have proposed a biomineralization-based DNA nanoprobe to transport nucleases and DNA sensors for enzyme-assisted imaging of microRNA in living cells.


Assuntos
Biomineralização , Sondas de DNA/química , DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Nanopartículas/química , Humanos , Estruturas Metalorgânicas/química , MicroRNAs/metabolismo
4.
Nat Commun ; 11(1): 978, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080196

RESUMO

In order to maintain tissue homeostasis, cells communicate with the outside environment by receiving molecular signals, transmitting them, and responding accordingly with signaling pathways. Thus, one key challenge in engineering molecular signaling systems involves the design and construction of different modules into a rationally integrated system that mimics the cascade of molecular events. Herein, we rationally design a DNA-based artificial molecular signaling system that uses the confined microenvironment of a giant vesicle, derived from a living cell. This system consists of two main components. First, we build an adenosine triphosphate (ATP)-driven DNA nanogatekeeper. Second, we encapsulate a signaling network in the biomimetic vesicle, consisting of distinct modules, able to sequentially initiate a series of downstream reactions playing the roles of reception, transduction and response. Operationally, in the presence of ATP, nanogatekeeper switches from the closed to open state. The open state then triggers the sequential activation of confined downstream signaling modules.


Assuntos
DNA/metabolismo , Transdução de Sinais , Trifosfato de Adenosina/metabolismo , Células Artificiais/química , Materiais Biomiméticos/química , Biomimética/métodos , Homeostase , Nanoestruturas/química , Biologia Sintética/métodos
5.
Anal Chem ; 92(5): 4154-4163, 2020 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-32050763

RESUMO

Peroxynitrite (ONOO-) is involved in neurodegenerative, inflammatory, cardiovascular disorders, cancers, and other pathological progress. However, current imaging methods for sensing ONOO- usually suffer from high background/autofluorescence for fluorescent probes and poor selectivity/short emission wavelength for chemiluminescent probes. Herein, we present a novel chemiluminescent molecule (oxygen-embedded quinoidal pentacene) responsive to ONOO- for the first time, on the basis of which we rationally construct a near-infrared nanoprobe for detecting ONOO- via chemiluminescence resonance energy transfer (CRET) mechanism. Notably, our nanoprobe exhibits good selectivity, ultrahigh sensitivity (nanomole level), low background noise, fast response, and high water solubility. Moreover, the near-infrared emission from CRET offers higher tissue penetration of the chemiluminescent signal. Finally, our nanoprobe is further successfully applied to detecting endogenous ONOO- in mice with abdominal inflammation, drug-induced hepatotoxicity, or tumor models in vivo. In summary, the self-luminescing nanoprobes can act as an alternative visualizable tool for illuminating the mechanism of ONOO- involved in the specific pathological process.

6.
Anal Chem ; 92(6): 4681-4688, 2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-32098468

RESUMO

Real-time imaging and quantification of adenosine triphosphate (ATP) fluctuation in cells are significant for understanding the relationship between energy metabolism and cell functions. However, few synthetic fluorescent probes have been reported to tackle this challenge due to lack of accurate fluorescence readout and suitable response concentration. Herein we designed and synthesized a ratiometric fluorescent probe (Rh6G-ACFPN) for quantitatively detecting the fluctuation of mitochondrial ATP in living cells. Rh6G-ACFPN selectively and reversibly responds to ATP with an ideal dissociation constant (Kd) of 4.65 mM (3-10 mM: the range of mitochondrial ATP concentrations). Live-cell imaging allows us to directly monitor the dynamic changes of mitochondrial ATP in high temporal resolution. Moreover, for the first time, mitochondrial ATP in normal and cancer cells lines was successfully quantified and discriminated. These results demonstrate the versatility of Rh6G-ACFPN as a useful imaging tool to elucidate the function of mitochondrial ATP in living cells.

7.
Top Curr Chem (Cham) ; 378(2): 21, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32030541

RESUMO

DNA nanostructures hold great promise for various applications due to their remarkable properties, including programmable assembly, nanometric positional precision, and dynamic structural control. The past few decades have seen the development of various kinds of DNA nanostructures that can be employed as useful tools in fields such as chemistry, materials, biology, and medicine. Aptamers are short single-stranded nucleic acids that bind to specific targets with excellent selectivity and high affinity and play critical roles in molecular recognition. Recently, many attempts have been made to integrate aptamers with DNA nanostructures for a range of biological applications. This review starts with an introduction to the features of aptamer-functionalized DNA nanostructures. The discussion then focuses on recent progress (particularly during the last five years) in the applications of these nanostructures in areas such as biosensing, bioimaging, cancer therapy, and biophysics. Finally, challenges involved in the practical application of aptamer-functionalized DNA nanostructures are discussed, and perspectives on future directions for research into and applications of aptamer-functionalized DNA nanostructures are provided.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , DNA/química , Nanoestruturas/química , Técnicas Eletroquímicas , Terapia Genética , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/terapia , Imagem Óptica/métodos , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/uso terapêutico
8.
Life Sci ; 248: 117461, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32097665

RESUMO

AIMS: To compare how OCT4A proteins interact with and regulate multiple OCT4A-octamer motifs (OMs) in different regions of the FOS gene expressed in somatic cancer cells versus pluripotent stem cells. MATERIALS AND METHODS: Two FOS reporter gene systems harboring predicted OMs or their mutational counterparts were introduced into HeLa and NCCIT cells with varying OCT4A protein levels. The transcription of dsGFP reflecting FOS expression was quantitated by RT-qPCR, the OCT4A-OMs binding and the correlation between OCT4A and FOS transcription was determined by ChIP-PCR and RNA-Seq, respectively. KEY FINDINGS: In NCCIT cells, abundant OCT4A proteins bound to and inhibited OM1 and OM2 at the promoter of the FOS gene. RA-induced OCT4A down-regulation transiently increased FOS transcription. In contrast, in HeLa cells that contain much lower levels of endogenous OCT4A proteins, OCT4A primarily bound to and activate OM1 thereby promoting FOS transcription. OCT4A KO significantly reduced FOS expression. Ectopically introduced OCT4A, at its leaked or induced expression level, promoted FOS transcription by binding to OM2/OM3 or OM1/OM3, respectively. Thus, the interaction of OCT4A proteins with different OMs is cellular context- and protein level-dependent, and such complicated OCT4A binding mode can only be reflected by a dsGFP-based reporter harboring the full-length FOS gene but not by that merely having the FOS promoter. SIGNIFICANCE: Our findings unravel an additional layer of regulatory mechanisms that account for the cellular context- and dose-related versatile functions of OCT4A protein, and further underscore the importance of precise modulation of OCT4A in the regenerative medicine and anticancer therapies.


Assuntos
Regulação da Expressão Gênica , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Motivos de Aminoácidos , Linhagem Celular Tumoral , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Fator 3 de Transcrição de Octâmero/metabolismo , Especificidade de Órgãos , Células-Tronco Pluripotentes/citologia , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transdução de Sinais , Transcrição Genética
10.
J Am Chem Soc ; 142(5): 2129-2133, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31955575

RESUMO

Labile heme (LH) is an important signaling molecule in virtually all organisms. However, specifically detecting LH remains an outstanding challenge. Herein, by learning from the bioactivation mechanism of artemisinin, we have developed the first LH-responsive small-molecule fluorescent probe, HNG, based on a 4-amino-1,8-naphthalimide (NG) fluorophore. HNG showed high selectivity for LH without interference from hemin, protein-interacting heme, and zinc protoporphyrin. Using HNG, the changes of LH levels in live cells were imaged, and a positive correlation of LH level with the degree of hemolysis was uncovered in hemolytic mice. Our study not only presents the first molecular probe for specific LH detection but also provides a strategy to construct probes with high specificity through a bioinspired approach.

11.
ACS Appl Mater Interfaces ; 12(5): 5403-5412, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31916740

RESUMO

The deficiency of reactive oxygen species (ROS) is the main reason for the current poor efficiency of tumor photodynamic therapy (PDT). To solve this problem, a simple light-triggered core-satellite nanoplatform (UPSD@Au) has been developed by loading Au nanoparticles on the surface of mesoporous silica-coated upconversion nanoparticles. Small molecules DC50 (C17H14BrF2N3OS) and photosensitizer (silicon phthalocyanine dihydroxide, SPCD) were loaded into the silica shell to improve ROS production. Meanwhile, PDT can be triggered through facile near-infrared laser irradiation given the occurrence of a moderate photothermal transfer process between upconversion nanoparticles and Au. The reasonable increment in temperature induced by Au resulted in the timely release of DC50. The inhibition of copper transfer by DC50 results in reduced ROS scavenging and thus improves light-triggered ROS accumulation. Notably, the expression levels of the human copper-trafficking proteins Atox1 and CCS in cancerous cells exceed those in normal cells, and thus enhanced ROS accumulation effect was achieved in cancerous cells. In vitro and in vivo results demonstrate that the synergism between DC50 and SPCD coloaded in the UPSD@Au nanoplatform increases the efficiency of PDT. The UPSD@Au platform represents an efficient codelivery method for hydrophobic small molecules and improves sensitization to specific cancer therapy.

12.
Chem Commun (Camb) ; 56(9): 1349-1352, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31904042

RESUMO

Herein, a novel two-photon ratiometric fluorescence assay was proposed for monitoring endogenous steroid sulfatase (STS) activity, which could be applied for the ratiometric imaging of STS activity in the endoplasmic reticulum of living cells and tissues and also could be used to distinguish estrogen-dependent tumor cells from other types of cells.


Assuntos
Corantes Fluorescentes/química , Naftalimidas/química , Esteril-Sulfatase/análise , Animais , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/toxicidade , Células HEK293 , Caracois Helix/enzimologia , Humanos , Limite de Detecção , Microscopia de Fluorescência/métodos , Simulação de Acoplamento Molecular , Naftalimidas/metabolismo , Naftalimidas/toxicidade , Fótons , Ligação Proteica , Esteril-Sulfatase/metabolismo
13.
Chem Commun (Camb) ; 56(13): 1956-1959, 2020 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-31956868

RESUMO

Herein, we report a pH stimulus-disaggregated BODIPY sensitizer (PTS) with low background-toxicity for achieving activated photodynamic/photothermal tumor therapy. Both the photodynamic and photothermal properties of PTS can be activated under acidic conditions, and PTS exhibits excellent antitumor properties, which is revealed by both in vitro and in vivo tests.


Assuntos
Compostos de Boro/química , Fármacos Fotossensibilizantes/química , Animais , Compostos de Boro/farmacologia , Compostos de Boro/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Luz , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/tratamento farmacológico , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Fototerapia , Transplante Heterólogo
14.
ACS Sens ; 5(1): 103-109, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31903754

RESUMO

DNA amplification is a useful technique for low-abundance biomarker detection and environmental monitoring because of its high signal-amplifying ability. However, intracellular application of DNA amplifiers remains challenging due to poor delivery efficiency and stability. Herein, we report an entropy-driven DNA amplifier-functionalized metal-organic framework (DNA amplifier-MOF) for the detection and imaging of multiple intracellular messenger RNAs (mRNAs). The DNA amplifier-MOF conjugate exhibits high cellular uptake, enhanced enzymatic stability, and good biocompatibility. Importantly, in the presence of phosphate ions, a surface-functionalized DNA amplifier can be released in cells with high efficiency, which facilitates the imaging of mRNA. This method is rapid and of high sensitivity/specificity, as validated in HepG2 and HL7702 cells for the imaging of TK1 and survivin mRNA, respectively. With further optimization, the strategy can become a powerful biotechnology tool for the detection of cancers at early stages and for elucidating biological processes.

15.
J Orthop Res ; 38(2): 258-268, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31429977

RESUMO

Liver kinase B1 (LKB1), a serine/threonine protein, is a key regulator in stem cell function and energy metabolism. Herein, we describe the role of LKB1 in modulating the differentiation of synovium-derived stem cells (SDSCs) toward chondrogenic, adipogenic, and osteogenic lineages. Human fetal SDSCs were transduced with CRISPR associated protein 9 (Cas9)-single-guide RNA vectors to knockout or lentiviral vectors to overexpress the LKB1 gene. Analyses including ICE (Inference of CRISPR Edits) data from Sanger sequencing and quantitative polymerase chain reaction (qPCR) as well as Western blot demonstrated successful knockout (KO) or overexpression (OE) of LKB1 in human fetal SDSCs without any detectable side effects in morphology, proliferation rate, and cell cycle. LKB1 KO increased CD146 expression; interestingly, LKB1 OE increased SSEA4 level. The qPCR data showed that LKB1 KO upregulated the levels of SOX2 and NANOG while LKB1 OE lowered the expression of POU5F1 and KLF4. Furthermore, LKB1 KO enhanced, and LKB1 OE inhibited, chondrogenic and adipogenic differentiation potential. However, perhaps due to the inherent inability to achieve osteogenesis, LKB1 did not obviously affect osteogenic differentiation. These data demonstrate that LKB1 plays a significant role in determining human SDSCs' adipogenic and chondrogenic differentiation, which might provide an approach for fine-tuning the direction of stem cell differentiation in tissue engineering and regeneration. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:258-268, 2020.

17.
Nano Lett ; 20(1): 176-183, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31777250

RESUMO

In chemodynamic therapy (CDT), real-time monitoring of reactive oxygen species (ROS) production is critical to reducing the nonspecific damage during CDT and feasibly evaluating the therapeutic response. However, CDT agents that can emit ROS-related signals are rare. Herein, we synthesize a semiconducting polymer nanoplatform (SPN) that can not only produce highly toxic ROS to kill cancer cells but also emit ROS-correlated chemiluminescent signals. Notably, the efficacy of both chemiluminescence and CDT can be significantly enhanced by hemin doping (∼10-fold enhancement for luminescent intensity). Such ROS-dependent chemiluminescence of SPN allows ROS generation within a tumor to be optically monitored during the CDT process. Importantly, SPN establishes an excellent correlation of chemiluminescence intensities with cancer inhibition rates in vitro and in vivo. Thus, our nanoplatform represents the first intelligent strategy that enables chemiluminescence-imaging-monitored CDT, which holds potential in assessing therapeutic responsivity and predicting treatment outcomes in early stages.

18.
Angew Chem Int Ed Engl ; 59(2): 695-699, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31628815

RESUMO

Fluorescence visualization (FV) in the near-infrared (NIR) window promises to break through the signal-to-background ratio (SBR) bottleneck of traditional visible-light-driven FV methods. However, straightforward NIR-FV has not been realized, owing to the lack of methods to readily transduce NIR responses into instrument-free, naked eye-recognizable outputs. Now, an initiation-input-transduction platform comprising a well-designed NIR fluorophore as the signal initiator and lanthanide-doped nanocrystals as the transducer for facile NIR-FV is presented. The analyte-induced off-on NIR signal serves as a sensitizing switch of transducer visible luminescence for naked-eye readout. The design is demonstrated for portable, quantitative detection of phosgene with significantly improved SBR and sensitivity. By further exploration of initiators, this strategy holds promise to create advanced NIR-FV probes for broad sensing applications.

19.
Angew Chem Int Ed Engl ; 59(5): 1891-1896, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31746514

RESUMO

Genetically encoded fluorescent proteins (FPs) have been used for metal ion detection. However, their applications are restricted to a limited number of metal ions owing to the lack of available metal-binding proteins or peptides that can be fused to FPs and the difficulty in transforming the binding of metal ions into a change of fluorescent signal. We report herein the use of Mg2+ -specific 10-23 or Zn2+ -specific 8-17 RNA-cleaving DNAzymes to regulate the expression of FPs as a new class of ratiometric fluorescent sensors for metal ions. Specifically, we demonstrate the use of DNAzymes to suppress the expression of Clover2, a variant of the green FP (GFP), by cleaving the mRNA of Clover2, while the expression of Ruby2, a mutant of the red FP (RFP), is not affected. The Mg2+ or Zn2+ in HeLa cells can be detected using both confocal imaging and flow cytometry. Since a wide variety of metal-specific DNAzymes can be obtained, this method can likely be applied to imaging many other metal ions, expanding the range of the current genetically encoded fluorescent protein-based sensors.

20.
Chem Commun (Camb) ; 56(3): 470-473, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31828262

RESUMO

By assembling DNAzyme on DNA nanowires through DNA hybridization, we have developed a novel accelerated DNAzyme-based fluorescent nanoprobe for fast, sensitive and selective detection of miRNA. Moreover, the strategy was successfully applied for in situ imaging of miRNA-21 in different cell lines.


Assuntos
DNA Catalítico/química , Corantes Fluorescentes/química , MicroRNAs/análise , Microscopia Confocal , Nanofios/química , Linhagem Celular Tumoral , Humanos , Limite de Detecção , MicroRNAs/metabolismo , Hibridização de Ácido Nucleico , Espectrometria de Fluorescência
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