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1.
Nat Commun ; 11(1): 4482, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901011

RESUMO

Intracellular trafficking governs receptor signaling, pathogenesis, immune responses and fate of nanomedicines. These processes are typically tracked by observing colocalization of fluorescent markers using confocal microscopy. However, this method is low throughput, limited by the resolution of microscopy, and can miss fleeting interactions. To address this, we developed a localization sensor composed of a quenched SNAP-tag substrate (SNAPSwitch) that can be conjugated to biomolecules using click chemistry. SNAPSwitch enables quantitative detection of trafficking to locations of interest within live cells using flow cytometry. Using SNAPSwitch, we followed the trafficking of DNA complexes from endosomes into the cytosol and nucleus. We show that antibodies against the transferrin or hyaluronan receptor are initially sorted into different compartments following endocytosis. In addition, we can resolve which side of the cellular membrane material was located. These results demonstrate SNAPSwitch is a high-throughput and broadly applicable tool to quantitatively track localization of materials in cells.


Assuntos
DNA/metabolismo , Sondas Moleculares/química , Nanopartículas/metabolismo , Proteínas/metabolismo , Animais , Transporte Biológico Ativo , Técnicas Biossensoriais/métodos , Química Click , Citometria de Fluxo , Corantes Fluorescentes , Células HEK293 , Humanos , Camundongos , Microscopia Confocal , Técnicas de Sonda Molecular , Sondas Moleculares/metabolismo , Células NIH 3T3
2.
ACS Chem Biol ; 15(9): 2331-2337, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32786258

RESUMO

We report on using the synthetic aminoadamantane-CH2-aryl derivatives 1-6 as sensitive probes for blocking M2 S31N and influenza A virus (IAV) M2 wild-type (WT) channels as well as virus replication in cell culture. The binding kinetics measured using electrophysiology (EP) for M2 S31N channel are very dependent on the length between the adamantane moiety and the first ring of the aryl headgroup realized in 2 and 3 and the girth and length of the adamantane adduct realized in 4 and 5. Study of 1-6 shows that, according to molecular dynamics (MD) simulations and molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculations, all bind in the M2 S31N channel with the adamantyl group positioned between V27 and G34 and the aryl group projecting out of the channel with the phenyl (or isoxazole in 6) embedded in the V27 cluster. In this outward binding configuration, an elongation of the ligand by only one methylene in rimantadine 2 or using diamantane or triamantane instead of adamantane in 4 and 5, respectively, causes incomplete entry and facilitates exit, abolishing effective block compared to the amantadine derivatives 1 and 6. In the active M2 S31N blockers 1 and 6, the phenyl and isoxazolyl head groups achieve a deeper binding position and high kon/low koff and high kon/high koff rate constants, compared to inactive 2-5, which have much lower kon and higher koff. Compounds 1-5 block the M2 WT channel by binding in the longer area from V27-H37, in the inward orientation, with high kon and low koff rate constants. Infection of cell cultures by influenza virus containing M2 WT or M2 S31N is inhibited by 1-5 or 1-4 and 6, respectively. While 1 and 6 block infection through the M2 block mechanism in the S31N variant, 2-4 may block M2 S31N virus replication in cell culture through the lysosomotropic effect, just as chloroquine is thought to inhibit SARS-CoV-2 infection.


Assuntos
Adamantano/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/prevenção & controle , Canais Iônicos/antagonistas & inibidores , Sondas Moleculares/química , Proteínas da Matriz Viral/antagonistas & inibidores , Adamantano/análogos & derivados , Adamantano/química , Adamantano/metabolismo , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação , Células Cultivadas , Cloroquina/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , Variação Genética , Humanos , Vírus da Influenza A/química , Vírus da Influenza A/genética , Influenza Humana/tratamento farmacológico , Cinética , Sondas Moleculares/metabolismo , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/prevenção & controle , Ligação Proteica , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
3.
Proc Natl Acad Sci U S A ; 117(30): 18110-18118, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32669427

RESUMO

Mechanical patterns control a variety of biological processes in plants. The microviscosity of cellular structures effects the diffusion rate of molecules and organelles, thereby affecting processes such as metabolism and signaling. Spatial variations in local viscosity are also generated during fundamental events in the cell life cycle. While crucial to a complete understanding of plant mechanobiology, resolving subcellular microviscosity patterns in plants has remained an unsolved challenge. We present an imaging microviscosimetry toolbox of molecular rotors that yield complete microviscosity maps of cells and tissues, specifically targeting the cytosol, vacuole, plasma membrane, and wall of plant cells. These boron-dipyrromethene (BODIPY)-based molecular rotors are rigidochromic by means of coupling the rate of an intramolecular rotation, which depends on the mechanics of their direct surroundings, with their fluorescence lifetime. This enables the optical mapping of fluidity and porosity patterns in targeted cellular compartments. We show how apparent viscosity relates to cell function in the root, how the growth of cellular protrusions induces local tension, and how the cell wall is adapted to perform actuation surrounding leaf pores. These results pave the way to the noninvasive micromechanical mapping of complex tissues.


Assuntos
Modelos Biológicos , Células Vegetais , Fenômenos Fisiológicos Vegetais , Viscosidade , Corantes Fluorescentes/química , Proteínas Motores Moleculares/metabolismo , Sondas Moleculares/química , Especificidade de Órgãos , Organelas/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(26): 14694-14702, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32554491

RESUMO

Innate immune cells destroy pathogens within a transient organelle called the phagosome. When pathogen-associated molecular patterns (PAMPs) displayed on the pathogen are recognized by Toll-like receptors (TLRs) on the host cell, it activates inducible nitric oxide synthase (NOS2) which instantly fills the phagosome with nitric oxide (NO) to clear the pathogen. Selected pathogens avoid activating NOS2 by concealing key PAMPs from their cognate TLRs. Thus, the ability to map NOS2 activity triggered by PAMPs can reveal critical mechanisms underlying pathogen susceptibility. Here, we describe DNA-based probes that ratiometrically report phagosomal and endosomal NO, and can be molecularly programmed to display precise stoichiometries of any desired PAMP. By mapping phagosomal NO produced in microglia of live zebrafish brains, we found that single-stranded RNA of bacterial origin acts as a PAMP and activates NOS2 by engaging TLR-7. This technology can be applied to study PAMP-TLR interactions in diverse organisms.


Assuntos
Encéfalo/enzimologia , DNA/química , Corantes Fluorescentes/química , Óxido Nítrico Sintase Tipo II , Animais , Encéfalo/metabolismo , Química Encefálica , DNA/metabolismo , Corantes Fluorescentes/metabolismo , Técnicas de Inativação de Genes , Camundongos , Microglia/química , Microglia/enzimologia , Microglia/metabolismo , Microscopia de Fluorescência , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Óxido Nítrico Sintase Tipo II/análise , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Fagossomos/química , Fagossomos/metabolismo , Peixe-Zebra
5.
Nat Commun ; 11(1): 2828, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32504003

RESUMO

The TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor are important constituents of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional initiation factor Bdp1 in the RNA polymerase (RNAP) III system, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain arising from DNA compaction and transcriptional activity on initiation complex formation. We made use of a DNA origami-based force clamp to follow the assembly of human initiation complexes in the RNAP II and RNAP III systems at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is sufficient to stabilise TBP on a strained promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of RNAP III.


Assuntos
DNA de Cadeia Simples/metabolismo , RNA Polimerase III/metabolismo , Imagem Individual de Molécula/métodos , Fator de Transcrição TFIIIB/metabolismo , Transcrição Genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/ultraestrutura , Transferência Ressonante de Energia de Fluorescência , Cinética , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Sondas Moleculares/ultraestrutura , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Estabilidade Proteica , RNA Polimerase III/química , Proteínas Recombinantes/metabolismo , Proteína de Ligação a TATA-Box/metabolismo
6.
J Cancer Res Clin Oncol ; 146(8): 1941-1951, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32447486

RESUMO

PURPOSE: Currently, the routine screening program has insufficient capacity for the early diagnosis of lung cancer. Therefore, a type of chitosan-molecular beacon (CS-MB) probe was developed to recognize the miR-155-5p and image the lung cancer cells for the early diagnosis. METHODS: Based on the molecular beacon (MB) technology and nanotechnology, the CS-MB probe was synthesized self-assembly. There are four types of cells-three kinds of animal models and one type of histopathological sections of human lung cancer were utilized as models, including A549, SPC-A1, H446 lung cancer cells, tumor-initiating cells (TICs), subcutaneous and lung xenografts mice, and lox-stop-lox(LSL) K-ras G12D transgenic mice. The transgenic mice dynamically displayed the process from normal lung tissues to atypical hyperplasia, adenoma, carcinoma in situ, and adenocarcinoma. The different miR-155-5p expression levels in these cells and models were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The CS-MB probe was used to recognize the miR-155-5p and image the lung cancer cells by confocal microscopy in vitro and by living imaging system in vivo. RESULTS: The CS-MB probe could be used to recognize the miR-155-5p and image the lung cancer cells significantly in these cells and models. The fluorescence intensity trends detected by the CS-MB probe were similar to the expression levels trends of miR-155 tested by qRT-PCR. Moreover, the fluorescence intensity showed an increasing trend with the tumor progression in the transgenic mice model, and the occurrence and development of lung cancer were dynamically monitored by the differen fluorescence intensity. In addition, the miR-155-5p in human lung cancer tissues could be detected by the miR-155-5p MB. CONCLUSION: Both in vivo and in vitro experiments demonstrated that the CS-MB probe could be utilized to recognize the miR-155-5p and image the lung cancer cells. It provided a novel experimental and theoretical basis for the early diagnosis of the disease. Also, the histopathological sections of human lung cancer research laid the foundation for subsequent preclinical studies. In addition, different MBs could be designed to detect other miRNAs for the early diagnosis of other tumors.


Assuntos
Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/genética , MicroRNAs/análise , Células A549 , Animais , Quitosana/química , Detecção Precoce de Câncer/métodos , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Camundongos Transgênicos , MicroRNAs/biossíntese , MicroRNAs/genética , Imagem Molecular/métodos , Sondas Moleculares/química , Nanotecnologia
7.
Nat Commun ; 11(1): 2399, 2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32404879

RESUMO

The ability to monitor molecules volumetrically throughout the body could provide valuable biomarkers for studies of healthy function and disease, but noninvasive detection of molecular targets in living subjects often suffers from poor sensitivity or selectivity. Here we describe a family of potent imaging probes that can be activated by molecules of interest in deep tissue, providing a basis for mapping nanomolar-scale analytes without the radiation or heavy metal content associated with traditional molecular imaging agents. The probes are reversibly caged vasodilators that induce responses detectable by hemodynamic imaging; they are constructed by combining vasoactive peptides with synthetic chemical appendages and protein blocking domains. We use this architecture to create ultrasensitive biotin-responsive imaging agents, which we apply for wide-field mapping of targets in rat brains using functional magnetic resonance imaging. We also adapt the sensor design for detecting the neurotransmitter dopamine, illustrating versatility of this approach for addressing biologically important molecules.


Assuntos
Imagem Molecular/métodos , Sondas Moleculares/metabolismo , Peptídeos/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Vasodilatadores/metabolismo , Animais , Biotina/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Células CHO , Cricetinae , Cricetulus , Dopamina/metabolismo , Células HEK293 , Humanos , Imagem por Ressonância Magnética/métodos , Sondas Moleculares/química , Neurotransmissores/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Ratos , Reprodutibilidade dos Testes , Vasodilatadores/química
8.
Br J Radiol ; 93(1111): 20200034, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32374626

RESUMO

Necrosis plays vital roles in living organisms which is related closely with various diseases. Non-invasively necrotic imaging can be of great values in clinical decision-making, evaluation of individualized treatment responses, and prediction of patient prognosis. This narrative review will demonstrate how the evolution of quinones for necrotic imaging has been promoted by searching for their active centers. In this review, we summarized the recent developments of various quinones with the continuous simplified π-conjugated cores in necrotic imaging and speculated their possible molecular mechanisms might be attributed to their intercalations with exposed DNA in necrotic tissues. We discussed their clinical challenges of necrotic imaging with quinones and their future translation studies deserved to be explored in personalized patient treatment.


Assuntos
Sondas Moleculares , Infarto do Miocárdio/patologia , Necrose/diagnóstico por imagem , Quinonas , Animais , Antraquinonas/química , Células/patologia , DNA/análise , Humanos , Sondas Moleculares/química , Infarto do Miocárdio/diagnóstico por imagem , Naftoquinonas/química , Quinonas/química , Quinonas/classificação , Ratos
9.
Biochim Biophys Acta Proteins Proteom ; 1868(8): 140427, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32283249

RESUMO

We show that the antibody, clone mAb(D38C6), of the α isoform of the catalytic subunit of PKA (PKAcα) inhibits the kinase-catalyzed phosphorylation with low-nanomolar inhibitory potency (Ki = 2.4 nM). This property of the antibody was established by its capacity to displace a synthetic small-molecule active site-binding (orthosteric) photoluminescent ARC-Lum(Fluo) probe from the complex with PKAcα. Likely, the competitiveness of association of the two binders with the protein is coming from two excluding conformations of PKAcα to which the binders bind. mAb(D38C6) possesses a linear peptide epitope and it binds to the disordered C-tail of unliganded inactive conformer of PKAcα. ARC-Lum(Fluo) probes bind to the ordered and active conformation of PKAcα with Phe327 residue from the C-tail taking part in the formation of the active core. Consecutive application of these competitive PKAcα binders was used to develop an immunoassay allowing the determination of PKAcα concentration in complex biological solutions. At first, PKAcα was captured from the solution by the isoform-specific antibody and thereafter a high-affinity ARC-Lum(Fluo) probe was used to displace PKAcα from the binary complex. The developed immunoassay could be used for quantification of small amounts (starting from 93 pg, 2.3 fmol) of PKAcα in cell lysates.


Assuntos
Anticorpos Monoclonais/química , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/análise , Imunoensaio , Sondas Moleculares/química , Peptídeos/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Especificidade de Anticorpos , Sítios de Ligação , Ligação Competitiva , Linhagem Celular Tumoral , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/química , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Células HeLa , Humanos , Cinética , Medições Luminescentes , Modelos Moleculares , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína
10.
Nat Commun ; 11(1): 1518, 2020 03 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.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , DNA Catalítico/química , Sondas Moleculares/química , Aptâmeros de Nucleotídeos/metabolismo , DNA Catalítico/metabolismo , MicroRNAs/metabolismo , Sondas Moleculares/metabolismo , Tamanho da Partícula , Precursores de RNA/metabolismo , Especificidade por Substrato
11.
Nat Chem Biol ; 16(5): 497-506, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32231343

RESUMO

We recently described glutathione peroxidase 4 (GPX4) as a promising target for killing therapy-resistant cancer cells via ferroptosis. The onset of therapy resistance by multiple types of treatment results in a stable cell state marked by high levels of polyunsaturated lipids and an acquired dependency on GPX4. Unfortunately, all existing inhibitors of GPX4 act covalently via a reactive alkyl chloride moiety that confers poor selectivity and pharmacokinetic properties. Here, we report our discovery that masked nitrile-oxide electrophiles, which have not been explored previously as covalent cellular probes, undergo remarkable chemical transformations in cells and provide an effective strategy for selective targeting of GPX4. The new GPX4-inhibiting compounds we describe exhibit unexpected proteome-wide selectivity and, in some instances, vastly improved physiochemical and pharmacokinetic properties compared to existing chloroacetamide-based GPX4 inhibitors. These features make them superior tool compounds for biological interrogation of ferroptosis and constitute starting points for development of improved inhibitors of GPX4.


Assuntos
Inibidores Enzimáticos/farmacologia , Nitrilos/química , Nitrilos/farmacologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/antagonistas & inibidores , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Animais , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Ferroptose/efeitos dos fármacos , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos SCID , Sondas Moleculares/química , Terapia de Alvo Molecular , Óxidos/química , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/química , Pró-Fármacos/química , Ratos Wistar , Selenocisteína/química , Selenocisteína/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade
12.
Nat Commun ; 11(1): 1250, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144257

RESUMO

Currently, there are no non-invasive tools to accurately diagnose wound and surgical site infections before they become systemic or cause significant anatomical damage. Fluorescence and photoacoustic imaging are cost-effective imaging modalities that can be used to noninvasively diagnose bacterial infections when paired with a molecularly targeted infection imaging agent. Here, we develop a fluorescent derivative of maltotriose (Cy7-1-maltotriose), which is shown to be taken up in a variety of gram-positive and gram-negative bacterial strains in vitro. In vivo fluorescence and photoacoustic imaging studies highlight the ability of this probe to detect infection, assess infection burden, and visualize the effectiveness of antibiotic treatment in E. coli-induced myositis and a clinically relevant S. aureus wound infection murine model. In addition, we show that maltotriose is an ideal scaffold for infection imaging agents encompassing better pharmacokinetic properties and in vivo stability than other maltodextrins (e.g. maltohexose).


Assuntos
Corantes Fluorescentes/administração & dosagem , Imagem Molecular/métodos , Miosite/diagnóstico por imagem , Infecção da Ferida Cirúrgica/diagnóstico por imagem , Trissacarídeos/administração & dosagem , Animais , Carbocianinas/administração & dosagem , Carbocianinas/química , Modelos Animais de Doenças , Estabilidade de Medicamentos , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Feminino , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Injeções Intravenosas , Medições Luminescentes/métodos , Camundongos , Microscopia de Fluorescência/métodos , Sondas Moleculares/administração & dosagem , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Miosite/microbiologia , Técnicas Fotoacústicas/métodos , Ratos , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/metabolismo , Infecção da Ferida Cirúrgica/microbiologia , Trissacarídeos/química , Trissacarídeos/metabolismo
13.
Food Chem ; 317: 126433, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32092613

RESUMO

Highly catalytic and stable N-doped carbon dots (N-CDs) were prepared rapidly by microwave procedure using glucose as precursor and ammonium sulfite as N-dopant. The reduction of AgNO3 by trisodium citrate (TCA) was slow to form nanosilver (AgNP), and the N-CDs exhibited strong catalysis of the AgNP reaction. The formed AgNPs were used as indicator in the presence of Vitoria blue B (VBB) molecule probe with a SERS peak at 1615 cm-1. With the increase of nancatalyst N-CDs concentration, the AgNP reaction speed up, and the SERS peak of VBB enhanced linearly due to formation of more AgNPs as substrate. In the presence of avidin (Ad), the SERS peak weakened. Upon addition of biotin, the SERS peak enhanced due to turn on the indicator nanoreaction. The enhanced SERS signal had a good linear relationship with the biotin concentration in range of 0.0006-0.021 ng/mL, with a detection limit of 0.3 pg/mL.


Assuntos
Biotina/análise , Análise de Alimentos/métodos , Prata/química , Análise Espectral Raman/métodos , Animais , Avidina/química , Carbono/química , Catálise , Citratos/química , Análise de Alimentos/instrumentação , Limite de Detecção , Nanopartículas Metálicas/química , Leite/química , Técnicas de Sonda Molecular/instrumentação , Sondas Moleculares/química , Compostos Orgânicos/química , Pontos Quânticos/química , Nitrato de Prata/química , Análise Espectral Raman/instrumentação
14.
J Chem Phys ; 152(6): 065104, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-32061231

RESUMO

Radical-pair reactions have been suggested to be sensitive to the direction of weak magnetic fields, thereby providing a mechanism for the magnetic compass in animals. Discovering the general principles that make radical pairs particularly sensitive to the direction of weak magnetic fields will be essential for designing bioinspired compass sensors and for advancing our understanding of the spin physics behind directional effects. The reference-probe model is a conceptual model introduced as a guide to identify radical-pair parameters for optimal directional effects. Radical pairs with probe character have been extensively shown to enhance directional sensitivity to weak magnetic fields, but investigations on the role of the reference radical are lacking. Here, we evaluate whether a radical has reference character and then study its relevance for optimal directional effects. We investigate a simple radical-pair model with one axially anisotropic hyperfine interaction using both analytical and numerical calculations. Analytical calculations result in a general expression of the radical-pair reaction yield, which in turn provides useful insights into directional effects. We further investigate the relevance of the reference character to robustness against variations of earth-strength magnetic fields and find that the reference character captures robust features as well. Extending this study to radical pairs with more hyperfine interactions, we discuss the interplay between reference character and optimal and robust directional effects in such more complex radical pairs.


Assuntos
Modelos Químicos , Sondas Moleculares/química , Animais , Anisotropia , Radicais Livres/química , Campos Magnéticos
15.
Chem Commun (Camb) ; 56(10): 1464-1480, 2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-31967621

RESUMO

This review discusses the advantages of using luminescent d6-transition-metal complexes as cell probes for optical microscopy. In particular it focusses on the Thomas group's use of specific complexes as "building blocks" toward the construction of biomolecular binding substrates, with DNA being a particular target. Using this approach, a range of new imaging probes for conventional optical microscopy, nanoscopy and transmission electron microscopy have been identified. Through selection of specific metal centres and by substitution of coordinated ligands we illustrate how new chemotherapeutics, photo-therapeutics, and theranostics have been identified and developed from the original architectures.


Assuntos
Complexos de Coordenação/química , DNA/química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Apoptose/efeitos dos fármacos , Bactérias/efeitos dos fármacos , Meios de Contraste/química , Meios de Contraste/metabolismo , Complexos de Coordenação/metabolismo , Complexos de Coordenação/toxicidade , DNA/metabolismo , Humanos , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Nanomedicina Teranóstica
16.
Nat Commun ; 11(1): 81, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900403

RESUMO

Live-cell Raman imaging based on bioorthogonal Raman probes with distinct signals in the cellular Raman-silent region (1800-2800 cm-1) has attracted great interest in recent years. We report here a class of water-soluble and biocompatible polydiacetylenes with intrinsic ultrastrong alkyne Raman signals that locate in this region for organelle-targeting live-cell Raman imaging. Using a host-guest topochemical polymerization strategy, we have synthesized a water-soluble and functionalizable master polydiacetylene, namely poly(deca-4,6-diynedioic acid) (PDDA), which possesses significantly enhanced (up to ~104 fold) alkyne vibration compared to conventional alkyne Raman probes. In addition, PDDA can be used as a general platform for multi-functional ultrastrong Raman probes. We achieve high quality live-cell stimulated Raman scattering imaging on the basis of modified PDDA. The polydiacetylene-based Raman probes represent ultrastrong intrinsic Raman imaging agents in the Raman-silent region (without any Raman enhancer), and the flexible functionalization of this material holds great promise for its potential diverse applications.


Assuntos
Células/citologia , Imagem Molecular/métodos , Sondas Moleculares/química , Polímero Poliacetilênico/química , Análise Espectral Raman/métodos , Alquinos/química , Células/química , Células HeLa , Humanos , Imagem Molecular/instrumentação , Sondas Moleculares/síntese química , Polímero Poliacetilênico/síntese química , Análise Espectral Raman/instrumentação
17.
PLoS One ; 15(1): e0227341, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31923258

RESUMO

Clan CA cysteine proteases, also known as papain-like proteases, play important roles throughout the malaria parasite life cycle and are therefore potential drug targets to treat this disease and prevent its transmission. In order to study the biological function of these proteases and to chemically validate some of them as viable drug targets, highly specific inhibitors need to be developed. This is especially challenging given the large number of clan CA proteases present in Plasmodium species (ten in Plasmodium falciparum), and the difficulty of designing selective inhibitors that do not cross-react with other members of the same family. Additionally, any efforts to develop antimalarial drugs targeting these proteases will also have to take into account potential off-target effects against the 11 human cysteine cathepsins. Activity-based protein profiling has been a very useful tool to determine the specificity of inhibitors against all members of an enzyme family. However, current clan CA proteases broad-spectrum activity-based probes either target endopeptidases or dipeptidyl aminopeptidases, but not both subfamilies efficiently. In this study, we present a new series of dipeptydic vinyl sulfone probes containing a free N-terminal tryptophan and a fluorophore at the P1 position that are able to label both subfamilies efficiently, both in Plasmodium falciparum and in mammalian cells, thus making them better broad-spectrum activity-based probes. We also show that some of these probes are cell permeable and can therefore be used to determine the specificity of inhibitors in living cells. Interestingly, we show that the choice of fluorophore greatly influences the specificity of the probes as well as their cell permeability.


Assuntos
Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/química , Malária/enzimologia , Animais , Antimaláricos/química , Permeabilidade da Membrana Celular , Humanos , Malária/diagnóstico por imagem , Malária/tratamento farmacológico , Sondas Moleculares/química , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Sulfonas , Triptofano
18.
Chem Commun (Camb) ; 56(9): 1317-1324, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31904034

RESUMO

G-quadruplexes are nucleic acids secondary structures that can be formed in guanine-rich sequences. More than 30 years ago, their formation was first observed in telomeric DNA. Since then, a number of other sequences capable of forming G-quadruplex structures have been described and increasing evidence supporting their formation in the context of living cells has been accumulated. To fully underpin the biological significance of G-quadruplexes and their potential as therapeutic targets, several chemical-biology tools and methods have been developed to map and visualise these nucleic acids secondary structures in human cells. In this review, we critically present the most relevant methods developed to investigate G-quadruplex prevalence in human cells and to study their biological functions, presenting the next key chemical-biology challenges that need to be addressed to fully unravel G-quadruplex mediated biology and their therapeutic potential.


Assuntos
DNA/química , Quadruplex G , Genoma Humano , Sondas Moleculares/química , RNA/química , DNA/genética , Humanos , Ligação de Hidrogênio , RNA/genética
19.
Org Biomol Chem ; 18(7): 1359-1368, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31984413

RESUMO

Gamma peptide nucleic acids (γPNAs), i.e., single-stranded PNA strands that are modified at the γ-position with (R)-diethylene glycol, and Invader probes, i.e., DNA duplexes with +1 interstrand zipper arrangements of 2'-O-(pyren-1-yl)methyl-RNA monomers, are two types of nucleic acid mimics that are showing promise for sequence-unrestricted recognition of double-stranded (ds) DNA targets. We recently demonstrated that recognition of dsDNA targets with self-complementary regions is challenging for single-stranded high-affinity probes like γPNAs due to their proclivity for secondary structure formation, but not so for Invader probes, which are engineered to form readily denaturing duplexes irrespective of the target sequence context. In the present study, we describe an approach that mitigates these limitations and improves the dsDNA-recognition properties of γPNAs in partially self-complementary target contexts. Chimeric probes between γPNAs and individual Invader strands are shown to form metastable duplexes that (i) are energetically activated for recognition of complementary mixed-sequence dsDNA target regions, (ii) reduce γPNA dimerization, and (iii) substantially improve the fidelity of the dsDNA-recognition process. Chimeric γPNA-Invader probes are characterized with respect to thermal denaturation properties, thermodynamic parameters associated with duplex formation, UV-Vis and fluorescence trends to establish pyrene binding modes, and dsDNA-recognition properties using DNA hairpin model targets.


Assuntos
DNA/química , Sondas Moleculares/química , Oligonucleotídeos/química , Ácidos Nucleicos Peptídicos/química , Conformação Molecular
20.
IET Nanobiotechnol ; 14(1): 19-24, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31935673

RESUMO

Ureaplasma urealyticum (uu) is one of the most common agents of urogenital infections and is associated with complications such as infertility, spontaneous abortion and other sexually transmitted diseases. Here, a DNA sensor based on oligonucleotide target-specific gold nanoparticles (AuNPs) was developed, in which the dispersed and aggregated states of oligonucleotide-functionalised AuNPs were optimised for the colorimetric detection of a polymerase chain reaction (PCR) amplicon of U. urealyticum DNA. A non-cross-linking approach utilising a single Au-nanoprobe specific of the urease gene was utilised and the effect of a PCR product concentration gradient evaluated. Results from both visual and spectral analyses showed that target-Au-nanoprobe hybrids were stable against aggregation after adding the inducer. Furthermore, when a non-target PCR product was used, the peak position shifted and salt-induced aggregation occurred. The assay's limit of detection of the assay was 10 ng with a dynamic range of 10-60 ng. This procedure provides a rapid, facile and low-cost detection format, compared to methods currently used for the identification of U. urealyticum.


Assuntos
Colorimetria/métodos , Ouro/química , Nanopartículas Metálicas/química , Reação em Cadeia da Polimerase/métodos , Ureaplasma urealyticum , Proteínas de Bactérias/genética , Limite de Detecção , Sondas Moleculares/química , Sondas Moleculares/genética , Tipagem Molecular/métodos , Ureaplasma urealyticum/genética , Ureaplasma urealyticum/isolamento & purificação , Urease/genética
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