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1.
Annu Rev Biophys ; 53(1): 275-297, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38346245

RESUMO

Genetically encoded fluorescent biosensors have revolutionized the study of cell signaling and metabolism, as they allow for live-cell measurements with high spatiotemporal resolution. This success has spurred the development of tailor-made biosensors that enable the study of dynamic phenomena on different timescales and length scales. In this review, we discuss different approaches to enhancing and developing new biosensors. We summarize the technologies used to gain structural insights into biosensor design and comment on useful screening technologies. Furthermore, we give an overview of different applications where biosensors have led to key advances over recent years. Finally, we give our perspective on where future work is bound to make a large impact.


Assuntos
Técnicas Biossensoriais , Transdução de Sinais , Técnicas Biossensoriais/métodos , Humanos , Animais , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo
2.
bioRxiv ; 2024 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-38370804

RESUMO

Fluorescent biosensors revolutionized biomedical science by enabling the direct measurement of signaling activities in living cells, yet the current technology is limited in resolution and dimensionality. Here, we introduce highly sensitive chemigenetic kinase activity biosensors that combine the genetically encodable self-labeling protein tag HaloTag7 with bright far-red-emitting synthetic fluorophores. This technology enables five-color biosensor multiplexing, 4D activity imaging, and functional super-resolution imaging via stimulated emission depletion (STED) microscopy.

3.
bioRxiv ; 2023 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-37503182

RESUMO

Genetically encoded fluorescent calcium indicators have revolutionized neuroscience and other biological fields by allowing cellular-resolution recording of physiology during behavior. However, we currently lack bright, genetically targetable indicators in the near infrared that can be used in animals. Here, we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains that can be genetically targeted to specific cell populations. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several dye-ligands that efficiently label the central nervous system in animals. When bound to a near-infrared dye-ligand, WHaloCaMP1a is more than twice as bright as jGCaMP8s, and shows a 7× increase in fluorescence intensity and a 2.1 ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a with near-infrared fluorescence emission to image Ca2+ responses in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae, and to quantitate calcium concentration using fluorescence lifetime imaging microscopy (FLIM).

4.
Nat Chem Biol ; 19(9): 1147-1157, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37291200

RESUMO

Fluorescent biosensors enable the study of cell physiology with spatiotemporal resolution; yet, most biosensors suffer from relatively low dynamic ranges. Here, we introduce a family of designed Förster resonance energy transfer (FRET) pairs with near-quantitative FRET efficiencies based on the reversible interaction of fluorescent proteins with a fluorescently labeled HaloTag. These FRET pairs enabled the straightforward design of biosensors for calcium, ATP and NAD+ with unprecedented dynamic ranges. The color of each of these biosensors can be readily tuned by changing either the fluorescent protein or the synthetic fluorophore, which enables simultaneous monitoring of free NAD+ in different subcellular compartments following genotoxic stress. Minimal modifications of these biosensors furthermore allow their readout to be switched to fluorescence intensity, fluorescence lifetime or bioluminescence. These FRET pairs thus establish a new concept for the development of highly sensitive and tunable biosensors.


Assuntos
Técnicas Biossensoriais , NAD , Proteínas Luminescentes/metabolismo , NAD/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Técnicas Biossensoriais/métodos
5.
J Am Chem Soc ; 145(5): 3075-3083, 2023 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-36716211

RESUMO

The specific and covalent labeling of the protein HaloTag with fluorescent probes in living cells makes it a powerful tool for bioimaging. However, the irreversible attachment of the probe to HaloTag precludes imaging applications that require transient binding of the probe and comes with the risk of irreversible photobleaching. Here, we introduce exchangeable ligands for fluorescence labeling of HaloTag (xHTLs) that reversibly bind to HaloTag and that can be coupled to rhodamines of different colors. In stimulated emission depletion (STED) microscopy, probe exchange of xHTLs allows imaging with reduced photobleaching as compared to covalent HaloTag labeling. Transient binding of fluorogenic xHTLs to HaloTag fusion proteins enables points accumulation for imaging in nanoscale topography (PAINT) and MINFLUX microscopy. We furthermore introduce pairs of xHTLs and HaloTag mutants for dual-color PAINT and STED microscopy. xHTLs thus open up new possibilities in imaging across microscopy platforms for a widely used labeling approach.


Assuntos
Corantes Fluorescentes , Ligantes , Microscopia de Fluorescência/métodos , Corantes Fluorescentes/metabolismo , Rodaminas
6.
Nat Chem Biol ; 19(3): 346-355, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36316571

RESUMO

Coenzyme A (CoA) is one of the central cofactors of metabolism, yet a method for measuring its concentration in living cells is missing. Here we introduce the first biosensor for measuring CoA levels in different organelles of mammalian cells. The semisynthetic biosensor is generated through the specific labeling of an engineered GFP-HaloTag fusion protein with a fluorescent ligand. Its readout is based on CoA-dependent changes in Förster resonance energy transfer efficiency between GFP and the fluorescent ligand. Using this biosensor, we probe the role of numerous proteins involved in CoA biosynthesis and transport in mammalian cells. On the basis of these studies, we propose a cellular map of CoA biosynthesis that suggests how pools of cytosolic and mitochondrial CoA are maintained.


Assuntos
Técnicas Biossensoriais , Proteínas , Animais , Ligantes , Corantes , Homeostase , Técnicas Biossensoriais/métodos , Coenzima A , Mamíferos
7.
ACS Chem Biol ; 17(6): 1321-1327, 2022 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-35584304

RESUMO

Fluorescence lifetime multiplexing requires fluorescent probes with distinct fluorescence lifetimes but similar spectral properties. Even though synthetic probes for many cellular targets are available for multicolor live-cell fluorescence microscopy, few of them have been characterized for their use in fluorescence lifetime multiplexing. Here, we demonstrate that, from a panel of 18 synthetic probes, eight pairwise combinations are suitable for fluorescence lifetime multiplexing in living mammalian cell lines. Moreover, combining multiple pairs in different spectral channels enables us to image four and with the help of self-labeling protein tags up to eight different biological targets, effectively doubling the number of observable targets. The combination of synthetic probes with fluorescence lifetime multiplexing is thus a powerful approach for live-cell imaging.


Assuntos
Corantes Fluorescentes , Mamíferos , Animais , Linhagem Celular , Fluorescência , Microscopia de Fluorescência/métodos
8.
Nat Methods ; 19(1): 65-70, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34916672

RESUMO

Self-labeling protein tags such as HaloTag are powerful tools that can label fusion proteins with synthetic fluorophores for use in fluorescence microscopy. Here we introduce HaloTag variants with either increased or decreased brightness and fluorescence lifetime compared with HaloTag7 when labeled with rhodamines. Combining these HaloTag variants enabled live-cell fluorescence lifetime multiplexing of three cellular targets in one spectral channel using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. Additionally, the brightest HaloTag variant showed up to 40% higher brightness in live-cell imaging applications.


Assuntos
Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Hidrolases/química , Linhagem Celular , Cristalografia por Raios X , Fluorescência , Humanos , Hidrolases/genética , Hidrolases/metabolismo , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Microscopia Confocal , Microscopia de Fluorescência/métodos , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Rodaminas/química
9.
Nat Commun ; 10(1): 4580, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31594948

RESUMO

Photoactivatable fluorophores are important for single-particle tracking and super-resolution microscopy. Here we present a photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. Using this photoactivatable fluorophore, we create probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophore's outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy.


Assuntos
Corantes Fluorescentes/efeitos da radiação , Microscopia Intravital/métodos , Rodaminas/efeitos da radiação , Silício/efeitos da radiação , Imagem Individual de Molécula/métodos , Animais , Células COS , Chlorocebus aethiops , Corantes Fluorescentes/química , Células HeLa , Humanos , Luz , Microscopia de Fluorescência/métodos , Processos Fotoquímicos/efeitos da radiação , Prótons , Rodaminas/química , Silício/química
10.
J Am Chem Soc ; 141(7): 2770-2781, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30550714

RESUMO

Super-resolution fluorescence microscopy is a powerful tool to visualize biomolecules and cellular structures at the nanometer scale. Employing these techniques in living cells has opened up the possibility to study dynamic processes with unprecedented spatial and temporal resolution. Different physical approaches to super-resolution microscopy have been introduced over the last years. A bottleneck to apply these approaches for live-cell imaging has become the availability of appropriate fluorescent probes that can be specifically attached to biomolecules. In this Perspective, we discuss the role of small-molecule fluorescent probes for live-cell super-resolution microscopy and the challenges that need to be overcome for their generation. Recent trends in the development of labeling strategies are reviewed together with the required chemical and spectroscopic properties of the probes. Finally, selected examples of the use of small-molecule fluorescent probes in live-cell super-resolution microscopy are given.


Assuntos
Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Animais , Linhagem Celular , Fluorescência , Corantes Fluorescentes/efeitos da radiação , Humanos , Luz
11.
Chemistry ; 23(57): 14345-14357, 2017 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-28967982

RESUMO

Malaria remains a major threat to mankind due to the perpetual emergence of resistance against marketed drugs. Twenty-one pyrazolopyran-based inhibitors bearing terminal biphenyl, aryl sulfonamide, or aryl sulfone motifs were synthesized and tested towards serine hydroxymethyltransferase (SHMT), a key enzyme of the folate cycle. The best ligands inhibited Plasmodium falciparum (Pf) and Arabidopsis thaliana (At) SHMT in target, as well as PfNF54 strains in cell-based assays in the low nanomolar range (18-56 nm). Seven co-crystal structures with P. vivax (Pv) SHMT were solved at 2.2-2.6 Šresolution. We observed an unprecedented influence of the torsion angle of ortho-substituted biphenyl moieties on cell-based efficacy. The peculiar lipophilic character of the sulfonyl moiety was highlighted in the complexes with aryl sulfonamide analogues, which bind in their preferred staggered orientation. The results are discussed within the context of conformational preferences in the ligands.

12.
Bioorg Med Chem ; 23(11): 2666-79, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25614112

RESUMO

Herein, we report on work towards the development of a new strategy for the synthesis of rare and biologically interesting indolizin-5(3H)-ones, which is based around the use of ring-closing metathesis to construct the carbocyclic ring system. This study has provided insights into the general stability of indolizin-5(3H)-ones and their tendency to exist as the tautomeric indolizin-5-ols. Furthermore, this approach has allowed access to other novel structurally related compounds based around unusual 6,5-azabicyclic scaffolds, which are also difficult to generate using typical methods. The azabicyclic compounds synthesized in this study reside in attractive regions of heterocyclic chemical space that are underexploited in current drug and agrochemical discovery efforts.


Assuntos
Compostos Aza/síntese química , Compostos Bicíclicos com Pontes/síntese química , Reação de Cicloadição , Descoberta de Drogas , Indolizinas/síntese química , Estrutura Molecular
13.
Chimia (Aarau) ; 67(10): 742-3, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24388145

RESUMO

A team of four students from Swiss high schools, selected in a national competition and accompanied by their mentors, represented their country at the 45(th) International Chemistry Olympiad (IChO) held in July 2013 in Moscow, Russia. The exceptional performances of Patrik Willi, Boris Stolz and Mario De Capitani in both practical and theoretical chemistry were rewarded by three bronze medals at the international level, reflecting their efforts during the year-long preparation prior to the competition.

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