Browse
Submit Data
Databases
API
Help

Dataset Information

7 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A photo-regulated aptamer sensor for spatiotemporally controlled monitoring of ATP in the mitochondria of living cells.

ABSTRACT: Fluorescent aptamer sensors have shown enormous potential for intracellular imaging of small molecule metabolites. Since metabolites distribute differently at different subcellular locations and their concentrations and locations fluctuate with time, methods are needed for spatiotemporally controlled monitoring of these metabolites. Built upon previous success in temporal control of aptamer-based sensors, we herein report an aptamer sensor containing a photocleavable linker and using DQAsomes to target mitochondria for spatiotemporally controlled monitoring of ATP in the mitochondria of living cells. The photocleavable modification on the DNA ATP aptamer sensor can prevent sensor activation before reaching mitochondria and the sensor can then be activated upon light irradiation. The sensor has a detection limit of 3.7 μM and high selectivity against other nucleotides, allowing detection of ATP concentration fluctuations in mitochondria induced by Ca²⁺ or oligomycin. This work represents the first successful delivery of a DNA aptamer sensor to mitochondria, providing a new platform for targeted delivery to subcellular organelles for monitoring energy producing processes, as well as mitochondrial dysfunction-related diseases in different cells.

SUBMITTER: Hong S

PROVIDER: S-EPMC8145946 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Heterogeneous Electrochemical Aptamer-Based Sensor Surfaces for Controlled Sensor Response.

Project description:Structure-switching sensors utilize recognition elements that undergo a conformation change upon target binding that is converted into a quantitative signal. Electrochemical, aptamer-based sensors achieve detection of analytes through a conformation change in an electrode-bound, oligonucleotide aptamer by measuring changes in electron transfer efficiencies. The analytical performance of these sensors is related to the magnitude of the conformation change of the aptamer. The goal of the present work is to develop a general method to predictably tune the analytical performance (sensitivity and linear range) of electrochemical, aptamer-based sensors by utilizing a mixture of rationally designed aptamer sequences that are specific for the same target but with different affinities on the same electrode surface. To demonstrate control over sensor performance, we developed heterogeneous sensors for two representative small molecule targets (adenosine triphosphate and tobramycin). We demonstrate that mixtures of modified sequences can be used to tune the affinity, dynamic range, and sensitivity of the resulting sensors predicted by a bi-Langmuir-type isotherm.

| S-EPMC5627764 | biostudies-literature

Electrochemical Aptamer-Based Sensor for Real-Time Monitoring of Insulin.

Project description:In this paper, we developed an electrochemical, aptamer-based (E-AB) for the real-time monitoring of insulin. The sensor utilizes a redox label-modified guanine-rich aptamer which folds into a G-quadruplex for specific recognition of insulin. To develop a reproducible E-AB sensor employing insulin aptamer probes for the detection of insulin, 10% sodium dodecyl sulfate (SDS) pretreatment is crucial as it disrupts interstrand G-quartets. After sensor pretreatment with 10% SDS, a more uniform sensor response is obtained. Upon introduction of the insulin target, binding-induced steric hindrance quantitatively reduces the efficiency of electron transfer of a distal-end redox label leading to the rapid signal change within ?60 s. Testing demonstrates that the E-AB insulin exhibits a limit of detection of 20 nM and can be used to discriminate against both glucagon and somatostatin in Krebs-Ringer bicarbonate buffer, typically used in perfusion experiments. These results demonstrate that this assay has potential for rapid, specific, and quantitative analysis of insulin.

| S-EPMC6404975 | biostudies-literature

Rationally designing aptamer sequences with reduced affinity for controlled sensor performance.

Project description:The relative ease of predicting the secondary structure of nucleic acid sequences lends itself to the design of sequences to perform desired functions. Here, we combine the utility of nucleic acid aptamers with predictable control over the secondary structure to rationally design sequences with controlled affinity towards a target analyte when employed as the recognition element in an electrochemical sensor. Specifically, we present a method to modify an existing high-gain aptamer sequence to create sequences that, when employed in an electrochemical, aptamer-based sensor, exhibit reduced affinity towards a small molecule analyte tobramycin. Sensors fabricated with the high-gain parent sequence saturate at concentrations much below the therapeutic window for tobramycin (7-18 µM). Accordingly, the rationale behind modifying this high-gain sequence to reduce binding affinity was to tune sensor performance for optimal sensitivity in the therapeutic window. Using secondary structure predictions and analysis of the NMR structure of an aminoglycoside RNA aptamer bound to tobramycin, we are able to successfully modify the aptamer sequence to tune the dissociation constants of electrochemical aptamer-based sensors between 0.17 and 3 µM. The guidelines we present represent a general strategy to lessening binding affinity of sensors employing aptamer-modified electrodes.

| S-EPMC4431182 | biostudies-literature

Rhodamine-based sensor for real-time imaging of mitochondrial ATP in living fibroblasts.

Project description:Mitochondria are essential for the production and maintenance of ATP in the eukaryotic cell. To image and monitor intracellular ATP level without cell breakage, biological and chemical sensors were developed in the last years. Here, we have internalized a rhodamine-based sensor RSL+ into living cells and monitored the mitochondrial ATP levels in cultured mouse embryonic fibroblasts. To evaluate the robustness of the sensor we imaged the changes of the mitochondrial ATP levels under non-physiological conditions upon incubation with FCCP, oligomycin, azide, deoxyglucose or phosphoenolpyruvate; all compounds that interfere with ATP homeostasis of the cell. The ATP sensor allowed us to determine the mitochondrial ATP levels in human skin fibroblasts where we observe a similar amount of ATP compared to mouse embryonic fibroblasts. We propose the RSL+ to be a valuable tool for the assessment of mitochondrial dysfunction in human cells derived from mitochondrial OXPHOS patients and for basic studies on bioenergetics metabolism.

| S-EPMC5656571 | biostudies-literature

Photo-click living strategy for controlled, reversible exchange of biochemical ligands.

Project description:A novel addition-fragmentation-chain transfer capable allyl sulfide functionalized PEG hydrogel is reported to allow controlled, reversible exchange of biochemical ligands. The exchange of biochemical ligands is achieved without permanent consumption of reactive functionalities. Demonstrated is the ability to exchange biochemical ligands multiple times using cytocompatible 720 nm two-photon light.

| S-EPMC4528616 | biostudies-literature

Development of an Anti-Idiotype Aptamer-Based Electrochemical Sensor for a Humanized Therapeutic Antibody Monitoring.

Project description:Therapeutic monoclonal antibodies (mAbs) are currently the most effective medicines for a wide range of diseases. Therefore, it is expected that easy and rapid measurement of mAbs will be required to improve their efficacy. Here, we report an anti-idiotype aptamer-based electrochemical sensor for a humanized therapeutic antibody, bevacizumab, based on square wave voltammetry (SWV). With this measurement procedure, we were able to monitor the target mAb within 30 min by employing the anti-idiotype bivalent aptamer modified with a redox probe. A fabricated bevacizumab sensor achieved detection of bevacizumab from 1-100 nM while eliminating the need for free redox probes in the solution. The feasibility of monitoring biological samples was also demonstrated by detecting bevacizumab in the diluted artificial serum, and the fabricated sensor succeeded in detecting the target covering the physiologically relevant concentration range of bevacizumab. Our sensor contributes to ongoing efforts towards therapeutic mAbs monitoring by investigating their pharmacokinetics and improving their treatment efficacy.

| S-EPMC10048959 | biostudies-literature

MIL/Aptamer as a Nanosensor Capable of Resisting Nonspecific Displacement for ATP Imaging in Living Cells.

Project description:Fluorescent probes physisorbed on nanomaterials have emerged as a kind of useful and facile sensing platform for biological important molecules. However, nonspecific displacement in the physisorption systems is a non-negligible problem for the intracellular analysis. MIL (Materials of Institut Lavoisier), a subclass of metal-organic frameworks (MOFs), has high porosity, large surface area, and intriguing three-dimensional (3D) nanostructure with promising biological and biomedical applications such as molecular detection and drug delivery. Herein, we report MIL/aptamer-FAM as a nanosensor capable of resisting nonspecific displacement for intracellular adenosinetriphosphate (ATP) sensing and imaging. In this approach, by virtue of the remarkable quenching capability, high affinity of aptamers, and dramatic capability of resisting nonspecific displacement of 3D MIL-100, the assay and imaging of ATP in living cells were realized. Our results demonstrated that the MIL/aptamer-FAM nanosensor not only shows high selectivity for the detection of ATP in buffer but also is able to act as a "signal-on" nanosensor for specific imaging of ATP in living cells. The strategy reported here opens up a new way to develop MOF-based nanosensors for intracellular delivery and metabolite detection.

| S-EPMC6648595 | biostudies-literature

Monitoring caspase-3 activation with a multimodality imaging sensor in living subjects.

Project description:Capsase-3 plays an important role in chemotherapy-induced apoptosis in many cancers. Herein, we applied a multimodality reporter vector to monitor caspase-3 activation indirectly in live cells and tumors of living animals undergoing apoptosis.A fusion protein (MTF) was constructed by combining three different reporter proteins, red fluorescent protein (mRFP1), firefly luciferase (FL), and HSV1-sr39 truncated thymidine kinase (TK), linked through a caspase-3 recognizable polypeptide linker. After cleavage by caspase-3, a significant gain in mRFP1, FL, and TK activity are observed by fluorescence-activated cell sorting and enzyme-based assays. A melanoma cell line (B16F10-mtf-hrl) stably expressing mtf (to measure caspase-3 activation) and hrl-IRES-gfp (to determine the decrease in a number of viable cells) vectors was generated to measure two independent molecular events upon treatment.Upon induction with 8 mumol/L staurosporine, the fusion protein showed a 2.8-fold increase in FL (P = 0.03), a 1.5-fold increase in TK (P = not significant), and a 2-fold increase in mRFP1 (P = 0.05) activity in 293T cells. Bioluminescence and micropositron emission tomography imaging of the apoptotic B16F10-mtf-hrl tumors showed a 2-fold higher FL activity (897 versus 416) and a 2-fold higher TK activity (10.3 versus 3.87) than control tumors when normalized with RL activity. Using a similar normalization approach, the time kinetics of caspase-3 activation by two protein kinase-C inhibitors was noninvasively monitored in living mice.This multimodality caspase sensor vector could effectively and noninvasively monitor caspase-3 activation from single live cells to a multicellular tumor environment and, thus, would be a valuable tool for drug screening in preclinical models and future patient cell based therapy.

| S-EPMC4155946 | biostudies-literature

Spatiotemporally controlled single cell sonoporation.

Project description:This paper presents unique approaches to enable control and quantification of ultrasound-mediated cell membrane disruption, or sonoporation, at the single-cell level. Ultrasound excitation of microbubbles that were targeted to the plasma membrane of HEK-293 cells generated spatially and temporally controlled membrane disruption with high repeatability. Using whole-cell patch clamp recording combined with fluorescence microscopy, we obtained time-resolved measurements of single-cell sonoporation and quantified the size and resealing rate of pores. We measured the intracellular diffusion coefficient of cytoplasmic RNA/DNA from sonoporation-induced transport of an intercalating fluorescent dye into and within single cells. We achieved spatiotemporally controlled delivery with subcellular precision and calcium signaling in targeted cells by selective excitation of microbubbles. Finally, we utilized sonoporation to deliver calcein, a membrane-impermeant substrate of multidrug resistance protein-1 (MRP1), into HEK-MRP1 cells, which overexpress MRP1, and monitored the calcein efflux by MRP1. This approach made it possible to measure the efflux rate in individual cells and to compare it directly to the efflux rate in parental control cells that do not express MRP1.

| S-EPMC3478613 | biostudies-literature

Monitoring of dynamic ATP level changes by oligomycin-modulated ATP synthase inhibition in SW480 cancer cells using fluorescent "On-Off" switching DNA aptamer.

Project description:Adenosine triphosphate (ATP) is the main energy source in cells and an important biomolecule participating in cellular reactions in living organisms. Since the ATP level changes dynamically reflecting the development of a debilitating disease or carcinogenesis, we have focused in this work on monitoring of the oligomycin (OMC)-modulated ATP synthase inhibition using a fluorescent-switching DNA aptamer designed for the detection of ATP (Apt(ATP)), as the model for studies of dynamic ATP level variation. The behavior of the ATP aptamer has been characterized using fluorescence spectroscopy. The Intramolecular fluorescence resonance energy transfer (iFRET) operates in the proposed aptamer from the FAM dye moiety to guanines of the aptamer G-quadruplex when the target ATP is present and binds to the aptamer changing its conformation. The iFRET process enables the detection of ATP down to the limit of detection, LOD?=?17 ?M, without resorting to any extra chemi-amplification schemes. The selectivity coefficients for relevant interferent triphosphates (UTP, GTP, and CTP) are low for the same concentration as that of ATP. We have demonstrated an efficient transfection of intact cells and OMC-treated SW480 colon cancer cells with Apt(ATP), using microscopic imaging, iFRET measurements, and cell viability testing with MTT method. The applicability of the switching DNA aptamer for the analysis of real samples, obtained by lysis of SW480 cells, was also tested. The proposed Apt(ATP) may be considered as a viable candidate for utilization in measurements of dynamic ATP level modulation in cells in different stages of cancer development and testing of new drugs in pharmacological studies. Graphical abstract.

| S-EPMC6834760 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data