Browse
Submit Data
Databases
API
Help

Dataset Information

25 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Nanomolar small-molecule detection using a genetically encoded 129Xe NMR contrast agent.

ABSTRACT: Genetically encoded magnetic resonance imaging (MRI) contrast agents enable non-invasive detection of specific biomarkers in vivo. Here, we employed the hyper-CEST 129Xe NMR technique to quantify maltose (32 nM to 1 mM) through its modulation of conformational change and xenon exchange in maltose binding protein (MBP). Remarkably, no hyper-CEST signal was observed for MBP in the absence of maltose, making MBP an ultrasensitive "smart" contrast agent. The resonance frequency of 129Xe bound to MBP was greatly downfield-shifted (?? = 95 ppm) from the 129Xe(aq) peak, which facilitated detection in E. coli as well as multiplexing with TEM-1 ?-lactamase. Finally, a Val to Ala mutation at the MBP-Xe binding site yielded 34% more contrast than WT, with 129Xe resonance frequency shifted 59 ppm upfield from WT. We conclude that engineered MBPs constitute a new class of genetically encoded, analyte-sensitive molecular imaging agents detectable by 129Xe NMR/MRI.

SUBMITTER: Roose BW

PROVIDER: S-EPMC5849143 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Nanomolar small-molecule detection using a genetically encoded 129Xe NMR contrast agent.

Roose B W BW Zemerov S D SD Dmochowski I J IJ

Chemical science 20170920 11

Genetically encoded magnetic resonance imaging (MRI) contrast agents enable non-invasive detection of specific biomarkers in vivo. Here, we employed the hyper-CEST 129Xe NMR technique to quantify maltose (32 nM to 1 mM) through its modulation of conformational change and xenon exchange in maltose binding protein (MBP). Remarkably, no hyper-CEST signal was observed for MBP in the absence of maltose, making MBP an ultrasensitive "smart" contrast agent. The resonance frequency of ...[more]

PMID: 29568427

Similar Datasets

Nanoparticle-Based Contrast Agents for 129Xe HyperCEST NMR and MRI Applications.

Project description:Spin hyperpolarization techniques have enabled important advancements in preclinical and clinical MRI applications to overcome the intrinsic low sensitivity of nuclear magnetic resonance. Functionalized xenon biosensors represent one of these approaches. They combine two amplification strategies, namely, spin exchange optical pumping (SEOP) and chemical exchange saturation transfer (CEST). The latter one requires host structures that reversibly bind the hyperpolarized noble gas. Different nanoparticle approaches have been implemented and have enabled molecular MRI with 129Xe at unprecedented sensitivity. This review gives an overview of the Xe biosensor concept, particularly how different nanoparticles address various critical aspects of gas binding and exchange, spectral dispersion for multiplexing, and targeted reporter delivery. As this concept is emerging into preclinical applications, comprehensive sensor design will be indispensable in translating the outstanding sensitivity potential into biomedical molecular imaging applications.

| S-EPMC6893250 | biostudies-literature

Development of an antibody-based, modular biosensor for 129Xe NMR molecular imaging of cells at nanomolar concentrations.

Project description:Magnetic resonance imaging (MRI) is seriously limited when aiming for visualization of targeted contrast agents. Images are reconstructed from the weak diamagnetic properties of the sample and require an abundant molecule like water as the reporter. Micromolar to millimolar concentrations of conventional contrast agents are needed to generate image contrast, thus excluding many molecular markers as potential targets. To address this limitation, we developed and characterized a functional xenon NMR biosensor that can identify a specific cell surface marker by targeted (129)Xe MRI. Cells expressing the cell surface protein CD14 can be spatially distinguished from control cells with incorporation of as little as 20 nM of the xenon MRI readout unit, cryptophane-A. Cryptophane-A serves as a chemical host for hyperpolarized nuclei and facilitates the sensitivity enhancement achieved by xenon MRI. Although this paper describes the application of a CD14-specific biosensor, the construct has been designed in a versatile, modular fashion. This allows for quick and easy adaptation of the biosensor to any cell surface target for which there is a specific antibody. In addition, the modular design facilitates the creation of a multifunctional probe that incorporates readout modules for different detection methods, such as fluorescence, to complement the primary MRI readout. This modular antibody-based approach not only offers a practical technique with which to screen targets, but one which can be readily applied as the xenon MRI field moves closer to molecular imaging applications in vivo.

| S-EPMC4136558 | biostudies-literature

Conditional, Genetically Encoded, Small Molecule-Regulated Inhibition of NF?B Signaling in RPE Cells.

Project description:PurposeNuclear factor ?B (NF?B) is a ubiquitously expressed, proinflammatory transcription factor that controls the expression of genes involved in cell survival, angiogenesis, complement activation, and inflammation. Studies have implicated NF?B-dependent cytokines or complement-related factors as being detrimentally involved in retinal diseases, thus making inhibition of NF?B signaling a potential therapeutic target. We sought to develop a conditional and reversible method that could regulate pathogenic NF?B signaling by the addition of a small molecule.MethodsWe developed a genetically based, trimethoprim (TMP)-regulated approach that conditionally inhibits NF?B signaling by fusing a destabilized dihydrofolate reductase (DHFR) domain to an inhibitor of NF?B, I?B?, in ARPE-19 cells. We then challenged ARPE-19 cells with a number of stimuli that have been demonstrated to trigger NF?B signaling, including LPS, TNF?, IL-1?, and A2E. Western blotting, electrophoretic mobility shift assay, quantitative PCR, ELISA, and NF?B reporter assays were used to evaluate the effectiveness of this DHFR-I?B? approach.ResultsThis destabilized domain approach, coupled with doxycycline-inducibility, allowed for accurate control over the abundance of DHFR-I?B?. Stabilization of DHFR-I?B? with TMP prevented IL-1?-, A2E-, LPS-, and TNF?-induced NF?B-mediated upregulation and release of the proinflammatory cytokines IL-1? and IL-6 from ARPE-19 cells (by as much as 93%). This strategy is dosable, completely reversible, and can be cycled "on" or "off" within the same cell population repeatedly to confer protection at desired time points.ConclusionsThese studies lay the groundwork for the use of destabilized domains in retinal pigment epithelium (RPE) cells in vivo and in this context, demonstrate their utility for preventing inflammatory signaling.

| S-EPMC5566385 | biostudies-literature

Nanomolar-Potency Small Molecule Inhibitor of STAT5 Protein.

Project description:We herein report the design and synthesis of the first nanomolar binding inhibitor of STAT5 protein. Lead compound 13a, possessing a phosphotyrosyl-mimicking salicylic acid group, potently and selectively binds to STAT5 over STAT3, inhibits STAT5-SH2 domain complexation events in vitro, silences activated STAT5 in leukemic cells, as well as STAT5's downstream transcriptional targets, including MYC and MCL1, and, as a result, leads to apoptosis. We believe 13a represents a useful probe for interrogating STAT5 function in cells as well as being a potential candidate for advanced preclinical trials.

| S-EPMC4234445 | biostudies-literature

Genetically Encoded Phase Contrast Agents for Digital Holographic Microscopy.

Project description:Quantitative phase imaging and digital holographic microscopy have shown great promise for visualizing the motion, structure, and physiology of microorganisms and mammalian cells in three dimensions. However, these imaging techniques currently lack molecular contrast agents analogous to the fluorescent dyes and proteins that have revolutionized fluorescence microscopy. Here we introduce the first genetically encodable phase contrast agents based on gas vesicles. The relatively low index of refraction of the air-filled core of gas vesicles results in optical phase advancement relative to aqueous media, making them a "positive" phase contrast agent easily distinguished from organelles, dyes, or microminerals. We demonstrate this capability by identifying and tracking the motion of gas vesicles and gas vesicle-expressing bacteria using digital holographic microscopy, and by imaging the uptake of engineered gas vesicles by mammalian cells. These results give phase imaging a biomolecular contrast agent, expanding the capabilities of this powerful technology for three-dimensional biological imaging.

| S-EPMC7685204 | biostudies-literature

Gadolinium Complexes as Contrast Agent for Cellular NMR Spectroscopy.

Project description:Aqua Gd3+ and Gd-DOTA (gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacete) complexes were studied as a contrast agent in cellular NMR (nuclear magnetic resonance) spectroscopy for distinguishing between intracellular and extracellular spaces. The contrast agents for this purpose should provide strong paramagnetic relaxation enhancement and localize in the extracellular space without disturbing biological functions. Cell membrane permeability to Gd complexes was evaluated from the concentrations of gadolinium complexes in the inside and outside of E. coli cells measured by the 1H-NMR relaxation. The site-specific binding of the complexes to E. coli cells was also analyzed by high-resolution solid-state 13C-NMR. The aqua Gd3+ complex did not enhance T1 relaxation in proportion to the amount of added Gd3+. This Gd3+ concentration dependence and the 13C-NMR indicated that its strong cytotoxicity should be due to the binding of the paramagnetic ions to cellular components especially at the lipid membranes. In contrast, Gd-DOTA stayed in the solution states and enhanced relaxation in proportion to the added amount. This agent exhibited strong T1 contrast between the intra- and extracellular spaces by a factor of ten at high concentrations under which the cells were viable over a long experimental time of days. These properties make Gd-DOTA suitable for selectively contrasting the living cellular space in NMR spectroscopy primarily owing to its weak interaction with cellular components.

| S-EPMC7312942 | biostudies-literature

Simultaneous detection of intracellular target and off-target binding of small molecule cancer drugs at nanomolar concentrations.

Project description:Background and purposeIn vitro assays that determine activities of drug candidates with isolated targets have only limited predictive value for activities in cellular assays. Poor membrane permeability and off-target binding are major reasons for such discrepancies. However, it still difficult to directly analyse off-target binding at the same time as target binding, on a subcellular level. Here, we present a combination of fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) as a solution to this problem.Experimental approachThe well-established dihydrofolate reductase inhibitor methotrexate and the kinase inhibitors PD173956 and purvalanol B were conjugated via polyethylene glycol linkers with the fluorophore Cy5. The cellular uptake and subcellular distribution of these compounds in single human cancer-derived cells were investigated by confocal laser scanning microscopy. In addition, molecular interactions inside the cell with the respective target proteins and off-target binding were detected simultaneously in the nanomolar range by FCCS and FCS, respectively, using cells expressing green fluorescent protein fusion proteins of dihydrofolate reductase and Abelson kinase 1.Key resultsLarge differences in the interaction patterns were found for these compounds. For methotrexate-Cy5, drug-target interactions could be detected and dissociation constants determined. In contrast, PD173956-Cy5 showed strong interactions with intracellular high-molecular weight structures, other than its target.Conclusions and implicationsThe combination of FCS and FCCS provides a powerful means to assess subcellular pharmacokinetics and dynamics of drug candidates at nanomolar concentrations.

| S-EPMC2936001 | biostudies-literature

Cryptophane Nanoscale Assemblies Expand 129Xe NMR Biosensing.

Project description:Cryptophane-based biosensors are promising agents for the ultrasensitive detection of biomedically relevant targets via 129Xe NMR. Dynamic light scattering revealed that cryptophanes form water-soluble aggregates tens to hundreds of nanometers in size. Acridine orange fluorescence quenching assays allowed quantitation of the aggregation state, with critical concentrations ranging from 200 nM to 600 nM, depending on the cryptophane species in solution. The addition of excess carbonic anhydrase (CA) protein target to a benzenesulfonamide-functionalized cryptophane biosensor (C8B) led to C8B disaggregation and produced the expected 1:1 C8B-CA complex. C8B showed higher affinity at 298 K for the cytoplasmic isozyme CAII than the extracellular CAXII isozyme, which is a biomarker of cancer. Using hyper-CEST NMR, we explored the role of stoichiometry in detecting these two isozymes. Under CA-saturating conditions, we observed that isozyme CAII produces a larger 129Xe NMR chemical shift change (? = 5.9 ppm, relative to free biosensor) than CAXII (? = 2.7 ppm), which indicates the strong potential for isozyme-specific detection. However, stoichiometry-dependent chemical shift data indicated that biosensor disaggregation contributes to the observed 129Xe NMR chemical shift change that is normally assigned to biosensor-target binding. Finally, we determined that monomeric cryptophane solutions improve hyper-CEST saturation contrast, which enables ultrasensitive detection of biosensor-protein complexes. These insights into cryptophane-solution behavior support further development of xenon biosensors, but will require reinterpretation of the data previously obtained for many water-soluble cryptophanes.

| S-EPMC6050516 | biostudies-literature

Bacterial spore detection and analysis using hyperpolarized 129Xe chemical exchange saturation transfer (Hyper-CEST) NMR.

Project description:Previously, we reported hyperpolarized 129Xe chemical exchange saturation transfer (Hyper-CEST) NMR techniques for the ultrasensitive (i.e., 1 picomolar) detection of xenon host molecules known as cryptophane. Here, we demonstrate a more general role for Hyper-CEST NMR as a spectroscopic method for probing nanoporous structures, without the requirement for cryptophane or engineered xenon-binding sites. Hyper-CEST 129Xe NMR spectroscopy was employed to detect Bacillus anthracis and Bacillus subtilis spores in solution, and interrogate the layers that comprise their structures. 129Xe-spore samples were selectively irradiated with radiofrequency pulses; the depolarized 129Xe returned to aqueous solution and depleted the 129Xe-water signal, providing measurable contrast. Removal of the outermost spore layers in B. anthracis and B. subtilis (the exosporium and coat, respectively) enhanced 129Xe exchange with the spore interior. Notably, the spores were invisible to hyperpolarized 129Xe NMR direct detection methods, highlighting the lack of high-affinity xenon-binding sites, and the potential for extending Hyper-CEST NMR structural analysis to other biological and synthetic nanoporous structures.

| S-EPMC4115335 | biostudies-literature

Genetically encoded fluorescent sensors for intracellular NADH detection.

Project description:We have developed genetically encoded fluorescent sensors for reduced nicotinamide adenine dinucleotide (NADH), which manifest a large change in fluorescence upon NADH binding. We demonstrate the utility of these sensors in mammalian cells by monitoring the dynamic changes in NADH levels in subcellular organelles as affected by NADH transport, glucose metabolism, electron transport chain function, and redox environment, and we demonstrate the temporal separation of changes in mitochondrial and cytosolic NADH levels with perturbation. These results support the view that cytosolic NADH is sensitive to environmental changes, while mitochondria have a strong tendency to maintain physiological NADH homeostasis. These sensors provide a very good alternative to existing techniques that measure endogenous fluorescence of intracellular NAD(P)H and, owing to their superior sensitivity and specificity, allow for the selective monitoring of total cellular and compartmental responses of this essential cofactor.

| S-EPMC3253140 | biostudies-literature