A Genetically Encoded β‐Lactamase Reporter for Ultrasensitive <sup>129</sup>Xe NMR in Mammalian Cells

Wang, Yanfei; Roose, Benjamin W.; Palovcak, Eugene; Carnevale, Vincenzo; Dmochowski, Ivan J.

doi:10.1002/anie.201604055

Cited by 52 publications

(60 citation statements)

References 49 publications

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“…Second, AQP1-dependent contrast can be detected at reasonably low concentrations (∼0.5 μM), which makes it a sensitive MRI reporter gene. Although CEST reporters operating on hyperpolarized xenon can achieve even higher molecular sensitivity2466, their use requires elaborate equipment for xenon hyperpolarization and administration, and in the case of gas vesicles, the expression of complex multi-gene clusters. In addition, successful CEST experiments require sophisticated pulse sequences, whereas diffusion-weighted imaging is implemented as a standard technique on clinical MRI scanners.…”

Section: Discussionmentioning

confidence: 99%

Non-invasive imaging using reporter genes altering cellular water permeability

Mukherjee

Davis

et al. 2016

Nat Commun

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Non-invasive imaging of gene expression in live, optically opaque animals is important for multiple applications, including monitoring of genetic circuits and tracking of cell-based therapeutics. Magnetic resonance imaging (MRI) could enable such monitoring with high spatiotemporal resolution. However, existing MRI reporter genes based on metalloproteins or chemical exchange probes are limited by their reliance on metals or relatively low sensitivity. Here we introduce a new class of MRI reporters based on the human water channel aquaporin 1. We show that aquaporin overexpression produces contrast in diffusion-weighted MRI by increasing tissue water diffusivity without affecting viability. Low aquaporin levels or mixed populations comprising as few as 10% aquaporin-expressing cells are sufficient to produce MRI contrast. We characterize this new contrast mechanism through experiments and simulations, and demonstrate its utility in vivo by imaging gene expression in tumours. Our results establish an alternative class of sensitive, metal-free reporter genes for non-invasive imaging.

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Section: Discussionmentioning

confidence: 99%

Non-invasive imaging using reporter genes altering cellular water permeability

Mukherjee

Davis

et al. 2016

Nat Commun

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“…62 We initially considered TEM-1 β-lactamase (bla) based on its well-established allosteric site whose size and hydrophobicity suggest it to be a good target for Xe exchange. We observed two saturation responses in the hyper-CEST z-spectrum for 80 μM bla: one free 129 Xe in solution peak centered at 195 ppm, and a second peak centered at 255 ppm that results from 129 Xe–bla interaction.…”

Section: Genetically-encoded Protein As a 129xe Nmr Reportermentioning

confidence: 99%

An Expanded Palette of Xenon-129 NMR Biosensors

Wang

Dmochowski

2016

Acc. Chem. Res.

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ConspectusMolecular imaging holds considerable promise for elucidating biological processes in normal physiology as well as disease states, by determining the location and relative concentration of specific molecules of interest. Proton-based magnetic resonance imaging (1H MRI) is nonionizing and provides good spatial resolution for clinical imaging but lacks sensitivity for imaging low-abundance (i.e., submicromolar) molecular markers of disease or environments with low proton densities. To address these limitations, hyperpolarized (hp) 129Xe NMR spectroscopy and MRI have emerged as attractive complementary methodologies. Hyperpolarized xenon is nontoxic and can be readily delivered to patients via inhalation or injection, and improved xenon hyperpolarization technology makes it feasible to image the lungs and brain for clinical applications.In order to target hp 129Xe to biomolecular targets of interest, the concept of “xenon biosensing” was first proposed by a Berkeley team in 2001. The development of xenon biosensors has since focused on modifying organic host molecules (e.g., cryptophanes) via diverse conjugation chemistries and has brought about numerous sensing applications including the detection of peptides, proteins, oligonucleotides, metal ions, chemical modifications, and enzyme activity. Moreover, the large (∼300 ppm) chemical shift window for hp 129Xe bound to host molecules in water makes possible the simultaneous identification of multiple species in solution, that is, multiplexing. Beyond hyperpolarization, a 106-fold signal enhancement can be achieved through a technique known as hyperpolarized 129Xe chemical exchange saturation transfer (hyper-CEST), which shows great potential to meet the sensitivity requirement in many applications.This Account highlights an expanded palette of hyper-CEST biosensors, which now includes cryptophane and cucurbit[6]uril (CB[6]) small-molecule hosts, as well as genetically encoded gas vesicles and single proteins. In 2015, we reported picomolar detection of commercially available CB[6] via hyper-CEST. Inspired by the versatile host–guest chemistry of CB[6], our lab and others developed “turn-on” strategies for CB[6]-hyper-CEST biosensing, demonstrating detection of protein analytes in complex media and specific chemical events. CB[6] is starting to be employed for in vivo imaging applications. We also recently determined that TEM-1 β-lactamase can function as a single-protein reporter for hyper-CEST and observed useful saturation contrast for β-lactamase expressed in bacterial and mammalian cells. These newly developed small-molecule and genetically encoded xenon biosensors offer significant potential to extend the scope of hp 129Xe toward molecular MRI.

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“…GVs allow xenon dissolved in surrounding media to exchange in and out of their gaseous compartment, producing MRI contrast through CEST at picomolar concentrations (Figure 4, e-f) 34 . Other proteins active as contrast agents for 129 Xe MRI and other hyperpolarized nuclei have also been reported 35,36 .…”

Section: Biomolecular Tools For Magnetic Resonance Imagingmentioning

confidence: 97%

Going Deeper: Biomolecular Tools for Acoustic and Magnetic Imaging and Control of Cellular Function

et al. 2017

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Most cellular phenomena of interest to mammalian biology occur within the context of living tissues and organisms. However, today’s most advanced tools for observing and manipulating cellular function – based on fluorescent or light-controlled proteins – work best in cultured cells, transparent model species or small, surgically accessed anatomical regions. Their reach into deep tissues and larger animals is limited by photon scattering. To overcome this limitation, we must design biochemical tools that interface with more penetrant forms of energy. For example, sound waves and magnetic fields easily permeate most biological tissues, allowing the formation of images and delivery of energy for actuation. These capabilities are widely used in clinical techniques such as diagnostic ultrasound, magnetic resonance imaging, focused ultrasound ablation and magnetic particle hyperthermia. Each of these modalities offers spatial and temporal precision that could be used to study a multitude of cellular processes in vivo. However, connecting these techniques to cellular functions such as gene expression, proliferation, migration and signaling requires the development of new biochemical tools that can interact with sound waves and magnetic fields as optogenetic tools interact with photons. Here, we discuss the exciting challenges this poses for biomolecular engineering, and provide examples of recent advances pointing the way to greater depth in in vivo cell biology.

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A Genetically Encoded β‐Lactamase Reporter for Ultrasensitive ¹²⁹Xe NMR in Mammalian Cells

Cited by 52 publications

References 49 publications

Non-invasive imaging using reporter genes altering cellular water permeability

Non-invasive imaging using reporter genes altering cellular water permeability

An Expanded Palette of Xenon-129 NMR Biosensors

Going Deeper: Biomolecular Tools for Acoustic and Magnetic Imaging and Control of Cellular Function

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A Genetically Encoded β‐Lactamase Reporter for Ultrasensitive 129Xe NMR in Mammalian Cells

Cited by 52 publications

References 49 publications

Non-invasive imaging using reporter genes altering cellular water permeability

Non-invasive imaging using reporter genes altering cellular water permeability

An Expanded Palette of Xenon-129 NMR Biosensors

Going Deeper: Biomolecular Tools for Acoustic and Magnetic Imaging and Control of Cellular Function

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Product

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A Genetically Encoded β‐Lactamase Reporter for Ultrasensitive ¹²⁹Xe NMR in Mammalian Cells