Structure-Based Design of Versatile Biosensors for Small Molecules Based on the PAS Domain of a Thermophilic Histidine Kinase

Cormann, Kai U.; Baumgart, Meike; Bott, Michael

doi:10.1021/acssynbio.8b00348

Cited by 9 publications

(7 citation statements)

References 52 publications

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“…Notably, the CitAP domain of the thermophilic bacterium Geobacillus thermoleovorans CitA SHK has recently been shown to be amenable to “binding pocket grafting” to convert its binding specificity to that of close homologues including l -malate, phthalate, and ethylmalonate. 56 Potentially, similar binding pocket grafting could be used to create a series of new biosensors based on the Citron1 and Citroff1 templates.…”

Section: Discussionmentioning

confidence: 99%

“…Together, these advantages mean that the advent of Citron1 and Citroff1 represent a major advance for the detection of citrate concentration dynamics in live mammalian cells using fluorescence imaging. Notably, the CitAP domain of the thermophilic bacterium Geobacillus thermoleovorans CitA SHK has recently been shown to be amenable to “binding pocket grafting” to convert its binding specificity to that of close homologues including l -malate, phthalate, and ethylmalonate . Potentially, similar binding pocket grafting could be used to create a series of new biosensors based on the Citron1 and Citroff1 templates.…”

Section: Discussionmentioning

confidence: 99%

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High-Performance Intensiometric Direct- and Inverse-Response Genetically Encoded Biosensors for Citrate

et al. 2020

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Motivated by the growing recognition of citrate as a central metabolite in a variety of biological processes associated with healthy and diseased cellular states, we have developed a series of high-performance genetically encoded citrate biosensors suitable for imaging of citrate concentrations in mammalian cells. The design of these biosensors was guided by structural studies of the citrate-responsive sensor histidine kinase and took advantage of the same conformational changes proposed to propagate from the binding domain to the catalytic domain. Following extensive engineering based on a combination of structure guided mutagenesis and directed evolution, we produced an inverse-response biosensor (Δ F / F min ≈ 18) designated Citroff1 and a direct-response biosensor (Δ F / F min ≈ 9) designated Citron1. We report the X-ray crystal structure of Citron1 and demonstrate the utility of both biosensors for qualitative and quantitative imaging of steady-state and pharmacologically perturbed citrate concentrations in live cells.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

High-Performance Intensiometric Direct- and Inverse-Response Genetically Encoded Biosensors for Citrate

et al. 2020

View full text Add to dashboard Cite

show abstract

“…Together, these advantages mean that the advent of Citron1 and Citroff1 represent a major advance for the detection of citrate concentration dynamics in live mammalian cells using fluorescence imaging. Notably, the CitAP domain of the thermophilic bacterium Geobacillus thermoleovorans CitA SHK has recently been shown to be amenable to "binding pocket grafting" to convert its binding specificity to that of close homologs including L-malate, phthalate, and ethylmalonate 52 . Potentially, similar binding pocket grafting could be used to create a series of new biosensors based on the Citron1 and Citroff1 templates.…”

Section: Discussionmentioning

confidence: 99%

High performance intensiometric direct- and inverse-response genetically encoded biosensors for citrate

Zhao

Shen

Wen

et al. 2020

Preprint

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Motivated by the growing recognition of citrate as a central metabolite in a variety of biological processes associated with healthy and diseased cellular states, we have developed a series of high-performance genetically encoded citrate biosensors suitable for imaging of citrate concentrations in mammalian cells. The design of these biosensors was guided by structural studies of the citrate-responsive sensor histidine kinase, and took advantage of the same conformational changes proposed to propagate from the binding domain to the catalytic domain. Following extensive engineering based on a combination of structure guided mutagenesis and directed evolution, we produced an inverse-response biosensor (ΔF/F min~1 8) designated Citroff1 and a direct-response biosensor (ΔF/F min~9 ) designated Citron1. We report the x-ray crystal structure of Citron1 and demonstrate the utility of both biosensors for qualitative and quantitative imaging of steady-state and pharmacologically-perturbed citrate concentrations in live cells.

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“…The field has developed a number of different molecular platforms for developing small molecule biosensors, including engineered transcription factors, enzymes, and nucleic acid aptamers; however, methods described to-date generally require an extensive application-specific development cycle for new biosensor components [3][4][5][6][7]].…”

Section: Introductionmentioning

confidence: 99%

Highly multiplexed selection of RNA aptamers against a small molecule library

Kaplan

Townshend

Smolke

2022

Preprint

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Applications of synthetic biology spanning human health, industrial bioproduction, and ecosystem monitoring often require small molecule sensing capabilities, typically in the form of genetically-encoded small molecule biosensors. Critical to the deployment of greater numbers of these systems are methods that support the rapid development of such biosensors against a broad range of small molecule targets. Here, we use a previously developed method for selection of RNA biosensors against unmodified small molecules (DRIVER) to perform a selection against a densely multiplexed mixture of small molecules, representative of those employed in high-throughput drug screening. Using a mixture of 5,120 target compounds randomly sampled from a large diversity drug screening library, we performed a 95-round selection and then analyzed the enriched RNA biosensor library using next generation sequencing (NGS). From our analysis, we identified RNA biosensors with at least 2-fold change in signal in the presence of at least 217 distinct target compounds with sensitivities down to 25 nM. Although many of these biosensors respond to multiple targets, clustering analysis indicated at least 150 different small-molecule sensing patterns. We also built a classifier that was able to predict whether the biosensors would respond to a new compound with an average precision of 0.82. Since the target compound library was designed to be representative of larger diversity compound libraries, we expect that the described approach can be used with similar compound libraries to identify aptamers against other small molecules with a similar success rate. The new RNA biosensors (or their component aptamers) described in this work can be further optimized and used in applications such as biosensing, gene control, or enzyme evolution. In addition, the data presented here provide an expanded compendium of new RNA aptamers compared to the 82 small molecule RNA aptamers published in the literature, allowing further bioinformatic analyses of the general classes of small molecules for which RNA aptamers can be found.

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Structure-Based Design of Versatile Biosensors for Small Molecules Based on the PAS Domain of a Thermophilic Histidine Kinase

Cited by 9 publications

References 52 publications

High-Performance Intensiometric Direct- and Inverse-Response Genetically Encoded Biosensors for Citrate

High-Performance Intensiometric Direct- and Inverse-Response Genetically Encoded Biosensors for Citrate

High performance intensiometric direct- and inverse-response genetically encoded biosensors for citrate

Highly multiplexed selection of RNA aptamers against a small molecule library

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