Harnessing a previously unidentified capability of bacterial allosteric transcription factors for sensing diverse small molecules in vitro

Cao, Jiaqian; Yao, Yongpeng; Fan, Keqiang; Tan, Gao-Yi; Xiang, Wensheng; Xia, Xuekui; Li, Shanshan; Wang, Weishan; Zhang, Lixin

doi:10.1126/sciadv.aau4602

Cited by 36 publications

(25 citation statements)

References 75 publications

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“…5c). CaT-SMelor has many unique advantages over previous aTF-based and protein-immobilization-assisted methods of small-molecule detection 4,5, (Table 1) and has great potential utility in the future development of a miniature portable device for the detection of small molecules or disease diagnoses.…”

Section: Discussionmentioning

confidence: 99%

“…The presence and binding of the target molecule usually induces a conformational change in the aTF DNA-binding domain, which then enhances or attenuates the double-stranded DNA (dsDNA)-binding capacity of the aTF by the direct binding of the aTF effector-binding domain 1 . Recently, we developed a bacterial-aTF-based in vitro biosensing platform, aTF-NAST (aTF-based nicked DNA-template-assisted signal transduction) by exploiting the competition between T4 DNA ligase and aTFs in binding to nicked DNA 4,5 . However, this is a relatively time-consuming, inconvenient and costly method of detecting small molecules.…”

Section: Introductionmentioning

confidence: 99%

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A CRISPR-Cas12a-derived biosensing platform for the highly sensitive detection of diverse small molecules

et al. 2019

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Besides genome editing, CRISPR-Cas12a has recently been used for DNA detection applications with attomolar sensitivity but, to our knowledge, it has not been used for the detection of small molecules. Bacterial allosteric transcription factors (aTFs) have evolved to sense and respond sensitively to a variety of small molecules to benefit bacterial survival. By combining the single-stranded DNA cleavage ability of CRISPR-Cas12a and the competitive binding activities of aTFs for small molecules and double-stranded DNA, here we develop a simple, supersensitive, fast and high-throughput platform for the detection of small molecules, designated CaT-SMelor ( C RISPR-Cas12a- and aT F-mediated s mall m ol e cu l e detect or ). CaT-SMelor is successfully evaluated by detecting nanomolar levels of various small molecules, including uric acid and p -hydroxybenzoic acid among their structurally similar analogues. We also demonstrate that our CaT-SMelor directly measured the uric acid concentration in clinical human blood samples, indicating a great potential of CaT-SMelor in the detection of small molecules.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A CRISPR-Cas12a-derived biosensing platform for the highly sensitive detection of diverse small molecules

et al. 2019

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“…Only very recently have aTFs been examined as biomolecular recognition elements for in vitro sensing. [ 10,28–31 ] Of the limited number of signal transduction mechanisms explored, only ours utilizes FRET. One homogeneous assay achieved nanomolar sensing using an amplified luminescent proximity homogeneous assay (AlphaScreen).…”

Section: Figurementioning

confidence: 99%

“…[ 28,31 ] Two other assay designs provide only an indirect measure of aTF–DNA binding by amplifying and quantifying (un)bound DNA. [ 29–31 ] Using existing methods of DNA detection facilitates incorporation into existing workflows but lacks the advantages of a real‐time, homogeneous assay without substantial gains in the LOD or linear dynamic range. [ 29–31 ] Our FRET‐based sensor maintains the rapidity and convenience of the homogeneous assay, while incorporating a ratiometric output and widely accessible fluorophores to facilitate broader adoption.…”

Section: Figurementioning

confidence: 99%

A Förster Resonance Energy Transfer‐Based Ratiometric Sensor with the Allosteric Transcription Factor TetR

Nguyen

Chern

Baer

et al. 2020

Small

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A recent description of an antibody‐free assay is significantly extended for small molecule analytes using allosteric transcription factors (aTFs) and Förster resonance energy transfer (FRET). The FRET signal indicates the differential binding of an aTF–DNA pair with a dose‐dependent response to its effector molecule, i.e., the analyte. The new sensors described here, based on the well‐characterized aTF TetR, demonstrate several new features of the assay approach: 1) the generalizability of the sensors to additional aTF–DNA–analyte systems, 2) sensitivity modulation through the choice of donor fluorophore (quantum dots or fluorescent proteins, FPs), and 3) sensor tuning using aTF variants with differing aTF–DNA binding affinities. While all of these modular sensors self‐assemble, the design reported here based on a recombinant aTF–FP chimera with commercially available dye‐labeled DNA uses readily accessible sensor components to facilitate easy adoption of the sensing approach by the broader community.

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“…Besides signal recognition, signal transduction is also an essential aspect for the development of biosensors. Various biosensing systems, including CRISPR-Cas12a-and allosteric transcription factors-mediated small molecule detector39 , allosteric transcription factors-based nicked DNA-template-assisted signal transduction40 , and quantum-dotallosteric transcription factors-FRET41 , have been reported. Other biosensors based on L-2-HG responding to LhgR and the corresponding transduction mechanisms could also be developed for detection of L-2-HG.…”

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confidence: 99%

A fluorescent l-2-hydroxyglutarate biosensor

Kang

Zhang

Gao

et al. 2020

Preprint

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Abstractl-2-Hydroxyglutarate (l-2-HG) plays important roles in diverse physiological processes, such as carbon starvation response, tumorigenesis, and hypoxic adaptation. Despite its importance and intensively studied metabolism, regulation of l-2-HG metabolism remains poorly understood and a regulator specifically responded to l-2-HG has never been identified. Based on the genomic neighborhood analysis of the gene encoding l-2-HG oxidase (LhgO), LhgR, which represses the transcription of lhgO, was identified in Pseudomonas putida W619 in this study. LhgR was demonstrated to recognize l-2-HG as its specific effector molecule, and this allosteric transcription factor was then used as a biorecognition element for construction of l-2-HG-sensing FRET sensor. The newly developed l-2-HG sensor can conveniently monitor the concentrations of l-2-HG in various biological samples. In addition to bacterial l-2-HG generation during carbon starvation, biological functions of the l-2-HG dehydrogenase and hypoxia induced l-2-HG accumulation were also revealed by using the l-2-HG sensor in human cells.

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Harnessing a previously unidentified capability of bacterial allosteric transcription factors for sensing diverse small molecules in vitro

Abstract: We develop a novel biosensing platform for the detection of diverse small molecules.

Cited by 36 publications

References 75 publications

A CRISPR-Cas12a-derived biosensing platform for the highly sensitive detection of diverse small molecules

A CRISPR-Cas12a-derived biosensing platform for the highly sensitive detection of diverse small molecules

A Förster Resonance Energy Transfer‐Based Ratiometric Sensor with the Allosteric Transcription Factor TetR

A fluorescent l-2-hydroxyglutarate biosensor

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