In Vitro and In Vivo Enzyme Activity Screening via RNA-Based Fluorescent Biosensors for S-Adenosyl-<scp>l</scp>-homocysteine (SAH)

Su, Yichi; Hickey, Scott F.; Keyser, Samantha G. L.; Hammond, Ming C.

doi:10.1021/jacs.6b01621

Cited by 81 publications

(83 citation statements)

References 52 publications

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“…This observation reveals an inherent limitation of RNA-based biosensors, which is that intercalating compounds may yield false-positive fluorescent changes. However, RNA-based biosensors have been successfully used to characterize inhibitors that specifically target proteins, including methyltransferases (Su et al, 2016) and RNA demethylases (Svensen and Jaffrey, 2016). …”

Section: Resultsmentioning

confidence: 99%

An RNA-Based Fluorescent Biosensor for High-Throughput Analysis of the cGAS-cGAMP-STING Pathway

Bose

Marcus

et al. 2016

Cell Chemical Biology

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Summary In mammalian cells, the second messenger (2′-5′, 3′-5′) cyclic GMP-AMP (2′, 3′-cGAMP), is produced by the cytosolic DNA sensor cGAMP synthase (cGAS), and subsequently bound by stimulator of interferon genes (STING) to trigger interferon response. Thus, the cGAS-cGAMP-STING pathway plays a critical role in pathogen detection, as well as pathophysiological conditions including cancer and autoimmune disorders. However, studying and targeting this immune signaling pathway has been challenging due to the absence of tools for high-throughput analysis. We have engineered an RNA-based fluorescent biosensor that responds to 2′, 3′-cGAMP. The resulting “mix-and-go” cGAS activity assay shows excellent statistical reliability as a high-throughput screening (HTS) assay and distinguishes between direct and indirect cGAS inhibitors. Furthermore, the biosensor enables quantitation of 2′, 3′-cGAMP in mammalian cell lysates. We envision this biosensor-based assay as a resource to study the cGAS-cGAMP-STING pathway in the context of infectious diseases, cancer immunotherapy, and autoimmune diseases.

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Section: Resultsmentioning

confidence: 99%

An RNA-Based Fluorescent Biosensor for High-Throughput Analysis of the cGAS-cGAMP-STING Pathway

Bose

Marcus

et al. 2016

Cell Chemical Biology

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“…[82] While these approaches used target-binding aptamers that were selected by SELEX, [83] which can be challenging, Kellenberger et al fused Spinach to variants of an atural GEMM-I riboswitch to produce ab iosensor for cyclic diguanosine monophosphate (GMP) and cyclic AMP-GMP in E. coli. [87] Spinach riboswitches involving strand displacement upon analyte binding were developed for thiamine 5'-pyrophosphate (TPP) and expressed and imaged in E. coli. [86] A modified Spinach2-S-adenosyl-l-homocysteine (SAH) riboswitch fusion was used to detect SAH in live E. coli and to screen inhibitors of methyltransferases in vitro.…”

Section: Methodsmentioning

confidence: 99%

“…[86] A modified Spinach2-S-adenosyl-l-homocysteine (SAH) riboswitch fusion was used to detect SAH in live E. coli and to screen inhibitors of methyltransferases in vitro. [87] Spinach riboswitches involving strand displacement upon analyte binding were developed for thiamine 5'-pyrophosphate (TPP) and expressed and imaged in E. coli. [88] Thea bove mentioned promiscuous aptamer DIR2s-Apt fused to the epidermal growth factor receptor (EGFR) represents ab ifunctional aptamer that was used to differentiate between cell-surface and internalized EGFR on mammalian cells by applying the two fluorogens (DIR-Pro and OTB-SO3) at different times.…”

Section: Methodsmentioning

confidence: 99%

RNA Structure and Cellular Applications of Fluorescent Light‐Up Aptamers

Neubacher

Hennig

2018

Angewandte Chemie

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The cellular functions of RNA are not limited to their role as blueprints for protein synthesis. In particular, noncoding RNA, such as, snRNAs, lncRNAs, miRNAs, play important roles. With increasing numbers of RNAs being identified, it is well known that the transcriptome outnumbers the proteome by far. This emphasizes the great importance of functional RNA characterization and the need to further develop tools for these investigations, many of which are still in their infancy. Fluorescent light‐up aptamers (FLAPs) are RNA sequences that can bind nontoxic, cell‐permeable small‐molecule fluorogens and enhance their fluorescence over many orders of magnitude upon binding. FLAPs can be encoded on the DNA level using standard molecular biology tools and are subsequently transcribed into RNA by the cellular machinery, so that they can be used as fluorescent RNA tags (FLAP‐tags). In this Minireview, we give a brief overview of the fluorogens that have been developed and their binding RNA aptamers, with a special focus on published crystal structures. A summary of current and future cellular FLAP applications with an emphasis on the study of RNA–RNA and RNA–protein interactions using split‐FLAP and Förster resonance energy transfer (FRET) systems is given.

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“…Since then, a variety of alternative fluorogenic dye/ RNA aptamer couples have been developed (Dolgosheina and Unrau 2016;Ouellet 2016;Pauff et al 2017). Similarly to fluorescent proteins, fluorescent RNAs have a wide application spectrum (e.g., live-cell gene expression imaging [Guet et al 2015;Zhang et al 2015], biosensing of proteins [Song et al 2013], nucleic acids [Sato et al 2015;Aw et al 2016;Huang et al 2017;Ong et al 2017], or even metabolites [Nakayama et al 2012;Paige et al 2012;Kellenberger et al 2013Kellenberger et al , 2015aSharma et al 2014;You et al 2015;Bose et al 2016;Hallberg et al 2016;Su et al 2016;Wang et al 2016]), and one can anticipate their significant impact on RNA biology.…”

Section: Introductionmentioning

confidence: 99%

Crystal structure and fluorescence properties of the iSpinach aptamer in complex with DFHBI

Fernández-Millán¹,

Autour²,

Ennifar³

et al. 2017

RNA

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Fluorogenic RNA aptamers are short nucleic acids able to specifically interact with small molecules and strongly enhance their fluorescence upon complex formation. Among the different systems recently introduced, Spinach, an aptamer forming a fluorescent complex with the 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), is one of the most promising. Using random mutagenesis and ultrahigh-throughput screening, we recently developed iSpinach, an improved version of the aptamer, endowed with an increased folding efficiency and thermal stability. iSpinach is a shorter version of Spinach, comprising five mutations for which the exact role has not yet been deciphered. In this work, we cocrystallized a reengineered version of iSpinach in complex with the DFHBI and solved the X-ray structure of the complex at 2 Å resolution. Only a few mutations were required to optimize iSpinach production and crystallization, underlying the good folding capacity of the molecule. The measured fluorescence half-lives in the crystal were 60% higher than in solution. Comparisons with structures previously reported for Spinach sheds some light on the possible function of the different beneficial mutations carried by iSpinach.

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In Vitro and In Vivo Enzyme Activity Screening via RNA-Based Fluorescent Biosensors for S-Adenosyl-l-homocysteine (SAH)

Cited by 81 publications

References 52 publications

An RNA-Based Fluorescent Biosensor for High-Throughput Analysis of the cGAS-cGAMP-STING Pathway

An RNA-Based Fluorescent Biosensor for High-Throughput Analysis of the cGAS-cGAMP-STING Pathway

RNA Structure and Cellular Applications of Fluorescent Light‐Up Aptamers

Crystal structure and fluorescence properties of the iSpinach aptamer in complex with DFHBI

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