RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messenger Cyclic di-AMP

Kellenberger, Colleen A.; Chen, Chen; Whiteley, Aaron T.; Portnoy, Daniel A.; Hammond, Ming C.

doi:10.1021/jacs.5b00275

Cited by 110 publications

(113 citation statements)

References 24 publications

(47 reference statements)

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“…These aptamers include those that bind triphenylmethanes [1], bisbenzimides [2], imidazoles [3], and the benzothiazolidene asymmetric cyanine dye, thiazole orange [4]. When linked to other aptamers that drive their ability to bind ligand, these light-up aptamers can report on the presence of analytes in vitro that range from metabolites to proteins depending on the linked aptamer’s specificity [5; 6]. …”

Section: Introductionmentioning

confidence: 99%

Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells

İlgü¹,

Ray²,

Bendickson

et al. 2016

Methods

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The regulation of RNA transcription is central to cellular function. Changes in gene expression drive differentiation and cellular responses to events such as injury. RNA trafficking can also have a large impact on protein expression and its localization. Thus, the ability to image RNA transcription and trafficking in real time and in living cells is a worthwhile goal that has been difficult to achieve. The availability of “light-up” aptamers that cause an increase in fluorescence of their ligands when bound by the aptamer have shown promise for reporting on RNA production and localization in vivo. Here we have investigated two light-up aptamers (the malachite green aptamer and the Spinach aptamers) for their suitability as reporters of RNA expression in vivo using two eukaryotic cell types, yeast and mammalian. Our analysis focused on the aptamer ligands, their contributions to background noise, and the impact of tandem aptamer strings on signal strength and ligand affinity. Whereas the background fluorescence is very low in vitro, this is not always true for cell imaging. Our results suggest the need for caution in using light-up aptamers as reporters for imaging RNA. In particular, images should be collected and analyzed by operators blinded to the sample identities. The appropriate control condition of ligand with the cells in the absence of aptamer expression must be included in each experiment. This control condition establishes that the specific interaction of ligand with aptamer, rather than nonspecific interactions with unknown cell elements, is responsible for the observed fluorescent signals. High background signals due to nonspecific interactions of aptamer ligands with cell components can be minimized by using IMAGEtags (Intracellular Multiaptamer GEnetic tags), which signal by FRET and are promising RNA reporters for imaging transcription.

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Section: Introductionmentioning

confidence: 99%

Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells

İlgü¹,

Ray²,

Bendickson

et al. 2016

Methods

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“…5c, d). The observed increase in relative fluorescence in the presence of 5HTP is comparable to robust cyclic dinucleotide sensors based upon natural riboswitch aptamer domains in live cells 10,45 .…”

Section: Resultsmentioning

confidence: 57%

Recurrent RNA motifs as scaffolds for genetically encodable small-molecule biosensors

et al. 2017

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Allosteric RNA devices are increasingly viewed as important tools capable of monitoring enzyme evolution, optimizing engineered metabolic pathways, facilitating gene discovery and regulators of nucleic acid-based therapeutics. A key bottleneck in the development of these platforms is the availability of small molecule binding RNA aptamers that robustly function in the cellular environment. While aptamers can be raised against nearly any desired target by in vitro selection, many cannot be easily integrated into devices or do not reliably function in a cellular context. Here, we describe a new approach using secondary and tertiary structural scaffolds derived from biologically active riboswitches and small ribozymes. Applied to neurotransmitter precursors 5-hydroxytryptophan and 3,4-dihydroxyphenylalanine, this approach yields easily identifiable and characterizable aptamers predisposed for coupling to readout domains to engineer nucleic acid sensory devices that function in vitro and in the cellular context.

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“…Jaffrey and co-workers developed Spinach into the first bifunctional sensor FLAP for S-adenosylmethionine (SAM) and adenosine 5'-diphosphate (ADP), which was genetically encoded in E. coli for real-time detection and imaging of the metabolites. [84,85] In 2015, Spinach2 was fused to cyclic di-AMPbinding riboswitches to visualize levels in L. monocytogenes and to screen for diadenylate cyclase activity of enzymes. [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.…”

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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RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messenger Cyclic di-AMP

Cited by 110 publications

References 24 publications

Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells

Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells

Recurrent RNA motifs as scaffolds for genetically encodable small-molecule biosensors

RNA Structure and Cellular Applications of Fluorescent Light‐Up Aptamers

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