Imaging Metabolite Dynamics in Living Cells Using a Spinach-Based

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Imaging Metabolite Dynamics in Living Cells Using a Spinach-Based Imaging metabolite dynamics in living cells using a PNAS PLUS Spinach-based riboswitch Mingxu You1, Jacob L. Litke1, and Samie R. Jaffrey2 Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10065 Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved April 24, 2015 (received for review March 6, 2015) Riboswitches are natural ligand-sensing RNAs typically that are 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) (6, 7). found in the 5′ UTRs of mRNA. Numerous classes of riboswitches Similar to the manner by which GFP induces fluorescence in HBI, have been discovered, enabling mRNA to be regulated by diverse Spinach binds to the otherwise nonfluorescent DFHBI and and physiologically important cellular metabolites and small mol- switches it to a highly fluorescent state. This property has allowed ecules. Here we describe Spinach riboswitches, a new class of ge- Spinach to be used to tag and image specific RNAs in living cells (6). netically encoded metabolite sensor derived from naturally occur- We recently converted the constitutively fluorescent Spinach– ring riboswitches. Drawing upon the structural switching mechanism DFHBI complex into sensors that fluoresce in proportion to the of natural riboswitches, we show that Spinach can be swapped for concentration of specific metabolites (5). These sensors comprise the expression platform of various riboswitches, allowing metabo- two domains: Spinach and a target-binding aptamer. The target- lite binding to induce Spinach fluorescence directly. In the case of the binding aptamer is inserted into a structurally critical stem of thiamine 5′-pyrophosphate (TPP) riboswitch from the Escherichia Spinach (5). Because the target-binding aptamer is unstructured coli thiM gene encoding hydroxyethylthiazole kinase, we show without the target molecule, the critical stem is disrupted, pre- that insertion of Spinach results in an RNA sensor that exhibits venting Spinach from folding and binding DFHBI. However, when fluorescence upon binding TPP. This TPP Spinach riboswitch binds the aptamer binds its target, the aptamer folds, enabling the folding TPP with affinity and selectivity similar to that of the endogenous of the critical stem in Spinach (Fig. S1A). This folding leads to riboswitch and enables the discovery of agonists and antagonists Spinach fluorescence that can be detected in vitro or in cells. Using of the TPP riboswitch using simple fluorescence readouts. Further- this approach, we created sensors by fusing Spinach to aptamers more, expression of the TPP Spinach riboswitch in Escherichia coli that bind S-adenosyl-methionine (SAM), ADP, and other metab- BIOCHEMISTRY enables live imaging of dynamic changes in intracellular TPP con- olites (5). These sensors allowed imaging of the dynamics and centrations in individual cells. Additionally, we show that other turnover of SAM and ADP in live bacterial cells (5). This approach riboswitches that use a structural mechanism similar to that of the was applied subsequently to detect and image cyclic di-GMP and TPP riboswitch, including the guanine and adenine riboswitches from various proteins in living cells (8, 9). These allosteric Spinach-based the Bacillus subtilis xpt gene encoding xanthine phosphoribosyl- sensors demonstrate the versatility of RNA as a platform for cre- transferase, and the S-adenosyl-methionine-I riboswitch from the ating genetically encoded sensors for imaging cellular metabolites. B. subtilis yitJ gene encoding methionine synthase, can be converted There are important technical challenges that must be ad- into Spinach riboswitches. Thus, Spinach riboswitches constitute a dressed when creating allosteric Spinach-based sensors. These novel class of RNA-based fluorescent metabolite sensors that exploit sensors rely on metabolite-binding aptamers, which typically the diversity of naturally occurring ligand-binding riboswitches. are generated using approaches such as systematic evolution of ligands by exponential enrichment (SELEX) (10, 11). SELEX- RNA probes | cellular imaging | riboswitch | fluorescence | metabolite generated aptamers typically exhibit poor selectivity and af- finity that are not desirable for sensors. Selectivity is important mong the most important tools for studying the dynamic Achanges in the intracellular level of signaling molecules and Significance metabolites in living cells are genetically encoded fluorescent sensors. Most sensors comprise two fluorescent proteins flanking Developing sensors to image cellular metabolites and signal- a central target-binding domain (1, 2). Binding of the target mol- ing molecules in living cells is challenging. Here we describe ecule induces conformational changes that reposition the fluo- Spinach riboswitches, a novel class of genetically encoded me- rescent proteins. This repositioning results in changes in FRET tabolite sensor based on riboswitches, a group of naturally oc- between the fluorescent proteins, which can be imaged by mi- curring ligand-binding RNAs. Spinach riboswitches use Spinach, croscopy in living cells (3). These sensors have provided fun- an RNA aptamer that binds and activates the fluorescence of an damentally novel insights into the dynamic changes in calcium, otherwise nonfluorescent small-molecule fluorophore. Drawing cyclic nucleotides, glucose, and other molecules during cellular upon structural insights into the mechanism of structural switch- signaling and disease states (4). ing in riboswitches, we show that Spinach can be swapped into Although genetically encoded sensors are powerful tools for various riboswitches, allowing metabolite binding to induce imaging cellular metabolites, only a small number of sensors have Spinach fluorescence directly. Expression of Spinach riboswitches been created because these sensors require a protein that binds in cells allows metabolite levels to be imaged in real time in live the target molecule of interest and which undergoes a target- bacterial cells. Spinach riboswitches thus provide a novel ap- induced conformational change that is sufficient to induce a proach to image cellular metabolites in living cells. change in FRET. However, proteins with these properties are not readily available for most physiologically important metabolites. Author contributions: M.Y. and S.R.J. designed research; M.Y. and J.L.L. performed re- As a result, sensors have not been generated for many important search; M.Y. and J.L.L. analyzed data; and M.Y., J.L.L., and S.R.J. wrote the paper. metabolites and signaling molecules. The authors declare no conflict of interest. Recently we described an alternative class of genetically encoded This article is a PNAS Direct Submission. sensors composed of RNA (5). These sensors are based on Spin- 1M.Y. and J.L.L. contributed equally to this work. ach, an “RNA mimic of green fluorescent protein” (6). Spinach is 2To whom correspondence should be addressed. Email: [email protected]. an RNA aptamer that binds fluorophores resembling the hydroxy- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. benzylidene imidazolinone (HBI) fluorophore in GFP, including 1073/pnas.1504354112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1504354112 PNAS Early Edition | 1of10 Downloaded by guest on October 8, 2021 because the cellular environment is highly complex and can sequence in the TPP-binding domain. However, TPP binding to contain unknown metabolites with structures similar to that of the riboswitch displaces the transducer sequence, allowing the the desired target molecule. In principle, aptamer selectivity transducer sequence to form one of the strands of the expression can be achieved using negative selection during SELEX (12); platform helix (Fig. 1A). As a result of helix formation, the ri- however, it is difficult to perform negative selection against all bosome’s access to the Shine–Dalgarno (SD) element is blocked, the diverse, structurally related metabolites in the cell, espe- thereby inhibiting translation (25–28). cially if some of them are unknown. Spinach has a structure similar to that of the helical expression Aptamer affinity also is critical for sensor function (13). If the platform of the TPP riboswitch. The fluorophore-binding domain aptamer has too high affinity for the target, the target will be fully of Spinach comprises a coaxial helical stack composed of two A- bound to the aptamer regardless of how its concentration changes form helices separated by an atypical G-quadruplex motif (29, 30). in cells. If the affinity is too low, very little target will be bound, The DFHBI-binding pocket is formed by the junction between the resulting in minimal fluorescence activation in cells. In most cases, upper helix (H1) and the G-quadruplex (29, 30). The helical aptamer binding affinities that are similar to the cellular metabolite structure is necessary to stabilize a U·A·Ubasetriplethatservesas concentration will produce sensors exhibiting fluorescence that the upper surface of the DFHBI-binding pocket (Fig. 1C). Because corresponds directly to the metabolite concentration (13). How- the helical structure is required for DFHBI binding to Spinach, we ever, it is difficult to obtain aptamers with a predetermined affinity reasoned the TPP riboswitch could regulate Spinach fluorescence if for a target molecule using SELEX (14). Thus, obtaining aptamers the transducer sequence controls the formation of the H1 helix. that have a suitable target-binding affinity
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