In-Gel Imaging of RNA Processing Using Broccoli Reveals Optimal Aptamer Expression Strategies

Filonov, Grigory S.; Kam, Christina W.; Song, Weiguo; Jaffrey, Samie R.

doi:10.1016/j.chembiol.2015.04.018

Cited by 145 publications

(199 citation statements)

References 32 publications

(53 reference statements)

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“…[25] We transcribed and purified a 102 nt Spinach construct (Table S1), and confirmed that the Spinach RNA yields a strong fluorescence light-up signal (250 fold) with DFHBI in vitro in folding buffer (37 °C, 1 h) (Figure S11a). Comparable results were obtained with F-30 Broccoli aptamers [26] as well (Figure S11b). …”

Section: Control Of Folding Of Rna Aptamerssupporting

confidence: 66%

RNA Cloaking by Reversible Acylation

2018

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We describe a selective and mild chemical approach to controlling RNA hybridization, folding, and enzyme interactions. Reaction of RNAs in aqueous buffer with an azide-substituted acylating agent (100-200 mM) yields several 2′-OH acylations per RNA strand in as little as 10 min. This poly-acylated (“cloaked”) RNA is strongly blocked from hybridization with complementary nucleic acids, from cleavage by RNA-processing enzymes, and from folding into active aptamer structures. Importantly, treatment with a water-soluble phosphine triggers a Staudinger reduction of the azide groups, resulting in spontaneous loss of acyl groups (“uncloaking”). This fully restores RNA folding and biochemical activity.

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Section: Control Of Folding Of Rna Aptamerssupporting

confidence: 66%

RNA Cloaking by Reversible Acylation

2018

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“…5a). To validate that RNA was being generated and to measure the kinetics of AR activation, we first deployed a fluorescent aptamer (F30-2xdBroccoli) 49 as the goi output of the rapa-T7 vector, allowing the visualization of RNA synthesis using fluorescence microscopy. Treatment of HEK293T cells transfected with the rapa-T7-F30-2xdBroccoli vector with 100 nM rapamycin for 30 min results in a robust enhancement in intracellular fluorescence (Fig.…”

Section: Resultsmentioning

confidence: 99%

Evolution of a split RNA polymerase as a versatile biosensor platform

2017

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Biosensors that transduce target chemical and biochemical inputs into genetic outputs are essential for bioengineering and synthetic biology. Current biosensor design strategies often suffer from a lack of signal-to-noise, requirements for extensive optimization for each new input, and poor performance in mammalian cells. Here we report the development of a proximity-dependent split RNA polymerase (RNAP) as a general platform for biosensor engineering. After discovering that interactions between fused proteins modulate the assembly of a split T7 RNAP, we optimized the split RNAP components for protein-protein interaction detection by phage-assisted continuous evolution (PACE). We then applied the resulting “activity-responsive RNAP” (AR) system to create light and small molecule activated biosensors, demonstrating the “plug-and-play” nature of the platform. Finally, we validated that ARs can interrogate multidimensional protein-protein interactions and trigger RNA nanostructure production, protein synthesis, and gene knockdown in mammalian systems, illustrating the versatility of ARs in synthetic biology applications.

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“…This approach to sensor design could be greatly improved by a number of anticipated advancements in RNA imaging (Filonov, Kam, Song, & Jaffrey, 2015) and new types of metabolite-recognizing riboswitches (Lynch & Gallivan, 2009; Weigand & Suess, 2007). By exploiting the diversity of naturally occurring riboswitches, it should be possible to image a large number of intracellular metabolites.…”

Section: Summary and Concluding Remarksmentioning

confidence: 99%

Developing Fluorogenic Riboswitches for Imaging Metabolite Concentration Dynamics in Bacterial Cells

Litke¹,

You²,

Jaffrey³

2016

Visualizing RNA Dynamics in the Cell

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Genetically encoded small molecule sensors are important tools for revealing the dynamics of metabolites and other small molecules in live cells over time. We recently developed RNA-based sensors that exhibit fluorescence in proportion to a small molecule ligand. One class of these RNA-based sensors are termed Spinach riboswitches. These are RNAs that are based on naturally occurring riboswitches, but have been fused to the Spinach aptamer. The resulting RNA is a fluorogenic riboswitch, producing fluorescence upon binding the cognate small molecule analyte. Here we describe how to design and optimize these sensors by adjusting critical sequence elements, guided by structural insights from the Spinach aptamer. We provide a step-wise procedure to characterize sensors in vitro and to express sensors in bacteria for live-cell imaging of metabolites. Spinach riboswitch sensors offer a simple method for fluorescence measurement of a wide range of metabolites for which riboswitches exist, including nucleotides and their derivatives, amino acids, cations, and anions.

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In-Gel Imaging of RNA Processing Using Broccoli Reveals Optimal Aptamer Expression Strategies

Cited by 145 publications

References 32 publications

RNA Cloaking by Reversible Acylation

RNA Cloaking by Reversible Acylation

Evolution of a split RNA polymerase as a versatile biosensor platform

Developing Fluorogenic Riboswitches for Imaging Metabolite Concentration Dynamics in Bacterial Cells

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