“Second-generation” fluorogenic RNA-based sensors

Mudiyanselage, Aruni P. K. K. Karunanayake; Wu, Rigumula; Leon-Duque, Mark A; Ren, Kewei; You, Mingxu

doi:10.1016/j.ymeth.2019.01.008

Cited by 27 publications

(20 citation statements)

References 121 publications

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“…All of these modified probes offer modularity and specificity, but they suffer from limited sensitivity, and have not been very successful in eukaryotic RNA imaging (Karunanayake Mudiyanselage, Wu, Leon‐Duque, Ren, & You, 2019). Chemical probes and sensors have also recently been developed that utilize a similar concept to image endogenous untagged RNAs using aptamers as quenchers (e.g., utilizing black hole quenchers; Yatsuzuka et al, 2018).…”

Section: Key Methodologies For Single‐molecule Rna Imagingmentioning

confidence: 99%

Methods for spatial and temporal imaging of the different steps involved in RNA processing at single‐molecule resolution

2020

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RNA plays a quintessential role as a messenger of information from genotype (DNA) to phenotype (proteins), as well as acts as a regulatory molecule (noncoding RNAs). All steps in the journey of RNA from synthesis (transcription), splicing, transport, localization, translation, to its eventual degradation, comprise important steps in gene expression, thereby controlling the fate of the cell. This lifecycle refers to the majority of RNAs (primarily mRNAs), but not other RNAs such as tRNAs. Imaging these processes in fixed cells and in live cells has been an important tool in developing an understanding of the regulatory steps in RNAs journey. Singlecell and single-molecule imaging techniques enable a much deeper understanding of cellular biology, which is not possible with bulk studies involving RNA isolated from a large pool of cells. Classic techniques, such as fluorescence in situ hybridization (FISH), as well as more recent aptamer-based approaches, have provided detailed insights into RNA localization, and have helped to predict the functions carried out by many RNA species. However, there are still certain processing steps that await high-resolution imaging, which is an exciting and upcoming area of research. In this review, we will discuss the methods that have revolutionized single-molecule resolution imaging in general, the steps of RNA processing in which these methods have been used, and new emerging technologies. This article is categorized under: RNA Export and Localization > RNA Localization RNA Methods > RNA Analyses in Cells RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions K E Y W O R D S RNA imaging, RNA processing, single-molecule FISH, splicing 1 | INTRODUCTION The synthesis of RNA is tightly regulated at multiple levels to ensure correct gene expression. In eukaryotes, pre-RNA molecules are transcribed from the parent DNA strand by RNA polymerase in the nucleus. For mRNAs, the pre-mRNA is modified by the addition of determined 5 0-G cap and a 3 0-poly-A tail, and introns are removed via splicing to produce a mature RNA

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Section: Key Methodologies For Single‐molecule Rna Imagingmentioning

confidence: 99%

Methods for spatial and temporal imaging of the different steps involved in RNA processing at single‐molecule resolution

2020

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“…Alternative design strategies have been proposed using natural riboswitches and circularly permutated molecules. However, since these sensors and their development are out of the scope of this chapter, the reader is redirected to some excellent recent reviews on the topic [127][128][129]. While for a long time the use of these sensors in mammalian cells was challenged by the short half-life of small RNAs in these cells, the recent development of the Tornado technology allows to express these sensors as highly stable circular RNAs [68].…”

Section: Detection Of Other Biological Molecules and Ionsmentioning

confidence: 99%

Development and Applications of Fluorogen/Light-Up RNA Aptamer Pairs for RNA Detection and More

Ryckelynck

2020

Methods in Molecular Biology

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The central role of RNA in living systems made it highly desirable to have non-invasive and sensitive technologies allowing for imaging the synthesis and the location of these molecules in living cells. This need motivated the development of small pro-e ce ec e ca ed e a bec e e ce b d e e ca e c dab e RNA ca ed-a a e. Ye , e development of these fluorogen/light-up RNA pairs is a long and thorough process starting with the careful design of the fluorogen and pursued by the selection of a specific and efficient synthetic aptamer. This chapter summarizes the main design and the selection strategies used up to now prior to introducing the main pairs. Then, the vast application potential of these molecules for live-cell RNA imaging and other applications will be presented and discussed.

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“…Table 1 summarizes the commonly used fluorogenic RNA complexes that can be used as potential reporters for the sensor development [ 89 ]. Right now, these fluorogenic RNA complexes are still far less versatile than the existing fluorescent protein toolbox.…”

Section: Reporting Systems In Germsmentioning

confidence: 99%

Intracellular Imaging with Genetically Encoded RNA-Based Molecular Sensors

et al. 2019

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Genetically encodable sensors have been widely used in the detection of intracellular molecules ranging from metal ions and metabolites to nucleic acids and proteins. These biosensors are capable of monitoring in real-time the cellular levels, locations, and cell-to-cell variations of the target compounds in living systems. Traditionally, the majority of these sensors have been developed based on fluorescent proteins. As an exciting alternative, genetically encoded RNA-based molecular sensors (GERMS) have emerged over the past few years for the intracellular imaging and detection of various biological targets. In view of their ability for the general detection of a wide range of target analytes, and the modular and simple design principle, GERMS are becoming a popular choice for intracellular analysis. In this review, we summarize different design principles of GERMS based on various RNA recognition modules, transducer modules, and reporting systems. Some recent advances in the application of GERMS for intracellular imaging are also discussed. With further improvement in biostability, sensitivity, and robustness, GERMS can potentially be widely used in cell biology and biotechnology.

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“Second-generation” fluorogenic RNA-based sensors

Cited by 27 publications

References 121 publications

Methods for spatial and temporal imaging of the different steps involved in RNA processing at single‐molecule resolution

Methods for spatial and temporal imaging of the different steps involved in RNA processing at single‐molecule resolution

Development and Applications of Fluorogen/Light-Up RNA Aptamer Pairs for RNA Detection and More

Intracellular Imaging with Genetically Encoded RNA-Based Molecular Sensors

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