Spinach-based fluorescent light-up biosensors for multiplexed and label-free detection of microRNAs

Zhao, Ying; Tu, Bin; Liu, Lan; Tang, Hao; Tang, Li‐Juan; Jiang, Jian‐Hui

doi:10.1039/c8cc00123e

Cited by 54 publications

(32 citation statements)

References 34 publications

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“…There are two potential explanations for the high analytical sensitivity of SENSR. First, concentrations of some components increased in the optimized SENSR condition compared with other studies that utilized SplintR ligase and fluorogenic aptamer for RNA detection 17,18 , improving the efficiencies of individual reaction steps (Supplementary Table 5). Second, the one-pot reaction scheme generated cooperation between ligation and transcription.…”

Section: Rapid and Sensitive Rna Detection By Sensrmentioning

confidence: 98%

“…Because of their specificity, the ligation-dependent methods are used for the detection of markers of genetic disorders 13,14 and pathogens 15,16 , typically combined with subsequent amplification and signal generation methods. In particular, the SplintR ligase can efficiently ligate two DNA probes using a target single-stranded RNA as a splint, enabling the sequence-specific detection of RNA molecules 17,18 . Because the reaction components of the ligation-dependent methods are relatively simple, we hypothesized that the ligation-dependent method could be exploited to establish a one-pot RNA detection method when combined with compatible amplification and signal generation methods in a single reaction mixture.…”

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confidence: 99%

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Sensitive fluorescence detection of SARS-CoV-2 RNA in clinical samples via one-pot isothermal ligation and transcription

et al. 2020

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The control of viral outbreaks requires nucleic acid diagnostic tests that are sensitive, simple and fast. Here, we report a highly sensitive and specific one-pot assay for the fluorescence-based detection of RNA from pathogens. The assay, which can be performed within 30-50 min of incubation time and can reach a limit of detection of 0.1-attomolar RNA concentration, relies on a sustained isothermal reaction cascade producing an RNA aptamer that binds to a fluorogenic dye. The RNA aptamer is transcribed by the T7 RNA polymerase from the ligation product of a promoter DNA probe and a reporter DNA probe that hybridize with the target single-stranded RNA sequence via the SplintR ligase (a Chlorella virus DNA ligase). In 40 nasopharyngeal SARS-CoV-2 samples, the assay reached positive and negative predictive values of 95 and 100%, respectively. We also show that the assay can rapidly detect a range of viral and bacterial RNAs.

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Section: Rapid and Sensitive Rna Detection By Sensrmentioning

confidence: 98%

mentioning

confidence: 99%

Sensitive fluorescence detection of SARS-CoV-2 RNA in clinical samples via one-pot isothermal ligation and transcription

et al. 2020

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“…Numerous fluorogenic aptamers have been explored, but only a few have actually made it into cells since FLAPs were initially designed for in vitro applications . Robust expression, correct folding, sufficient brightness, and low photobleaching are only a few FLAP properties that determine their applicability in cells .…”

Section: Cellular Applications Of Flapsmentioning

confidence: 99%

RNA Structure and Cellular Applications of Fluorescent Light‐Up Aptamers

Neubacher

Hennig

2018

Angew Chem Int Ed

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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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“…[72][73][74] Robust expression, correct folding,sufficient brightness,and low photobleaching are only af ew FLAP properties that determine their applicability in cells. [72][73][74] Robust expression, correct folding,sufficient brightness,and low photobleaching are only af ew FLAP properties that determine their applicability in cells.…”

Section: Cellular Applications Of Flapsmentioning

confidence: 99%

“…Numerous fluorogenic aptamers have been explored, but only afew have actually made it into cells since FLAPs were initially designed for in vitro applications. [72][73][74] Robust expression, correct folding,sufficient brightness,and low photobleaching are only af ew FLAP properties that determine their applicability in cells. [63] Usually,newly developed FLAPs intended for cellular applications are tested in live cells (bacteria or cultured mammalian cells) either in isolation or as atag to an RNAofinterest (FLAP-tag, Figure 9A).…”

Section: Cellular Applications Of Flapsmentioning

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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Spinach-based fluorescent light-up biosensors for multiplexed and label-free detection of microRNAs

Abstract: A novel Spinach-based fluorescent light-up biosensor utilizing the T7 in vitro transcription process to generate unmodified Spinach sequences for multiplexed microRNA detection has been developed.

Cited by 54 publications

References 34 publications

Sensitive fluorescence detection of SARS-CoV-2 RNA in clinical samples via one-pot isothermal ligation and transcription

Sensitive fluorescence detection of SARS-CoV-2 RNA in clinical samples via one-pot isothermal ligation and transcription

RNA Structure and Cellular Applications of Fluorescent Light‐Up Aptamers

RNA Structure and Cellular Applications of Fluorescent Light‐Up Aptamers

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