Monitoring and visualizing microRNA dynamics during live cell differentiation using microRNA-responsive non-viral reporter vectors

Nakanishi, Hiizu; Miki, Kenji; Komatsu, Kaoru R.; Umeda, Mikio; Mochizuki, Megumi; Inagaki, Azusa; Yoshida, Yoshinori; Saito, Hirohide

doi:10.1016/j.biomaterials.2017.02.033

Cited by 25 publications

(13 citation statements)

References 49 publications

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“…To achieve cellular state-dependent translational activation and repression in RNA circuits, we focused on miRNA-responsive mRNAs that we had previously used to sort or visualize specific cell types 21,26,[32][33][34] . Thus, we designed CaVT mRNA that contains a complementary sequence to miR-21-5p or miR-302a-5p, two miRNAs highly expressed in HeLa and human iPS cells (hiPSCs, 201B7 strain), respectively.…”

Section: And 5)mentioning

confidence: 99%

Caliciviral protein-based artificial translational activator for mammalian gene circuits with RNA-only delivery

Nakanishi

Saito

2020

Nat Commun

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Synthetic RNA-based gene circuits enable sophisticated gene regulation without the risk of insertional mutagenesis. While various RNA binding proteins have been used for translational repression in gene circuits, the direct translational activation of synthetic mRNAs has not been achieved. Here we develop Caliciviral VPg-based Translational activator (CaVT), which activates the translation of synthetic mRNAs without the canonical 5′-cap. The level of translation can be modulated by changing the locations, sequences, and modified nucleosides of CaVT-binding motifs in the target mRNAs, enabling the simultaneous translational activation and repression of different mRNAs with RNA-only delivery. We demonstrate the efficient regulation of apoptosis and genome editing by tuning translation levels with CaVT. In addition, we design programmable CaVT that responds to endogenous microRNAs or small molecules, achieving both cell-state-specific and conditional translational activation from synthetic mRNAs. CaVT will become an important tool in synthetic biology for both biological studies and future therapeutic applications.

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Section: And 5)mentioning

confidence: 99%

Caliciviral protein-based artificial translational activator for mammalian gene circuits with RNA-only delivery

Nakanishi

Saito

2020

Nat Commun

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“…The use of these materials could enhance the capability of our devices to separate cells. Alternatively, our devices could be encoded into the genome to ensure their uptake by the cells (31). In this case, the devices continuously detect changes in the cell during a certain process (e.g., differentiation) rather than taking a snapshot of the moment.…”

Section: Discussionmentioning

confidence: 99%

Numerical operations in living cells by programmable RNA devices

2019

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Integrated bioengineering systems can make executable decisions according to the cell state. To sense the state, multiple biomarkers are detected and processed via logic gates with synthetic biological devices. However, numerical operations have not been achieved. Here, we show a design principle for messenger RNA (mRNA) devices that recapitulates intracellular information by multivariate calculations in single living cells. On the basis of this principle and the collected profiles of multiple microRNA activities, we demonstrate that rationally programmed mRNA sets classify living human cells and track their change during differentiation. Our mRNA devices automatically perform multivariate calculation and function as a decision-maker in response to dynamic intracellular changes in living cells.

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“…This protocol might allow mature hiPSCs to be easily discerned from partially reprogrammed colonies and might enable the preparation of hiPSCs potentially applicable for medical applications. Recently, a non-integrating episomal vector was exploited to construct a fluorescence-based imaging system to evaluate miRNA activity 46 . Although this system allowed long-term monitoring of miR-302a-5p expression during hiPSC differentiation, episomal vectors may be rapidly cleared from highly proliferating cells 46 .…”

Section: Discussionmentioning

confidence: 99%

“…Recently, a non-integrating episomal vector was exploited to construct a fluorescence-based imaging system to evaluate miRNA activity 46 . Although this system allowed long-term monitoring of miR-302a-5p expression during hiPSC differentiation, episomal vectors may be rapidly cleared from highly proliferating cells 46 . By contrast, the replication of the SeVdp genome is remarkably stable, even in hiPSCs (Supplementary Fig.…”

Section: Discussionmentioning

confidence: 99%

Sensitive and long-term monitoring of intracellular microRNAs using a non-integrating cytoplasmic RNA vector

Sano

Ohtaka

Iijima

et al. 2017

Sci Rep

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MicroRNAs (miRNAs) are small noncoding RNAs that modulate gene expression at the post-transcriptional level. Different types of cells express unique sets of miRNAs that can be exploited as potential molecular markers to identify specific cell types. Among the variety of miRNA detection methods, a fluorescence-based imaging system that utilises a fluorescent-reporter gene regulated by a target miRNA offers a major advantage for long-term tracking of the miRNA in living cells. In this study, we developed a novel fluorescence-based miRNA-monitoring system using a non-integrating cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp). Because SeVdp vectors robustly and stably express transgenes, this system enabled sensitive monitoring of miRNAs by fluorescence microscopy. By applying this system for cellular reprogramming, we found that miR-124, but not miR-9, was significantly upregulated during direct neuronal conversion. Additionally, we were able to isolate integration-free human induced pluripotent stem cells by long-term tracking of let-7 expression. Notably, this system was easily expandable to allow detection of multiple miRNAs separately and simultaneously. Our findings provide insight into a powerful tool for evaluating miRNA expression during the cellular reprogramming process and for isolating reprogrammed cells potentially useful for medical applications.

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Monitoring and visualizing microRNA dynamics during live cell differentiation using microRNA-responsive non-viral reporter vectors

Cited by 25 publications

References 49 publications

Caliciviral protein-based artificial translational activator for mammalian gene circuits with RNA-only delivery

Caliciviral protein-based artificial translational activator for mammalian gene circuits with RNA-only delivery

Numerical operations in living cells by programmable RNA devices

Sensitive and long-term monitoring of intracellular microRNAs using a non-integrating cytoplasmic RNA vector

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