Integrating Lineage Tracing and Single-Cell Analysis

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Integrating Lineage Tracing and Single-Cell Analysis

research highlights SENSORS AND PROBES Pinpointing RNA mutations Riboregulator-based sensors are designed to diferentiate single-nucleotide RNA variants in live cells. ingle-nucleotide variations within hybridization occurs between a pair of The researchers hope that SNIPRs research highlights RNA molecules have critical roles in perfectly matched sequences, the SNIPR will enable detection of drug-resistance Scell biology and disease progression. activates the translation of the output mutations for diseases such as malaria, Yet, point mutations remain a challenge to protein. In contrast, when a mutant tuberculosis and HIV, and improve identify, particularly in live cells and cell- target is present, the free-energy penalty understanding of the evolution of drug free systems at physiological temperatures. induced by the resulting sequence resistance. Green also remarks that “SNIPRs The Green and Yan groups at Arizona State mismatch prevents SNIPR activation, have been engineered to work with the University have described riboregulator- thus stopping expression of the translational machinery of prokaryotes, so based sensors, termed single-nucleotide- output protein. they cannot currently be used in eukaryotic specific programmable riboregulators Beyond detection of sequence variants, cells or eukaryotic cell-free systems, but we (SNIPRs), to detect transcript point SNIPRs also allow for detection of do hope to develop such tools in the future.” variations in live prokaryotic cells and cell- chemically modified bases that can induce free reactions. conformational changes in SNIPRs. Lei Tang Green comments that that “they Moreover, SNIPRs are compatible with [riboregulators] are genetically encoded, not only live cells but also paper-based Published online: 2 April 2020 and they can be readily programmed to colorimetric assays that can be used for https://doi.org/10.1038/s41592-020-0805-0 detect different target RNAs.” SNIPRs the identification of virus strains. To make Research paper are engineered to retain a docking site SNIPRs easy to use, Green and Yan have also Hong, F. et al. Precise and programmable detection of that binds the target RNA and forms a developed an algorithm for designing mutations using ultraspecifc riboregulators. Cell 180, target–SNIPR duplex. When target–SNIPR SNIPR sequences. 1018–1032 (2020). We Create Solutions Ultra Precise Motion Control - D.C. Servo APPLIED SCIENTIFIC motors down to 20 nm, piezos down INSTRUMENTATION to 1 nm, and low drift XYZ stages. Microscopy - Automation, modular microscopes, autofocus complete light www.asiimaging.com sheet systems, and components. [email protected] (800) 706-2284 OEM - Custom designed systems (541) 461-8181 to user specifications. NATURE METHODS | VOL 17 | APRIL 2020 | 359–365 | www.nature.com/naturemethods 365.

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Engineering a Circular Riboregulator in Escherichia Coli
bioRxiv preprint doi: https://doi.org/10.1101/008987; this version posted September 25, 2014. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Engineering a circular riboregulator in Escherichia coli William Rostain1,2, Shensi Shen2, Teresa Cordero1, Guillermo Rodrigo2,3 and Alfonso Jaramillo1,2,* 1 School of Life Sciences, University of Warwick, Coventry, CV4 7AL, United Kingdom. 2 Institute of Systems and Synthetic Biology, CNRS - Université d’Evry val d’Essonne, 91000 Évry, France. 3 Instituto de Biologia Molecular y Celular de Plantas, CSIC – Universidad Politécnica de Valencia, 46022 Valencia, Spain. * Corresponding author. School of Life Sciences, University of Warwick. Gibbet Hill Road, Coventry, CV4 7AL, United Kingdom. Tel: +44 (0)24 765 73432, E-mail: [email protected] Type: Letter Running title: Circular Riboregulator. Keywords: Biotechnology, Riboregulators, Splicing, Synthetic Biology. 1 bioRxiv preprint doi: https://doi.org/10.1101/008987; this version posted September 25, 2014. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Circular RNAs have recently been shown to be important gene expression regulators in mammalian cells. However, their role in prokaryotes remains elusive. Here, we engineered a synthetic riboregulator that self-splice to produce a circular molecule, exploiting group I permuted intron-exon (PIE) sequences. We demonstrated that the resulting circular riboregulator can activate gene expression, showing increased dynamic range compared to the linear form. We characterized the system with a fluorescent reporter and with an antibiotic resistance marker.
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