Protein Scaffold-Activated Protein Trans-Splicing in Mammalian Cells

Selgrade, Daniel; Lohmueller, Jason; Lienert, Florian; Silver, Pamela A.

doi:10.1021/ja401689b

Cited by 31 publications

(31 citation statements)

References 34 publications

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“…33 These rapamycin-binding domains have been used for many applications including ligand-induced protein splicing 34−36 and regulation of gene expression. 37−39 The two domains do not interact in the absence of rapamycin, and upon the addition of rapamycin, a stable tertiary complex forms with K d ≈ 2.5 nM.…”

Section: Resultsmentioning

confidence: 99%

Modular Extracellular Sensor Architecture for Engineering Mammalian Cell-based Devices

et al. 2014

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Engineering mammalian cell-based devices that monitor and therapeutically modulate human physiology is a promising and emerging frontier in clinical synthetic biology. However, realizing this vision will require new technologies enabling engineered circuitry to sense and respond to physiologically relevant cues. No existing technology enables an engineered cell to sense exclusively extracellular ligands, including proteins and pathogens, without relying upon native cellular receptors or signal transduction pathways that may be subject to crosstalk with native cellular components. To address this need, we here report a technology we term a Modular Extracellular Sensor Architecture (MESA). This self-contained receptor and signal transduction platform is maximally orthogonal to native cellular processes and comprises independent, tunable protein modules that enable performance optimization and straightforward engineering of novel MESA that recognize novel ligands. We demonstrate ligand-inducible activation of MESA signaling, optimization of receptor performance using design-based approaches, and generation of MESA biosensors that produce outputs in the form of either transcriptional regulation or transcription-independent reconstitution of enzymatic activity. This systematic, quantitative platform characterization provides a framework for engineering MESA to recognize novel ligands and for integrating these sensors into diverse mammalian synthetic biology applications.

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Section: Resultsmentioning

confidence: 99%

Modular Extracellular Sensor Architecture for Engineering Mammalian Cell-based Devices

et al. 2014

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“…S2 and see SI Appendix, Supplementary Results) and thus designed a variation of this experiment that could hone in on the translation process itself. We used split firefly luciferase fused to split inteins (27)(28)(29) (Fig. 5A), which relies on protein splicing to produce a functional luciferase protein after the two halves are brought together by Pum binding to mRNA.…”

Section: Significancementioning

confidence: 99%

Programmable RNA-binding protein composed of repeats of a single modular unit

Adamala

Martin-Alarcon

Boyden

2016

Proc. Natl. Acad. Sci. U.S.A.

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The ability to monitor and perturb RNAs in living cells would benefit greatly from a modular protein architecture that targets unmodified RNA sequences in a programmable way. We report that the RNA-binding protein PumHD (Pumilio homology domain), which has been widely used in native and modified form for targeting RNA, can be engineered to yield a set of four canonical protein modules, each of which targets one RNA base. These modules (which we call Pumby, for Pumilio-based assembly) can be concatenated in chains of varying composition and length, to bind desired target RNAs. The specificity of such Pumby-RNA interactions was high, with undetectable binding of a Pumby chain to RNA sequences that bear three or more mismatches from the target sequence. We validate that the Pumby architecture can perform RNA-directed protein assembly and enhancement of translation of RNAs. We further demonstrate a new use of such RNA-binding proteins, measurement of RNA translation in living cells. Pumby may prove useful for many applications in the measurement, manipulation, and biotechnological utilization of unmodified RNAs in intact cells and systems.

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“…Inteins and split ( trans -acting) inteins that are activated in response to small molecules, light and protein inputs have been generated, enabling the post-translational control of circuits 65–67 . There are also several systems available that use light to induce protein-protein interactions in a reversible manner.…”

Section: Tools and Basic Circuitsmentioning

confidence: 99%

Synthetic biology in mammalian cells: next generation research tools and therapeutics

Lienert

Lohmueller

Garg

et al. 2014

Nat Rev Mol Cell Biol

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253

216

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Recent progress in DNA manipulation and gene circuit engineering has greatly improved our ability to programme and probe mammalian cell behaviour. These advances have led to a new generation of synthetic biology research tools and potential therapeutic applications. Programmable DNA-binding domains and RNA regulators are leading to unprecedented control of gene expression and elucidation of gene function. Rebuilding complex biological circuits such as T cell receptor signalling in isolation from their natural context has deepened our understanding of network motifs and signalling pathways. Synthetic biology is also leading to innovative therapeutic interventions based on cell-based therapies, protein drugs, vaccines and gene therapies.

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Protein Scaffold-Activated Protein Trans-Splicing in Mammalian Cells

Cited by 31 publications

References 34 publications

Modular Extracellular Sensor Architecture for Engineering Mammalian Cell-based Devices

Modular Extracellular Sensor Architecture for Engineering Mammalian Cell-based Devices

Programmable RNA-binding protein composed of repeats of a single modular unit

Synthetic biology in mammalian cells: next generation research tools and therapeutics

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