Construction of integrated gene logic-chip

Masubuchi, Takeya; Endo, Masayuki; Iizuka, Ryo; Iguchi, Ayaka; Yoon, Dong Hyun; Sekiguchi, Tetsushi; Qi, Hao; Iinuma, Ryosuke; Miyazono, Yuya; Shoji, Shuichi; Funatsu, Takashi; Sugiyama, Hiroshi; Harada, Yoshie; Ueda, Takuya; Tadakuma, Hisashi

doi:10.1038/s41565-018-0202-3

Cited by 44 publications

(35 citation statements)

References 39 publications

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“…The DNA origami technique has found broad applicability as an experimental tool for spatial organization of native multi-protein systems, such as amyloid fibrils 21 , membrane fusion proteins 22 , nucleosomes 23,24 , and intrinsically disordered proteins 25 . Additionally, these platforms have been used to engineer localized genetic circuits 26 , to study confinement-induced enzyme activity 27 , and to investigate scaffolded metabolic cascades [28][29][30][31] . Although these studies have elegantly applied the programmability of DNA nanotechnology to facilitate organization of e.g.…”

Section: Introductionmentioning

confidence: 99%

A DNA-based synthetic apoptosome

Bj¹,

Aj²,

Gumí-Audenis³

et al. 2019

Preprint

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Living cells are able to regulate key cellular processes by physically assembling signaling components on dedicated molecular platforms. The spatial organization of proteins in these higher-order signaling complexes facilitates proximity-driven activation and inhibition events, allowing tight regulation of the flow of information. Here, we employ the programmability and modularity of DNA origami as a controllable molecular platform for studying protein-protein interactions involved in intracellular signaling. Specifically, we engineer a synthetic, DNA origami-based version of the apoptosome, a large multi-protein signaling complex that regulates apoptosis by co-localization of multiple caspase-9 monomers. Our in vitro characterization using both wildtype caspase-9 monomers and inactive mutants tethered to a DNA origami platform directly demonstrates that enzymatic activity is induced by proximity-driven dimerization with asymmetric, half-of-sites reactivity. Additionally, experimental results supported by a detailed thermodynamic model reveal a multivalent activity enhancement in tethered caspase-9 oligomers of three and four enzymes, partly originating from a statistical increase in the number of active catalytic units in higherorder enzyme clusters. Our results offer fundamental insights in caspase-9 activity regulation and demonstrate that DNA origami provides a modular platform to construct and characterize higher-order signaling complexes. The engineered DNA-based protein assembly platform has the potential to be broadly applied to inform the function of other important multi-enzyme assemblies involved in inflammation, innate immunity, and necrosis.

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Section: Introductionmentioning

confidence: 99%

A DNA-based synthetic apoptosome

Bj¹,

Aj²,

Gumí-Audenis³

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…The DNA origami technique has found broad applicability as an experimental tool for spatial organisation of native multi-protein systems, such as amyloid fibrils 21 , membrane fusion proteins 22 , nucleosomes 23,24 , and intrinsically disordered proteins 25 . Additionally, these platforms have been used to engineer localized genetic circuits 26 , to study confinement-induced enzyme activity 27,28 , and to investigate scaffolded metabolic cascades [29][30][31][32][33] . Although these studies have elegantly applied the programmability of DNA nanotechnology to facilitate organisation of e.g.…”

mentioning

confidence: 99%

Proximity-induced caspase-9 activation on a DNA origami-based synthetic apoptosome

et al. 2020

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Living cells regulate key cellular processes by spatial organisation of catalytically active proteins in higher-order signalling complexes. These act as organising centres to facilitate proximityinduced activation and inhibition of multiple intrinsically weakly associating signalling components, which makes elucidation of the underlying protein-protein interactions challenging. Here we show that DNA origami nanostructures provide a programmable molecular platform for the systematic analysis of signalling proteins by engineering a synthetic DNA origami-based version of the apoptosome, a multi-protein complex that regulates apoptosis by co-localizing multiple caspase-9 monomers. Tethering of both wildtype and inactive caspase-9 variants to a DNA origami platform demonstrates that enzymatic activity is induced by proximity-driven dimerization with half-of-sites reactivity, and additionally, reveals a multivalent activity enhancement in oligomers of three and four enzymes. Our results offer fundamental insights in Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

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“…Hisashi Tadakuma (Osaka University, now ShanghaiTech University) presented their recent achievements regarding the transcription nano-chip, where RNA polymerase and the target gene were integrated on DNA origami scaffold (Masubuchi et al 2018). DNA origami allowed them to precisely control the molecular layout with nano-meter resolution ( Fig.…”

Section: Main Textmentioning

confidence: 99%