Precision Templated Bottom-Up Multiprotein Nanoassembly through Defined Click Chemistry Linkage to DNA

Marth, Gabriella; Hartley, Andrew M.; Reddington, Samuel C.; Sargisson, Lauren L.; Parcollet, Marlène; Dunn, Katherine E.; Jones, D. Dafydd; Stulz, Eugen

doi:10.1021/acsnano.7b01711

Cited by 37 publications

(37 citation statements)

References 40 publications

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“…The small bioorthogonal azide moiety was used for conjugation to a bicyclononyne-functionalized oligonucleotide (BCN-DNA) using strain-promoted azide-alkyne cycloaddition ( Fig. 1d) [39][40][41] . The oligonucleotide was designed with a 10-nucleotide (nt) single-stranded linker separating the enzyme and a 15-nt anti-handle used for hybridization to the handles on the DNA origami surface (Supplementary Table 2 and Supplementary Fig.…”

Section: Resultsmentioning

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: Resultsmentioning

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 small bioorthogonal azide moiety was used for conjugation to a bicyclononyne-functionalized oligonucleotide (BCN-DNA) using strain-promoted azide-alkyne cycloaddition (Fig. 1d) [41][42][43] . The oligonucleotide consists of a 10-nucleotide (nt) single-stranded linker separating the enzyme and a 15-nt anti-handle used for hybridization to the handles (Supplementary Table 2 and Supplementary Fig.…”

Section: Activity Of Dna-functionalized Caspase-9mentioning

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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“…Antibodies such as IgG can also be conjugated to DNA origami for medical applications (Agasti et al, 2017;Kuzuya et al, 2011). Chemical modifications, such as SNAP-tag (Derr et al, 2012;Hariadi et al, 2015;Hariadi, Appukutty, & Sivaramakrishnan, 2016), click-chemistry (Marth et al, 2017), and amine N-hydroxysuccinimide-ester conjugation (Agasti et al, 2017), have been employed to conjugate DNA to proteins that carry out complex functions. For example, the Shih, Reck-Peterson, and Sivaramakrishman labs use SNAP-tagged molecular motors such as myosin, dynein and kinesin that bind to benzylguanine-labeled amine-modified ssDNA on SST-based DNA nanotubes to determine the motile behavior of actin and microtuble-based motor assemblies (Figure 3b) (Derr et al, 2012;Hariadi et al, 2015;Hariadi et al, 2016).…”

Section: Protein Sensingmentioning

confidence: 99%

Mix‐and‐match nanobiosensor design: Logical and spatial programming of biosensors using self‐assembled DNA nanostructures

Liu¹,

Kumar²,

Taylor³

2018

WIREs Nanomed Nanobiotechnol

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The evergrowing need to understand and engineer biological and biochemical mechanisms has led to the emergence of the field of nanobiosensing. Structural DNA nanotechnology, encompassing methods such as DNA origami and single-stranded tiles, involves the base pairing-driven knitting of DNA into discrete one-, two-, and three-dimensional shapes at nanoscale. Such nanostructures enable a versatile design and fabrication of nanobiosensors. These systems benefit from DNA's programmability, inherent biocompatibility, and the ability to incorporate and organize functional materials such as proteins and metallic nanoparticles. In this review, we present a mix-and-match taxonomy and approach to designing nanobiosensors in which the choices of bioanalyte and transduction mechanism are fully independent of each other. We also highlight opportunities for greater complexity and programmability of these systems that are built using structural DNA nanotechnology. This article is categorized under: Implantable Materials and Surgical Technologies > Nanomaterials and Implants Diagnostic Tools > Biosensing Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

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Precision Templated Bottom-Up Multiprotein Nanoassembly through Defined Click Chemistry Linkage to DNA

Cited by 37 publications

References 40 publications

A DNA-based synthetic apoptosome

A DNA-based synthetic apoptosome

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

Mix‐and‐match nanobiosensor design: Logical and spatial programming of biosensors using self‐assembled DNA nanostructures

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