DNA origami scaffold for studying intrinsically disordered proteins of the nuclear pore complex

Ketterer, Philip; Ananth, Adithya N.; Trip, Diederik S. Laman; Mishra, Ankur; Bertosin, Eva; Ganji, Mahipal; Torre, Jaco van der; Onck, Patrick; Dietz, Hendrik; Dekker, Cees

doi:10.1038/s41467-018-03313-w

Cited by 123 publications

(122 citation statements)

References 46 publications

Supporting

Mentioning

120

Contrasting

Order By: Relevance

“…Figure 4d shows a frequency plot of translocation events vs dwell time. We note that the distribution of dwell times observed for the different DNA origami structures are significantly narrower than those found in previous studies with DNA nanostructures, [25] DNA concatemers, [26] and DNA origami, [27] where prolonged dwell times were reported. The dwell time provides an indication of how long the DNA origami spends in the nanopore, and can therefore be used and an indicative measure of the speed of translocation.…”

Section: Resultscontrasting

confidence: 83%

“…In particular, the differentiation of DNA cubes from RNA rings was demonstrated using variations of the peak dwell time and the peak ion current through a 50 nm silicon nitride nanopore membrane. [27] However, despite these remarkable successes, the relationship between the structure of the DNA origami and the corresponding ion current during translocation remains largely unexplored, which hinders the adoption of DNA origami as carrier and unique identifier in nanopipette applications. [26] Further work using DNA origami as a molecular breadboard to isolate nuclear pore complexes enabled the differentiation between wild type and mutant forms by analysis of their characteristic ion current traces.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Analysis of 2D DNA Origami with Nanopipettes

et al. 2018

View full text Add to dashboard Cite

Nanopipettes are a useful tool for the detection and analysis of biological molecules at the single-molecule level. Here, we employ nanopipettes fabricated from glass capillaries to identify differently structured 2D DNA origami through the distinctive amplitude and dwell time of the ion current peak resulting from the translocation of the origami through the nanopipette pore. We demonstrate that the ion current peak originating from frame-like DNA origami comprises two individual peaks in contrast to solid tiles of similar size that only lead to a single peak in the ion current. Interestingly, the fine details of the shape of the double-peak characteristic of the translocation of DNA origami frames are correlated with the structural features of the DNA origami. In particular, the size of the central cavity governs the lag time between the two constituent peaks of the double-peak. This work demonstrates the ability of glass nanopipettes to identify and characterize differently structured DNA origami of similar size, advancing the potential of nanopipettes for high-sensitivity single-molecule studies.

show abstract

Section: Resultscontrasting

confidence: 83%

Section: Introductionmentioning

confidence: 99%

Analysis of 2D DNA Origami with Nanopipettes

et al. 2018

View full text Add to dashboard Cite

show abstract

“…DNA origami-based nanostructures, in particular, are well-suited as synthetic platforms as their unique addressability allows for precise assembly of multiple non-identical proteins with nanometer accuracy [18][19][20] . 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] .…”

Section: Introductionmentioning

confidence: 99%

A DNA-based synthetic apoptosome

Bj¹,

Aj²,

Gumí-Audenis³

et al. 2019

Preprint

View full text Add to dashboard Cite

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.

show abstract

“…macromolecules bound with NTRs) caught in transit [21]. Therefore, to better understand the behaviour and function of the FG-nups within this complex system, mimetic NPCs made using DNA origami have recently been created [5,22].…”

Section: Introductionmentioning

confidence: 99%

Quantification of biomolecular dynamics inside real and synthetic nuclear pore complexes using time-resolved atomic force microscopy

Stanley

Akpinar

Shen

et al. 2019

Preprint

View full text Add to dashboard Cite

Over the past decades, atomic force microscopy (AFM) has emerged as an increasingly powerful tool to study the dynamics of biomolecules at nanometre length scales. However, the more stochastic the nature of such biomolecular dynamics, the harder it becomes to distinguish them from AFM measurement noise. Rapid, stochastic dynamics are inherent to biological systems comprising intrinsically disordered proteins. One role of such proteins is in the formation of the transport barrier of the nuclear pore complex (NPC): the selective gateway for macromolecular traffic entering or exiting the nucleus. Here, we use AFM to observe the dynamics of intrinsically disordered proteins from two systems: the transport barrier of native NPCs, and the transport barrier of a mimetic NPC made using a DNA origami scaffold. Analysing data recorded with 50-200 ms temporal resolution, we highlight the importance of drift correction and appropriate baseline measurements in such experiments. In addition, we describe an auto-correlation analysis to quantify time scales of observed dynamics and to assess their veracityan analysis protocol that lends itself to the quantification of stochastic fluctuations in other biomolecular systems. The results reveal the surprisingly slow rate of stochastic, collective transitions inside mimetic NPCs, highlighting the importance of FG-nup cohesive interactions.

show abstract

DNA origami scaffold for studying intrinsically disordered proteins of the nuclear pore complex

Cited by 123 publications

References 46 publications

Analysis of 2D DNA Origami with Nanopipettes

Analysis of 2D DNA Origami with Nanopipettes

A DNA-based synthetic apoptosome

Quantification of biomolecular dynamics inside real and synthetic nuclear pore complexes using time-resolved atomic force microscopy

Contact Info

Product

Resources

About