Real-Time Assembly of Viruslike Nucleocapsids Elucidated at the Single-Particle Level

Marchetti, Margherita; Kamsma, Douwe; Vargas, Ernesto; García-Reynoso, José Agustín; Schoot, Paul van der; Vries, Renko de; Wuite, Gijs J. L.; Roos, Wouter H.

doi:10.1021/acs.nanolett.9b02376

Cited by 41 publications

(48 citation statements)

References 54 publications

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“…Note that in earlier papers we have shown that VLP lengths obtained using AFM on dried complexes are consistent with those obtained using other methods, in particular, cryo-electron microscopy and static light scattering, 7 as well as AFM in liquid. 20…”

Section: Resultsmentioning

confidence: 99%

Nature of Amorphous Hydrophilic Block Affects Self-Assembly of an Artificial Viral Coat Polypeptide

et al. 2019

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Consensus motifs for sequences of both crystallizable and amorphous blocks in silks and natural structural analogues of silks vary widely. To design novel silklike polypeptides, an important question is therefore how the nature of either the crystallizable or the amorphous block affects the self-assembly and resulting physical properties of silklike polypeptides. We address herein the influence of the amorphous block on the self-assembly of a silklike polypeptide that was previously designed to encapsulate single DNA molecules into rod-shaped viruslike particles. The polypeptide has a triblock architecture, with a long N-terminal amorphous block, a crystallizable midblock, and a C-terminal DNA-binding block. We compare the self-assembly behavior of a triblock with a very hydrophilic collagen-like amorphous block (GXaaYaa)132 to that of a triblock with a less hydrophilic elastin-like amorphous block (GSGVP)80. The amorphous blocks have similar lengths and both adopt a random coil structure in solution. Nevertheless, atomic force microscopy revealed significant differences in the self-assembly behavior of the triblocks. If collagen-like amorphous blocks are used, there is a clear distinction between very short polypeptide-only fibrils and much longer fibrils with encapsulated DNA. If elastin-like amorphous blocks are used, DNA is still encapsulated, but the polypeptide-only fibrils are now much longer and their size distribution partially overlaps with that of the encapsulated DNA fibrils. We attribute the difference to the more hydrophilic nature of the collagen-like amorphous block, which more strongly opposes the growth of polypeptide-only fibrils than the elastin-like amorphous blocks. Our work illustrates that differences in the chemical nature of amorphous blocks can strongly influence the self-assembly and hence the functionality of engineered silklike polypeptides.

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Section: Resultsmentioning

confidence: 99%

Nature of Amorphous Hydrophilic Block Affects Self-Assembly of an Artificial Viral Coat Polypeptide

et al. 2019

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“…In a different single-particle approach, a dsDNA strand was stretched between two beads in a double optical tweezers set-up 72 and then exposed to a solution of fluorescently labelled synthetic polypeptide strands, which played the role of model capsid proteins. Different assembly stages of the resulting VLP were visualized in real time with millisecond temporal resolution (Fig.…”

Section: Viral Self-assembly Dynamicsmentioning

confidence: 99%

“…Optical tweezers adapted with permission from ref. 72 . Interferometric scattering microscopy adapted with permission from ref.…”

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confidence: 99%

Physics of viral dynamics

2021

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“…The dual‐trap optical tweezers with 3‐color confocal fluorescence microscopy set‐up [ 28 ] (C‐trap, LUMICKS, Amsterdam, The Netherlands), was used for the single‐cell kinetic experiments. A 5‐channel microfluidic cell (LUMICKS) mounted on an automated XY‐stage was used to isolate individual cells under different conditions.…”

Section: Methodsmentioning

confidence: 99%

Single Cell Reactomics: Real‐Time Single‐Cell Activation Kinetics of Optically Trapped Macrophages

et al. 2021

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Macrophages are well known for their role in immune responses and tissue homeostasis. They can polarize towards various phenotypes in response to biophysical and biochemical stimuli. However, little is known about the early kinetics of macrophage polarization in response to single biophysical or biochemical stimuli. Our approach, combining optical tweezers, confocal fluorescence microscopy, and microfluidics, allows us to isolate single macrophages and follow their immediate responses to a biochemical stimulus in real‐time. This strategy enables live‐cell imaging at high spatiotemporal resolution and omits surface adhesion and cell–cell contact as biophysical stimuli. The approach is validated by successfully following the early phase of an oxidative stress response of macrophages upon phorbol 12‐myristate 13‐acetate (PMA) stimulation, allowing detailed analysis of the initial macrophage response upon a single biochemical stimulus within seconds after its application, thereby eliminating delay times introduced by other techniques during the stimulation procedure. Hence, an unprecedented view of the early kinetics of macrophage polarization is provided.

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Real-Time Assembly of Viruslike Nucleocapsids Elucidated at the Single-Particle Level

Cited by 41 publications

References 54 publications

Nature of Amorphous Hydrophilic Block Affects Self-Assembly of an Artificial Viral Coat Polypeptide

Nature of Amorphous Hydrophilic Block Affects Self-Assembly of an Artificial Viral Coat Polypeptide

Physics of viral dynamics

Single Cell Reactomics: Real‐Time Single‐Cell Activation Kinetics of Optically Trapped Macrophages

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