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Review
. 2024 Dec 27;5(1):17-27.
doi: 10.1021/jacsau.4c00986. eCollection 2025 Jan 27.

DNA Nanotags for Multiplexed Single-Particle Electron Microscopy and In Situ Electron Cryotomography

Yuanfang Chen  1   2 , Yiqian Huang  1   2 , Yuhe R Yang  1   2
Affiliations
Review

DNA Nanotags for Multiplexed Single-Particle Electron Microscopy and In Situ Electron Cryotomography

Yuanfang Chen et al. JACS Au. .

Abstract

DNA nanostructures present new opportunities as Nanotags for electron microscopy (EM) imaging, leveraging their high programmability, unique shapes, biomolecule conjugation capability, and stability compatible with standard cryogenic sample preparation protocols. This perspective highlights the potential of DNA Nanotags to enable high-throughput multiplexed EM analysis and facilitate in situ particle identification for cryogenic electron tomography (cryo-ET). Meanwhile, applying Nanotags in live-cell environments requires the efficient cellular uptake of intact structures and successful cytosolic migration. Promising strategies such as employing direct cytosolic delivery platforms and expressing RNA-based Nanotags in situ are discussed, while more systematic studies are needed to fully understand the intracellular trafficking and achieve precise localization of DNA Nanotags.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Application of DNA Nanotags to cryo-EM and cryo-ET. (A) “Molecular support” (left) and “molecular goniometer” (middle) rotate DNA binding proteins for cryo-EM reconstruction. DNA origami “signposts” for cellular membrane tagging (right). (B) Conceptual workflow of high throughput cryo-EM-based drug screening using DNA Nanotags (top). Representative structures with distinctive features,,, (bottom). Scale bar: 20 nm. The first panel is reproduced with permission from ref (34). Copyright 2011 American Association for the Advancement of Science. The second panel is reproduced with permission from ref (36). Copyright 2012 American Association for the Advancement of Science. The third panel is reproduced with permission from ref (28). Copyright 2008 Springer Nature. The fourth panel is reproduced from ref (75). Available under a CC-BY license. Copyright 2012 The Authors. (C) Schematic illustration of in situ cryo-ET application of DNA Nanotags. The unique shape of Nanotags can localize POIs in a crowded cellular environment. (D) Focused refinement of protein reconstructions. (E) Micrographs and 3D reconstruction results of DNA origami “signposts”-labeled membrane proteins. Reproduced from ref (19). Available under a CC-BY license. Copyright 2021 The Authors.
Figure 2
Figure 2
Strategies to improve the efficiency of delivering DNA nanostructures via the endocytosis-lysosome escape pathway.
Figure 3
Figure 3
Strategies to direct cytosolic delivery across the cellular membrane or expressing Nanotags in situ.
See this image and copyright information in PMC

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