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DNA Nanotags for Multiplexed Single-Particle Electron Microscopy

and In Situ Electron Cryotomography
Published as part of JACS Au special issue “DNA Nanotechnology for Optoelectronics and Biomedicine”.
Yuanfang Chen,# Yiqian Huang,# and Yuhe R. Yang*

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ABSTRACT: DNA nanostructures present new opportunities as

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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.
KEYWORDS: DNA nanostructures, nanotags, electron microscopy, cryo-electron tomography

■ INTRODUCTION
Cryo-electron microscopy (cryo-EM) has emerged as a
Moving forward, in situ characterization requires the
identification of targets within a native cellular environment
formidable technique in determining the three-dimensional crowded with cytoskeletons, cytoplasmic inclusions, organelles,
(3D) structures of biomacromolecules at near-atomic reso- etc.11 This complexity poses an even greater challenge
lution.1 However, this powerful technology faces significant concerning target localization. To address these challenges,
throughput limitations: each sample requires a unique grid, strategies have been developed to localize targets among
and data acquisition for individual cryo-EM grids is time- intercellular milieu and noises by tagging proteins of interest
intensive, often spanning several hours or even days.2 Enabling (POIs) with detectable tags.12 Current strategies can be
the concurrent characterization of mixed particle species on a categorized into density tags and size tags. Electron microscope
single grid holds a good chance to accelerate the research and (EM)-visible heavy atoms such as gold13−15 or iron16−18 can
development (R&D) cycle of new pharmaceuticals. For be attached to, and localize target proteins via immuno-
example, one could simultaneous analysis of multiple drugs labeling or genetically encoded metallothionein or ferritin,
against G protein-coupled receptors (GPCRs) and their while iron-rich media required for ferritin-tagging is often toxic
binding pockets,3,4 as well as one specific drug ligand against to living cells. Nevertheless, the strongly scattered electrons by
GPCRome-wide targets, particularly understudied and orphan metal atoms may give a risk of obscuring biological samples
receptors. This type of multiplexed imaging offers structural during tomography reconstruction.19 On the other hand, size
insights toward functional selectivity and signaling bias of each tags use proteins with adequate sizes for visual detection in EM
drug molecule to impart activations in distinct pathways.5 imaging. For example, an iron-free FerriTag enables nanoscale
Although a prototype instrument was developed to dispense mapping of Hip1R distribution surrounding clathrin-coated
each individual gold nanoparticle sample from a 96-well plate
to defined locations on a TEM grid using a piezoelectric Received: October 18, 2024
capillary dispensing system,6 the time required to dispense Revised: December 17, 2024
multiple specimens (∼4 h for delivering 96 spots) is Accepted: December 17, 2024
incompatible with cryo-EM grid preparation of biological
samples.7−10 To date, achieving high-throughput multiplexed
cryo-EM imaging remains challenging.
© XXXX The Authors. Published by
American Chemical Society https://doi.org/10.1021/jacsau.4c00986
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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 features28,34,36,75 (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.

membrane.20 The encapsulin-derived genetically encoded customizable shape, offers a promising approach as novel cryo-
multimeric particles (GEMs) with recognizable yet symmetric electron tomography (cryo-ET) tags, providing precise local-
structural signatures can be exploited to localize particles with ization and enhanced visibility.19
precision on the scale of 10−25 nm.21 Importantly, both iron- The field of DNA nanotechnology was first pioneered by
free FerriTag and GEM tags can be automatically identified by Nadrian C. Seeman in the early 1980s, who proposed using
convolutional neural networks (CNNs). However, neither DNA to create periodic molecular scaffolds that could
density tags nor size tags offer asymmetric structures that can
crystallize guest molecules.23,24 By exploiting their programm-
precisely pinpoint the location of tagged proteins, instead
ability, scientists design and assemble DNA molecules into
indicating only a roughly spherical region around the tag. This
lack of confident particle picking can result in a high number of precise and predictable shapes and patterns.25−28 In 2006, Paul
invalid false positive particle images, potentially leading to Rothemund introduced the concept of DNA origami,29 a
overfitting and model bias during data processing,22 which technique that allows the folding of a long single-stranded
hinders reliable structure reconstruction through subtomogram DNA (ssDNA) into complex shapes with the help of short
averaging. Here, DNA nanostructure, with its unique and staple strands. This strategy enabled the robust creation of
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intricate two-dimensional and later three-dimensional nano- identifiers, marking the types of proteins in a mixed sample and
scale objects.30,31 offering novel possibilities for the high-throughput screening of
Several distinct merits empower DNA nanostructures to protein complexes by multiplexed cryo-EM imaging (Figure
serve as an effective Nanotag for cryo-EM: (1) The highly 1B, top).
distinguishable shapes of DNA nanostructures can serve as Admittedly, the strong contrast of DNA molecules can cause
unambiguous indicators for other biomolecules under EM. misalignment of the target protein, and blur the particle
Hundreds of elaborate structures with defined densities,32 averages, as already observed in both negative staining EM (ns-
curvatures,33,34 and even intricate shapes such as Chinese EM)57−59 and cryo-EM52,60,61 analysis of DNA nanostructure-
characters35,36 can be easily created following simple yet robust protein complexes. Therefore, providing the necessary spacing
strategies (Figure 1B, bottom). Through rational design, one for POI on Nanotags would be beneficial. For example,
can engineer asymmetric objects that display different patterns Aissaoui et al. reported a V-shaped DNA origami scaffold
from different perspectives under transmission electron designed to accommodate POIs, where the proteins are
microscopy (TEM),32,36 providing valuable information for anchored at the center of the “V” by a long double-stranded
orientation assignment algorithms. Excitingly, computer-aided DNA (dsDNA), effectively isolating them from the frame-
methods have emerged to automate the generation of complex work.55 Adopting this strategy, an ns-EM data set of RNAP
DNA origami objects, including DEADLUS,37 TALOS,38 protein (∼450 kDa) with approximately 4,000 particles yielded
DNAxiS,38 and 4vHelix.39 (2) DNA nanostructures can be a 25 Å resolution map. Notably, despite its persistence length
coupled with lipids,40 peptides41 and proteins41 via diverse of ∼50 nm,62 the nonnegligible flexibility of dsDNA63 cannot
conjugation methods. It is noteworthy that the conjugation be considered as a rigid linker for hybridizing protein and
between DNA and proteins does not necessarily depend on Nanotag. Considering the challengeable cryo-EM reconstruc-
genetically fused protein tags. Oligonucleotides are possible to tion of multidomain proteins64 (which are connected by short
be attached to minimally modified proteins,41 preserving their amino acid sequences65), using a flexible dsDNA spacer would
natural state. (3) DNA nanostructures can remain structurally likely introduce additional conformational heterogeneity, and
intact under sample preparation conditions for most cryo-EM consequently compromise the POI resolutions.
applications. Current research demonstrated that DNA Novel data processing methods can be expected to weaken
nanostructures can withstand common protein buffers (e.g., or subtract Nanotag signals in a high-throughput manner after
tris-buffered saline (TBS)42 and phosphate-buffered saline identifying their contextual information (Figure 1D). In recent
(PBS) buffer43,44) with the addition of trace stabilizing cations years, Zhang’s group has developed a series of algorithms that
during cryo-EM sample preparation.45 It can also tolerate some can automatically estimate and weaken unwanted signals in
detergents, such as n-Dodecyl-β-D-maltoside (DDM) and macromolecular complexes to improve the alignment reliability
lauryl maltose neopentyl glycol (LMNG), that are often of target proteins.66−68 For example, their approach searched
indispensable for membrane protein samples (Yang group the microtubule networks by multicurve fitting, modeled the
unpublished results). For in situ cryo-ET applications, various tubulin-lattice signals, and then removed them. The removal of
modification strategies have been developed, such as lipid the superior tubulin-lattice signal improved the alignment and
bilayer coating,46 protein coating,47 polymer coating,48 and UV reconstruction of outer-arm dynein (OAD).66 With the shape
welding,49 that allow DNA nanostructures to endure cell information on predefined DNA nanostructures, these signal
cultivation conditions for at least several hours,50 which is removal methods should be readily implementable in Nanotag
within the typical time frame for cryogenic sample prepara- systems to suppress the DNA signals. Another approach,
tion.51 Together, the DNA nanostructure meets the primary termed “composite masks”,69 allows for focused refinement of
requirements for use as Nanotags in EM bioimaging. In fact, a small region while preserving the low-frequency signals from
DNA nanostructures have already been utilized in cryo- detergent micelle. They used a tight mask to separate POI
EM52−55 and cryo-ET19 to assist in the characterization of from micelle signals, as well as a loose mask to low-pass filter
biological samples (Figure 1A), illustrating their compatibility the micelles to reduce noise and overfitting, thereby
with structural biology. This Perspective first delves into the constraining the global alignment during 3D refinement. This
critical challenges associated with the application of this idea can be applied to Nanotag-attached proteins, where the
emerging DNA Nanotag strategy in multiplexed single-particle orientation assignment of POI particles is often interdependent
cryo-EM imaging. Further, we provide the prospect of using with Nanotag scaffolds.
Nanotags within a cellular environment for in situ cryo-ET, and
discuss the two main technical questions, namely cell-entering
of Nanotags and their intracellular trafficking.
■ IN SITU PARTICLE PICKING
As an emerging bioimaging technique, cryo-ET allows for the

■ MULTIPLEXED SINGLE-PARTICLE EM
DNA nanotechnology was initially proposed as a tool for X-ray
examination of macromolecules and complexes in situ,
preserving their native states and interactions within the
cellular context. In order to determine high-resolution 3D
structural analysis according to Seeman’s pioneering vision.23 models, hundreds of thousands of particle subvolumes need to
In the following decades, cryo-EM technology emerged and be picked from tomograms. Particle picking can be performed
became a mainstream technique in structural biology,56 when manually, or by template-matching methods in which a known
the potential of DNA nanostructures in cryo-EM started to be (or manually picked) structure serves as a template.70
realized.52 It has been noticed that the programmable shape of Alternatively, template-free methods can extract candidate
DNA nanostructures can provide priori knowledge, such as particles based solely on their size.71 Nevertheless, picking
orientation angles of target protein, to the single particle particles in a crowded cellular landscape remains challenging.72
analysis (SPA) algorithms.53,54 However, only DNA-binding On the one hand, the total electron dose used is limited to
proteins can be accurately rotated by these scaffolds. For a protect the sample from radiation damage, resulting in a very
more generic purpose, DNA Nanotags can serve as particle low signal-to-noise ratio (SNR) for each tilt frame.73 On the
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Figure 2. Strategies to improve the efficiency of delivering DNA nanostructures via the endocytosis-lysosome escape pathway.

other hand, macromolecules are densely packed and developed to improve uptake efficiency47,48,77−95 and promote
surrounded by various other structures within cells, making it endosomal escape.96−99 Alternative approaches focus on direct
difficult to identify and isolate individual particles, particularly cytosolic delivery across the cell membrane,100−104 offering the
in low SNR tomograms. Advanced computational approaches possibility to better preserve the structural integrity of DNA
based on deep neural network74 are increasingly employed for Nanotags. This procedure necessitates specific modifications to
the automated picking of large particles with identifiable redirect a portion of the Nanotags from primary endocytosis-
shapes21 such as DNA Nanotags. based pathways.
It has been pointed out that the phosphorus in DNA Strategies to improve the efficiency of delivering DNA
molecule backbones scatters elastically ∼4× more electrons nanostructures via the Endocytosis-Lysosome Escape pathway
than the major elements in proteins, such as carbon, oxygen, or can be categorized into four groups: First, modulating the
nitrogen.19 DNA Nanotag composed of densely packed DNA structural features (Figure 2A). Numerous studies have
molecules may thus generate a stronger contrast in a low SNR demonstrated that a higher degree of structural rigidity,77
tomogram context. Combined with its distinctive shapes, DNA compactness,80 and smaller aspect-ratio79 are associated with
Nanotag holds the potential to provide precise and confident enhanced uptake of bare DNA nanostructures.78−81 Second,
positioning of particles of interest within the crowded and modifying the surface charge (Figure 2B). Coating layers such
heterogeneous cellular environment (Figure 1C). Based on this as oligolysine-PEG,48 polyethylenimine (PEI),82 virus capsid,47
idea, the signpost origami tags (SPOTs),19 which bind to GFP- and preadsorbed protein corona83 can mask the negatively
tagged membrane proteins on the cell surface (Figure 1E, left), charged DNA backbones, reducing electrostatic repulsion from
shed new light on developing a generally applicable tagging the plasma membrane. Third, assisting with lipids (Figure 2C).
platform for cryo-ET bioimaging. Despite the low-resolution Noncoated DNA nanostructures with single-stranded probes
subvolume averaging results of target gB protein, two classes can hybridize with cell-anchored cholesterol-ssDNA, facilitat-
have been obtained that broadly resemble two known ing direct interaction with the cell membrane.87−89 Alter-
conformations of gB (Figure 1E, right), inferring that the natively, DNA nanostructures can be coated with cationic
presence of SPOTs is compatible with the subvolume lipids via electrostatic interactions84 or with noncationic lipids
alignment and classification process. Encouraged by the through hydrophobic interactions using the Frame-Guided
extracellular leaflet labeling SPOTs, DNA Nanotags that target Assembly (FGA) strategy,85,86 both of which have been shown
cytoplasmic leaflets, organelle membranes, and cytoplasmic to enhance cellular uptake. Lastly, receptor targeting (Figure
biomolecular complexes can be anticipated. 2D). Reported ligand−receptor pairs can facilitate receptor-

■ CYTOSOLIC ENTRY OF NANOTAGS

The major challenges in utilizing DNA Nanotags for in situ
mediated endocytic pathways, with ligands such as trans-
ferrin,90 folate,91,92 aptamers,93,94 and tumor-penetrating
peptide,95 etc. having been explored.
imaging are 2-fold: first, getting them across the cellular Next, after DNA Nanotags are initially captured by early
membrane into the cytosol; second, enabling intracellular lysosomes during endocytosis, endosomal escape is crucial for
migration to designated subcellular organelles. DNA Nanotags, delivery to the cytosol. Various endosomal escape peptides
as nanometric particles, predominantly enter cells via have been explored,96 among which hemagglutinin (HA)
endocytosis.76 Consequently, numerous strategies have been fusogenic peptide97 and aurein 1.298 are applied to DNA
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structures. These peptides can be loaded through chemical

conjugation or electrostatic adsorption, with the latter allowing
for higher peptide coverage on the nanostructures.99
The strategies described above have significantly enhanced
the internalization of DNA nanostructures via endocytosis. For
instance, transferrin-modified DNA nanostructures have shown
an uptake efficiency up to 22-fold higher than the unmodified
ones in KB cells.90 Modifications with HA fusogenic peptide
have achieved endosomal escape rates as high as 61%.97
However, several key points merit attention. First, the cellular
uptake of DNA nanostructures significantly depends on the
cell types. Generally, immune cells and cancer cells exhibit a
higher efficiency of internalization compared to other cells.79
Second, current experimental approaches applied to assess
cellular uptake efficiency typically include fluorescence
confocal microscopy, flow cytometry, and quantitative PCR
(qPCR). These methods frequently employ DNA nanostruc- Figure 3. Strategies to direct cytosolic delivery across the cellular
tures labeled with single or dual fluorescent dyes to determine membrane or expressing Nanotags in situ.
spatial localization or through FRET (Förster Resonance
Energy Transfer) to evaluate interdye distances and thus infer efficient delivery of mRNA and plasmid DNA (pDNA) into
the structural integrity. However, structural characterization different cell lines including AC10, H1299, and HEK293T.104
techniques such as transmission electron microscopy (TEM) The PLV particles, ∼ 80 nm in size, align well with the size
or atomic force microscopy (AFM) are rarely used to evaluate range of the DNA nanostructures. Moreover, this delivery
the structural integrity of DNA nanostructures postcell lysis. mechanism does not necessitate modifications of the DNA
Ideally, for in situ EM imaging applications, DNA Nanotags nanostructures. These attributes, collectively, establish PLVs as
should preserve their intricate structural features upon reaching a highly promising platform for the cytosolic entry of DNA
their intended cellular destinations. Consequently, strategies Nanotags.
promoting direct cytosolic uptake, which circumvent endo-
somal pathways, are of considerable value. These methods
prevent DNA Nanotags from exposure to degradative
■ IN SITU EXPRESSION OF NANOTAGS
Expressing Nanotags in situ presents another solution to
conditions (e.g., low pH) within endo/lysosomes, where circumvent the difficulties associated with intracellular delivery.
significant disintegration can occur.105 Physical approaches Here, structures constructed with only one single-stranded
that enable the direct delivery of DNA nanostructures to the nucleic acid hold the potential to be intracellularly replicated
cytoplasm include electrotransfection,100 nanoneedles,101 and and expressed once the nanostructure-forming “pseudogenes”
“cell squeezing” technique.102 Electrotransfection, commonly are transfected (Figure 3C). To fabricate such single-stranded
used for plasmids, requires a spermidine buffer to maintain the nanostructures with size and shape diversities, universal
structural integrity of DNA nanostructures at high voltages. strategies, termed single-stranded origami (ssOrigami), have
The latter two methods increase membrane permeability by emerged.
inducing mechanical stress and creating membrane pores, The key challenge of folding a compact single-stranded
heavily relying on specialized instruments. In addition, while structure is to avoid kinetic traps imposed by knots during
studies report minimal impact on cell survival, the structural designing the strand routing.109 Geary et al. inserted extra 180°
integrity of DNA nanostructures postdelivery remains kissing loops between each antiparallel double-crossover (DX)
uncertain. Thiol-mediated103,106,107 uptake holds promise for to decrease the topological complexity.109,110 Instead of
cytosolic entry at more native cell states (Figure 3A). Yan et al. associating helices using antiparallel crossovers in traditional
developed a DNA nanodevice, CytoDirect, which integrates multistranded origami strategy,29 Yin and co-workers em-
disulfide moieties and human epidermal growth factor receptor ployed paranemic crossovers (PX) in their ssOrigami strategy
2 (HER2) affibodies.103 The HER2 affibody, functioning as a to prevent the long single-stranded DNA from being
targeting domain, brings the disulfide bonds on the DNA knotted.111 Additionally, other single-stranded origami design
nanodevice into the proximity of the thiol groups on the cell languages implementing 90° kinks and branched kissing-loops
surface. After internalization, intracellular endogenous gluta- were proposed to build wireframe structures.112,113 Notably, all
thione (GSH) cleaves these disulfide bonds, enabling the rapid of these works demonstrated that long single-stranded DNA or
release of the nanodevice from the cell membrane into the RNA can be replicated both in vitro and in vivo, but the
cytosol. It is noted that no experimental data regarding the assembly involves differentiated annealing steps. PX-based
structural integrity of the released nanodevices have been origami necessitates a thermo-annealing program starting at 85
reported. °C, which is unavailable during cell cultivation. Albeit, RNA
Although research on the direct cytosolic uptake of DNA origami from Geary et al. realized the isothermal cotranscrip-
nanostructures is limited, recent advancements in mRNA tional folding in vitro at 37 °C,109 and wireframe RNA
vaccine delivery systems offer valuable insights. Lewis et al. structures were further shown to successfully fold within living
developed a proteolipid vehicle (PLV) that facilitates cells, as confirmed by AFM images of cell lysates.112,113 We
membrane fusion between PLVs and the cell membrane by believe that this discrepancy likely arises from the lack of self-
incorporating fusion-associated small transmembrane (FAST) complementary sequences within each of the two half RNA
proteins104 (Figure 3B). Due to FAST proteins being fragments in PX-based designs, resulting in the inability to
compatible across various cell types,108 PLVs have achieved form correctly folded local hairpins or stems during tran-
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scription. As a result, complete denaturation is favored to melt that smaller nanoparticles encounter less resistance from the
all secondary structures, allowing for the pairing of two half matrix and therefore diffuse more rapidly. Additionally,
fragments. Continuous efforts in structural designing to passivating the nanoparticle surface to reduce nonspecific
minimize spurious local kinetic traps during cotranscriptional interactions with cellular components is essential for ensuring
in situ folding will be beneficial for creating more delicate the particles reach their intended targets efficiently.134
Nanotags. Inspiration can also be drawn from the mechanisms Nevertheless, the migration behavior of DNA nanostructures
of protein folding, as proteins are similarly folded by a single has not yet been systematically studied. While trends observed
chain of polypeptides within living cells.111 in other types of nanoparticles are helpful, the unique
To specifically label a POI, a variety of ligands can be properties of DNA Nanotags, such as strong negative charge
attached to the Nanotags. Options include peptides,114 and delicate programmability of shapes (e.g., varying twist
antibodies,58 self-labeling protein tags,115 small molecular angles and handedness33), introduce factors that could affect
ligands,116 and bio-orthogonal functional groups.117 These their intracellular migration. Studying DNA Nanotag migration
modifications, however, are not applicable to in situ expressed directly within living cells could better mirror real-world
and folded RNA Nanotags. Alternatively, short, single-stranded conditions. Alternatively, simplified analog networks, such as
nucleic acid aptamers can serve as affinity labels for DNA supramolecular hydrogels135 or extracellular matrices,136
Nanotags.118 A caveat for using aptamers is that the can be employed for single-factor analyses. Real-time
conditional aptamer discovery pipeline requires intensive observation techniques, such as single-particle tracking
efforts, with no guarantee that high-affinity aptamers can (SPT),137,138 offer valuable insights into dynamics information
always be obtained for any given POI.119 Exploiting existing by extracting key parameters, including mean square displace-
aptamers that target recombinant protein tags120,121 is a simple ment (MSD) and diffusion coefficients,139 enabling the
yet efficient solution that circumvents the de novo selection of characterization of particle diffusion rates. Additional exper-
unique aptamers. For example, the “SPOTs” target sfGFP- imental results with simulations could help identify trends that
fused gB by integrating a high-affinity GFP-binding aptamer.19 aid the rational design of DNA Nanotags and optimize their
However, other attempts, such as using aptamer against migration for in situ cryo-ET applications.
hexahistidine tags, were less successful,19 highlighting the need
for more robust aptamers against common protein fusion tags.
Another concern is that off-target Nanotags may be
■ SUMMARY AND OUTLOOK
DNA nanostructures offer unique shapes than “blob-like”
misidentified as correctly targeted ones during data processing, protein particles and demonstrate compatibility with standard
compromising their labeling accuracy and increasing the false- cryo-EM protocols, including the ability to conjugate with
positive rate. Besides genetically regulating the expression level biomolecules and withstand various sample preparation
of the Nanotag-carrying plasmid, constructing a target- conditions. Building on these advantages, this article explores
responsive allosteric nanostructure could be one feasible an emerging application of DNA nanostructures: DNA
solution to screen the positively tagged Nanotags in the Nanotags�molecular markers specifically designed to label
cellular matrix. For example, ligand-responsive aptamer switch proteins.
architectures can elicit conformational change and strand By parallelly labeling protein complexes with distinct
displacement of nucleic acid molecules.122 These aptamers can Nanotags, these markers enable the identification of multiple
trigger the conformational change of origami nanodevi- targets within a single EM grid, facilitating high-throughput
ces,123,124 allowing the accurate recognition and separation of multiplexed EM imaging. EM-visible Nanotags are also
target particles in cryo-ET data sets. anticipated to accurately localize target particles from low

■ INTRACELLULAR MIGRATION AND TARGETING

Targeted delivery of DNA nanostructures to specific organelles
SNR cryo-ET data sets of crowded cellular landscapes.
However, the successful integration of DNA Nanotags into
in situ applications requires overcoming challenges related to
is primarily achieved by attaching organelle-targeting peptides cellular uptake and intracellular trafficking. Platforms for the
(OTPs) such as nuclear targeting sequence (NTS) peptides, direct cytosolic delivery of mRNA and plasmids present
N-(βA)VVVKKKRKVVC, and mitochondrial targeting se- promising strategies for delivering DNA Nanotags. Addition-
quence (MTS) peptides, N-(βA)LLYRSSCLTRTAPKF- ally, the potential for in situ expression of ssOrigami offers an
RISQRLSLM,101 to these constructs. The distribution of innovative alternative. For the second challenge, although
DNA nanocages within subcellular organelles was observed current reports on the intracellular migration of DNA
through the colocalization of dye-labeled DNA nanostructures nanostructures are limited, insights can be gleaned from
and specifically stained cellular organelles.101 Alternatively, simulations and the in vivo diffusion behavior of other
caveolae-mediated endocytosis (CvME) allows endocytic nanoparticles. Another concern is the stimulation of immune
carriers to either fuse with early endosomes or transfer directly pathways by DNA, including TLR9,140 cGAS/STING141 and
to the ER via caveosomes,125 potentially enabling ER delivery AIM2.142 However, the extent of the intracellular immune
without the need for related OTPs.126,127 response elicited by DNA nanostructures has not been
Given that the cytoplasm is a highly complex, multiphase explored. Consequently, the potential impact of Nanotags on
system with spatial heterogeneity,128 enhancing the diffusion of the native cellular environment should be carefully evaluated to
Nanotags becomes particularly critical for achieving rapid determine whether they affect the intended targets of interest.
cytosolic trafficking. Typically, nanoparticle diffusion is Despite these challenges, future optimizations toward
influenced by the intrinsic properties of both the surrounding assembly and modification processes of DNA Nanotags will
matrix128 (such as pore size, solid content, viscosity, etc.) and be necessary to ensure robust stability and simpler usage,
the nanoparticles themselves (such as size,129 aspect paving the way for the development of ready-to-use protein-
ratio,130,131 etc.), as well as their mutual interactions.128 Both labeling kits for widespread adoption in structural biology
theoretical models132,133 and experimental evidence129 suggest laboratories. In addition to their applications in structural
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biology, DNA Nanotags can serve as valuable tools for Multiplexed TEM Specimen Preparation and Analysis of Plasmonic
investigating various physiological mechanisms. For instance, Nanoparticles. Microsc Microanal 2015, 21 (4), 1017−1025.
tomograms reconstructed with “signposts” (Figure 1E) high- (7) Jain, T.; Sheehan, P.; Crum, J.; Carragher, B.; Potter, C. S.
light the potential application of DNA Nanotags in mapping Spotiton: a prototype for an integrated inkjet dispense and
the distribution of exposed protein epitopes143 on the surface vitrification system for cryo-TEM. J. Struct Biol. 2012, 179 (1), 68−
75.
of the protein corona, cell, and other structures.
(8) Razinkov, I.; Dandey, V.; Wei, H.; Zhang, Z.; Melnekoff, D.;

■ AUTHOR INFORMATION
Corresponding Author
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Yuhe R. Yang − CAS Key Laboratory of Nanosystem and E. T.; Rice, W. J.; Kahn, P. A.; Potter, C. S.; Carragher, B. Spotiton:
Hierarchical Fabrication, CAS Center for Excellence in New features and applications. J. Struct Biol. 2018, 202 (2), 161−169.
Nanoscience, National Center for Nanoscience and (10) Dandey, V. P.; Budell, W. C.; Wei, H.; Bobe, D.; Maruthi, K.;
Kopylov, M.; Eng, E. T.; Kahn, P. A.; Hinshaw, J. E.; Kundu, N.; et al.
Technology of China, CAS, Beijing 100190, China;
Time-resolved cryo-EM using Spotiton. Nat. Methods 2020, 17 (9),
University of Chinese Academy of Sciences, Beijing 100049, 897−900.
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■ ACKNOWLEDGMENTS
This work was supported by National Key R&D Program of
2024. DOI: 10.1101/2024.09.10.612178.
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