Construction of DNA aggregates in cell milieu for bio‐interference

Precise assembly of biomolecules into functional aggregate structures represents a key characteristic of nature living cells, and is critical for the cellular processes. The construction of artificial aggregates in living cells by responding to intracellular specific stimuli has been applied in elucidating molecular mechanisms of naturally cellular processes and interfering with cellular processes, which is potential and significant in biomedicine. DNA (deoxyribonucleic acid) features sequence programmability, precise assembly and versatility, and therefore is regarded as the potential candidate for constructing versatile functional aggregates in living cells. In this review, we summarize our recent efforts of employing DNA to in situ construct versatile aggregates in living cells via responding intracellular triggers, and the subsequent bio‐interference of the DNA aggregates. Finally, we discuss the remaining challenges and opportunities in the field, and envision that rational design and construction of versatile DNA aggregates in living system would be a promising solution for precision and personalized therapeutics.

contain an array of enzymes capable of breaking down all types of biological polymer, such as proteins, nucleic acids, carbohydrates, and lipids [7] ; peroxisomes are small, mono-membrane-enclosed organelles that contain enzymes involved in a variety of metabolic reactions, including several aspects of energy metabolism [8] ; mitochondria are bi-layer membrane-bound cell organelles that generate most of the chemical energy needed to power the cell's biochemical reactions. [9]In normal cells, molecular aggregates are usually created on demand and behave dynamically to meet the precise requirement of the system at any given time, and the abnormal aggregates generation usually results in the cellular dysfunction. [10]Aggregates in living systems provided a reactive environment and material basis for various metabolic activities. [11]nspired by the nature's evolution of function through structure formation, researchers have attempted to construct dynamic assembly systems of aggregates using exogenous molecules for cellular regulation. [12,13]For example, employing carbon nanozyme to accommodate multiple enzyme-like activities that mimics natural peroxisome.The nanozyme-based artificial peroxisome exhibits stable and multiple peroxisomal-like activities, including catalase, uricase, superoxide dismutase, peroxidase, and oxidase [14] ; assembling tri-enzyme (MenF, MenD, and MenH) on Mi3-a globular protein cage-to construct multienzyme assemblies for enhanced biocatalysis, which enhance cascade reactions in living cells [15] ; employing phospholipid molecule as building module to construct grana-like cisternae stacks via the reorganization of stacked micro-sized phospholipid bicelles to mimic grana functions. [16]Constructing aggregates inside living cells achieved intervention and regulation for cell fate and allowed to investigated the mechanisms of disease onset at the subcellular level, contributing to emerging solutions for precise therapeutics at the subcellular level. [17]Moreover, from the perspective of nanocarriers design, in contrast to small molecules, construction of intracellular artificial aggregates would force the carried small drug molecules accumulation in the cellular target site, which was proved to solve the circumvention of limitations that small molecule drugs face, such as drug resistance in cancer cells. [18]In general, the bottom-up approach of constructing artificial aggregates inside cells would be significant for elucidating molecular mechanisms of naturally occurring cellular processes and interfering with cellular processes, which would be a potential and novel strategy for treatment of diseases. [19]Integrating artificial aggregates in a biological context is in return an important milestone in materials science, as these material systems expand the boundaries of biomedicine. [20]n recent years, deoxyribonucleic acid (DNA) has been developed as a favored and biocompatible biomaterial to construct building-block for the fabrication of versatile functional materials, owing to its precise and programable synthesis. [21]Among various stimuli-responsive materials, DNA nanomaterials have proven to be a strong candidate for achieving dynamic, precise, and controlled intracellular aggregates due to the structural programmability, high biocompatibility and wide range of functionalization of DNA, [22] thus providing new insights into the biomedical development of precise regulation of cell fate.To be specific, DNA strand can be rationally designed and accurately synthesized from four deoxynucleotide monomers (adenine A, guanine G, cytosine C, and thymine T), and then the principle of Watson-Crick base pairing directs the formation of unique intermolecular hydrogen bonding between A and T, C, and G, resulting to the specific double helix structure of DNA, which is the molecular basis of the precise assembly of DNA; Moreover, the variety of DNA conformations and the dynamic molecular structures endows DNA assemblies with appealing responsiveness and functions.Besides, the sequence and structure accuracy of DNA make sure the precise regulation on the function of DNA materials by rationally changing the sequence of bases in DNA molecules.
Taken together, DNA is potential to meet the requirements of the precise construction of aggregates inside living cells, and recently we have tried to fabricating various dynamic DNA intelligent nanosystems for aggregates construction in living cells.In this review, we summarize our recent works based on the issue of DNA aggregates on micro/nano-scales for interference with cell processes (Figure 1).

STRATEGIES FOR CONSTRUCTING INTRACELLULAR DNA AGGREGATES
To successfully construct DNA aggregates inside living cells, the design of DNA assembly precursors for DNA aggregates should be precisely tailored to a specific intracellular microenvironment.In this review, we showcase double-strands DNA (dsDNA), branched DNA, DNA polyhedrons and DNA nanocomplex as assembly precursors to fabricate functional DNA aggregates within mammalian cells via endogenous stimuli mediation (Figure 2).Targeted endogenous stimuli include pH [27,28] and metal ions, [24] and those stimuli have been widely exploited in state-of-the-art prodrugs and delivery systems in biomedicine. [29]

Intracellular triggers for DNA molecules aggregation
In a living cell, various biomolecules including macromolecules, small molecules and ions are dispersed in the confined space, and the content and distribution usually different from that in the extracellular environment.Furthermore, the intracellular complicated physiological environment is orderly organized in multiple internal subcompartments on demand for well cell operation.For example, abundant K + distributes in cytoplasm to maintain the cellular osmotic pressure, and intracellular concentration of K + is about 30 times higher than that in extracellular environment. [30]Intracellular antioxidant reduced glutathione (GSH) spreads in cytoplasm as well, which plays a critical role in catabolizing peroxides catabolizing peroxides with the concentration of 2-10 mM in normal cells, about 100-1000 fold higher than that of extracellular environment. [31]Lysosome contains abundant hydrogen ions (H + ) to maintain the acidic environment (about pH 5.0) for the degradation of obsoleting biomacromolecules by the involved a vast amount of hydrolases. [17,32,33]All the differentiation of intracellular physiological environment from extracellular environment could be employed as an endogenous trigger for construction of aggregates, and herein, we mainly focus on the triggers for construction of DNA aggregates, including acidic pH, K + .

Acidic pH
Intracellular acidic environment can trigger DNA molecules containing cytosine-rich DNA sequences aggregation by formation of i-motif structure.In acidic environment, cytosinerich DNA sequences can be hemi-protonated and formed cytosine-cytosine + (C:C + ) base pairs, and these hemiprotonated C:CH + base pairs stack upon each other to fold into a stable tetraplex i-motif structure. [34]Therefore, incorporating two separated part cytosines into one DNA sequence (such as CCCCTAACCCC) allows the inter-sequence i-motif formation, following the assembly of DNA sequence in acidic environment.
Integration of cytosine-rich sequences into DNA nanomodules allows the intracellular acidic environment induced aggregation.The DNA nanomodules taken up by cells experience an increasingly acidic environment as they progress through the endocytic pathway. [35]Endocytic vesicles are the F I G U R E 1 Illustration of construction of DNA aggregates inside living cells, including aggregation triggers, aggregation precursors, and biological application.Reproduced with permission. [23]Copyright 2020, Wiley-VCH. [24]Copyright 2022, Nature Publishing Group. [25]Copyright 2022, American Chemical Society. [26]Copyright 2022, Wiley-VCH.
least acidic, whereas lysosomes can reach pH values as low as 4.5-4.7.Therefore, when DNA modules are transported from early endosomes to the lysosome (the cytosolic pH is slightly more acidic), they will undergo a pH gradient from 6.3 to 4.7, which stimulate conformational switching of pH-responsive components of i-motif. [26]The pH of environmental differentiation between subcellular regions offers the possibility of spatiotemporally controlled aggregation of i-motif structures, allowing i-motif-based DNA material systems to precisely assemble or disassemble at specific subcellular sites.

K +
Inside living cells, the concentration of intracellular K + is about 140 mM, which is about 30 times that of extracellular K + , and is used to balance osmotic pressure and transmit signals. [11]The significant difference in the concentration of K + between the intra-(140 mM) and extra-cellular (3-5 mM) environments facilitates the specifical formation of G-quadruplexes (GQ) in cells.Taking advantage of the significant difference in K + content, GQ structures can be introduced in DNA material systems for the realization of intracellular K + responsive assembly of DNA aggregates.GQ within specific repetitive G-rich sequences [36] are typically formed by four tracts of three Gs (G3) with intervening loops sequences of variable composition and length. [37]Sequences with longer (G4-7), shorter (G2), or uneven G-tracts also form GQ structures. [38]GQ can form within one strand (intramolecular) or from multiple strands (intermolecular). [39]GQ dynamics show that compared with double-stranded DNA, the structure of GQ is ion-dependent, and different cations have varying effects on its formation and stabilization. [40,41]Among many cations, K + is the most effective monovalent cation for promoting the formation and stabilizing GQ. [42,43] Therefore, incorporating two separated part guanine into one DNA sequence (such as TTGGGTTGGG) allows the inter-sequence GQ formation, following the assembly of DNA aggregates in presence of K + .

Long linear dsDNA
The linear dsDNA is commonly utilized as the basic unit for organizing various nanomaterials due to the easiest form to design and synthesize. [44]In particular, the dsDNA is designed with two sticky terminal sequences at each end for the further assembly.As for the preparation of DNA aggregates, the employment of long linear dsDNA is indispensable.However, the length of linear DNA synthesize is limited by the synthetic capability of solid-phase synthesis.Therefore, polymerase chain reaction (PCR), one of the methods of enzymatic extension, comes into being. [45]owever, the two ends of conventional PCR products are blunt, which limits their further assembly.To achieve the sticky ends-hanging PCR products, we previous rationally designed and synthesized a chemically cross-linked dsDNA containing two sticky ends to be adopted as primers for PCR. [23]To be specific, dsDNA primer was designed with high content of thymine base (T).Upon initiation of UV illu-mination, T base could react with 4′,5′,8′-trimethylpsoralen to form interstrand covalent cross-linked T-psoralen-T sites inside dsDNA, and consequently the dsDNA were chemically "locked" and can withstand the high temperature. [46]o be used as the primer of PCR, one of sticky end of the chemically cross-linked dsDNA were designed as the forward primer sequence or reverse primer sequence for the following PCR, and to endowing the PCR products assembly capacity, the other sticky end can be designed as the sequences that can be assembled (such as cytosine-rich sticky end that we designed).Therefore, by using the two chemically "locked" dsDNA as primers for PCR, a long linear dsDNA with assemble sticky ends was obtained.We designed and synthesized a 402 base-pair (bp) long linear DNA module with cytosine-rich sticky ends via the chemically "locked" dsDNA primers-based PCR, and the long linear DNA had an acidic stimuli specifically mediated topological transformation from nanoparticles to DNA aggregates. [23]2.2 Branched DNA Compared with linear DNA, the complex topologies and functional modules of branched DNA spanned DNA structures from multiple dimensions. [47]The branched DNA precursors as a building-block was traditionally composed of multiple-armed DNA strands derived from branched points (nucleoside or a non-nucleoside unit). [48]One of the topological structures of branched DNA precursors was single double-helix as an arm of branches.Li and coworkers first synthesized Y-shaped branched DNA (Y-DNA) as repetitive units of dendrimer-like DNA. [49]The Y-DNA with single double-helix as an arm was self-assembled from three single-stranded DNA (ssDNA) that were partially complementary to each other.In addition to Y-DNA, multibranched DNA structures surrounding a branched point have been prepared by Li's synthetic method [50] and the different number and length of branches affect equilibrium phase behaviors of branched DNA precursors. [51,52]Each arm of branched DNA precursors usually contains a double-stranded region and an extended sticky end.The crossed double-stranded regions are used to tighten complementary ssDNA to maintain the rigidity and stability of the structures.In addition, the flexible sticky ends could elongate branched DNA into DNA aggregates or connect multiple functional or responsive elements.We designed X-shaped DNA (X-DNA) and Y-DNA. [24]X-DNA contained four sticky ends (SEs), and Y-DNA contained two SEs as well as one i-motif sequence and the formation of DNA aggregate I via complementary base pairing of sticky ends (SE1 and SE2) between X-DNA and Y-DNA.In the presence of H + , DNA aggregate II was formed through cross-linking of i-motif sequence.

DNA polyhedrons
The tetrahedral DNA nanostructure (TDN), which has also recently been called 'tetrahedral framework nucleic acids', was successfully synthesized as the simplest DNA cube and first presented by Turberfield and co-workers in 2005. [53][56] Four single strands of DNA form one TDN.Each single strand contains three blocks of sequence, each of which is complementary to the sequence of one of the other single strands.Therefore, four triangles of DNA helices shape into a tetrahedral structure after hybridization.At each vertex of TDNs represent single oligonucleotides.Utilizing the programmability and unique modification vertexes of TDNs, through the introduction of response module, such as i-motif and GQ structures at vertexes of TDNs, TDNs can form aggregates with response assembly.For example, we constructed DNA-TDNs that three vertexes of TDNs were tethered with guaninerich sequences that promoted K + -mediated aggregation of tetrahedrons. [25]2.4DNA Nanocomplex In order to expand the "tool-box" of structure and function of DNA assembly precursors, DNA nanocomplex was synthesized by combining DNA with other polymers to construct more functional DNA aggregation. [57]DNA nanocomplex-assisted aggregates stand for composite structures consisting of other nanomaterials, in which DNA not only act as cross-linkers or for regulating the aggregation states of these aggregates, but also have functional roles to regulate cell fate.For example, via precipitation polymerization of NIPAM (N-isopropylacrylamide), 4-MAPBA ((4-methacrylamidophenyl) boronic acid), Bis (N,N-methylene diacrylamide), and Acrydite-DNA, the DNA cross-linked polymeric nanoframeworks were constructed.In these nanoframeworks, DNA was introduced as cross-linker of the polymer chain and could trigger hybridization chain reaction (HCR) of H1 and H2 hairpin monomers that were featured with single-stranded toeholds at their 3′ and 5′ ends, respectively.Therefore, by introducing response or functional modules in H1 or H2, functional DNA aggregates can be obtained. [58]

BIO-INTERFERENCE OF INTRACELLULAR DNA AGGREGATES
As we have mentioned before, endogenic molecular aggregates are usually created on demand to meet the precise requirement of the system at any given time in living cells, and the abnormal aggregates generation usually results in the cytopathy.Construction of exogenous aggregates in living systems allows intervention and regulation to cell fate, which contributes to investigate the molecular mechanisms of disease and further emerges solutions for precise therapeutics at the subcellular level.In recent, our laboratory has tried to construct various exogenous DNA aggregates for cell behavior interference such as cell cytoskeleton interference [23] and cell redox homeostasis interference, [24] or organelle interference such as mitochondrial [25] and lysosome interference. [26]

Interfering with cell cytoskeleton
The cytoskeleton is a class of mesh-like structures formed by the polymerization of protein fibers, which play a crucial role in maintaining cellular morphology, localizing organelles, material transport and signaling. [59,60]In the confined space of the cell, aggregates formed in response to intracellular stimuli disrupt the assembly process by binding cytoskeletal proteins such as actin, microtubulin and waveform proteins, thereby interfering with cellular metabolic responses and inducing autophagy and even apoptosis. [61,62]We developed dynamic assembly of organelle-like DNA aggregates with topological transformation within lysosomes. [23]A long chain of dsDNA (C-monomer) with cytosine-rich sticky ends was synthesized by PCR as the basic building block (Figure 3A), and the long chain was compressed into uniform spherical nanoparticles with a particle size of about 100 nm using high concentration of Mg 2+ (C-nanoparticle) (Figure 3B).When the nanoparticles were internalized into cells via lysosomal pathway, the nanoparticles first entered the acidic lysosomes.In the acidic environment, the Mg 2+compressed nanoparticles dissolved into a monomeric state, and further assembled into DNA aggregates through the intermolecular i-motif structures formation (Figure 3C).Cellular experiments demonstrated that the topological transformation from DNA long chain monomers to aggregates can interfere with the cytoskeleton of U87GM, as manifested by structural reorganization and orientation changes of actin filaments (Figure 3D).Scratch experiments demonstrated that the deformation of the U87GM cytoskeleton improved the migration ability and the scratch healing rate was significantly accelerated compared to the nonassembled group (Figure 3E).The dynamic self-assembled structures regulate cell behaviors via stimuli mediated topological transformation inside living cells, which will provide important implications for understanding on cellular behaviors, and is of great significance for the development of nanobiomedicines.

Interfering with cell redox homeostasis
Reactive oxygen species (ROS) are one of the byproducts of cellular metabolism and are involved in the transduction of a variety of intracellular signaling molecules, and the production and clearance of ROS at normal metabolic levels are in dynamic equilibrium. [63,64]The balance of intracellular ROS levels is maintained by a variety of organelles, including the endoplasmic reticulum, mitochondria, and peroxisomes. [65]mong them, peroxisomes are a class of multifunctional organelles widely found in eukaryotic cells, containing a large number of oxidoreductases, which can be used to eliminate excessive intracellular accumulation of ROS to alleviate oxidative stress and thus prevent the occurrence of various diseases. [8,66]rtificial organelles are biologically active intracellular nano-reactors that can enhance or replace the corresponding functions of natural organelles. [67]Considering the unique advantage of DNA molecules in the construction of dynamic assembly systems, we developed a DNA-human cerium oxide nanocomplex to achieve in situ construction of intracellular artificial peroxisome (AP). [24]By rational design of dendritic DNA, DNA building units with complementary sticky ends were synthesized to form DNA assemblies (DAS) through base complementary pairing.Then, cerium oxide nanoparticles assemble with the DAS through coordination with the phosphate backbone and electrostatic interactions Reproduced with permission. [23]opyright 2020, Wiley-VCH.
to form DNA-ceria nanocomplex (DCNC) with uniform, suitable dimensions for cellular uptake (Figure 4A).i-motif structures formed between the dendrimer DNAs in the intracellular acidic lysosomal environment are able to drive the assembly of nanoscale DCNCs into micron-scale aggregates and exhibit durable intracellular retention.Ceria has been reported to be the mimetics of enzymes due to its unique redox properties. [68,69]Inside living cells, cerium oxide exhibits catalytic activity as an active center that mimics the activities of superoxide dismutase, catalase and peroxidase, efficiently breaks down the intracellular excess accumulation of ROS to maintain redox homeostasis, and protects mitochondrial activity and cytoskeletal function from damage by oxidative stress (Figure 4B).Bio-effects of AP showed that AP possessed strong ability of ROS elimination, and maintained redox homeostasis via the regulation of intracellular ROS level (Figure 4C) and resistance of GSH consumption through ROS elimination (Figure 4D).CLSM images showed that the cytoskeleton in AP-treated group showed clear actin filaments, suggesting that with the protection of AP, cells maintained the structural integrity under oxidative stress (Figure 4E).Transwell assay results showed that the level of cell migration of AP-treated group was equal to that observed in blank group, indicating AP protected cells migration form the presence of high concentration of H 2 O 2 (Figure 5F).And fluorescence microscopy images of living/dead cell assay showed that propidium iodide (PI)-stained dead cell of APtreated group was lowest (Figure 4G).Compared with other strategies for constructing artificial organelles, our strategy achieved the in situ dynamic assembly to form functional DNA aggregates (AP) in living cells.

Interfering with mitochondria
Organelles are dynamic compartments within cells with unique functions that provide the environment and material basis for the normal conduct of various intracellular metabolic reactions and are important for the maintenance of cellular life activities.Organelle dysfunction affects cellular metabolic behavior and life processes and is closely related to the development of a range of diseases. [70]Therefore, rational regulation of organelle function is a promising tool for disease intervention and treatment, which has attracted great attention from researchers in recent years. [71,72]Mitochondria are energy transit stations within living cells and play an important regulatory role in cellular physiological metabolism and life processes, which are directly related Reproduced with permission. [24]Copyright 2022, Nature Publishing Group.
to the development and progression of metastatic solid tumors. [73]Dynamic intracellular aggregates can induce the release of cytochrome C by targeting mitochondria and altering their membrane potential, which in turn triggers a series of apoptotic cascade responses. [74] devised a self-assembly strategy based on TDNs to interfere with mitochondrial function and thus regulate cellular energy metabolism and migratory behavior. [25]One vertex of the DNA tetrahedra was modified with triphenylphosphine (TPP) for intracellular targeting to the mitochondrial surface.Reproduced with permission. [25]Copyright 2022, American Chemical Society.
A guanine-rich (G) sequence was designed on the other vertexes to fold into a G-tetrahedral structure in response to high levels of intracellular K + , thereby forming DNA aggregates on the mitochondrial surface (Figure 5A).The results of measuring the mitochondrial membrane potential of MCF-7 cells using the Mitochondrial Membrane Potential Kit showed that the red-green fluorescence intensity of TDNs-G-TPP-treated MCF-7 cells was significantly reduced compared with the control group, indicating that the mitochondrial surface self-assembly behavior effectively reduced the mitochondrial membrane potential (Figure 5B).Bio-transmission electron microscopy showed that the morphology of mitochondria became swollen due to the inhibition of mitosis of mitochondria by the surface-wrapped DNA assemblies (Figure 5C).Impairment of mitochondrial morphology and function directly affected cellular aerobic respiration and adenosine triphosphate (ATP) production (Figure 5D), which in turn affected other intracellular metabolic activities involved in energy currency ATP, such as cell migration.Transwell experiments showed that the percentage of migrating cells in the TDNs-G-TPP-treated group was significantly higher than that in the control group, demonstrating the decrease in cell migration ability (Figure 5E,F).Thus, the design of dynamic aggregate systems targeting mitochondria using DNA molecules can achieve precise effects on mitochondrial physiological functions while localizing the assembly on the mitochondrial surface, providing a feasible solution for the precise regulation of cellular physiological activities. .Reproduced with permission. [26]Copyright 2022, Wiley-VCH.

Interfering with lysosome
Lysosomes act as cellular recycling bins and are involved in a variety of intracellular biological processes, including degradation of metabolic waste and endocytic substrates, plasma membrane repair, and signal transduction. [75,76]Abnormal lysosomal function can lead to the development of various diseases, such as lysosomal accumulation disease, neurodegenerative diseases and metabolic diseases. [77,78]Therefore, the rational regulation of lysosome production or function using DNA dynamic assembly system will directly affect the intracellular homeostatic environment and cellular metabolic behavior, which may be a potential means of disease treatment.We constructed a NIPAM-DNA aggregates that can dynamically assemble within lysosomes to disrupt the lysosomal microenvironment. [26]The NIPAM-DNA nanoframework is mediated by the lysosomal engagement pathway to be taken up into cells and assembled into micron-sized aggregates by the i-motif structure driven by abundant pro-tons in lysosomes (Figure 6A).The dramatic increase in the size of the endocytosed aggregates interferes with the lysosomal efflux process, which manifests as a prolonged retention of the material in the lysosome (Figure 6B).On the other hand, the consumption of protons by the assembly process lowers the pH of the lysosomal microenvironment (Figure 6C,D), which in turn inhibits the activity of hydrolytic enzymes in the lysosomes and diminishes the degradation of the endocytosed material, which will facilitate the delivery of nucleic acid drugs such as siRNA.Fluorescence images of the effect of lysosome interference on gene silencing efficiency of siActin showed the weaker fluorescence signals were observed in NIPAM-DNA aggregates siActin-treated cells (Figure 6E,F).This work takes full advantage of the programmability and design refinement of DNA nanostructures to enable dynamic aggregate of nanomaterials coupled with biological processes within the lysosome, thus achieving precise regulation of organelle function, which is expected to guide the development of novel nanomedical material systems.

CONCLUSIONS AND PERSPECTIVES
Construction of DNA aggregates inside living cells via DNA precise assembly has been proved a successful strategy for cellular behaviors regulation.In this review, we summarized the recent advances in intracellular in situ construction of DNA aggregates mediated by cellular endogenous triggers, and the subsequent bio-interference of the DNA aggregates.
The unique cellular endogenous environment such as acidic lysosome or affluent K + , provides feasibility for triggering DNA precursors assembly in living cells.Multiple DNA precursor forms including long linear dsDNA, branched DNA, DNA tetrahedron and DNA nanocomplex have been utilized to construct DNA aggregates for bio-interference (Figure 7).The artificial DNA aggregates could interfere with the cellular behaviors or the subcellular organelle function by special function design in DNA materials.
In spite of the reported advances of DNA aggregates construction in living cells for bio-interference, more potential strategies are still to be explored to dedicate for more intelligent and versatile DNA aggregation systems for precision medicine (Figure 7).
1. Develop other more DNA precursors form.[86][87] All those DNA nanomaterials are potential to be rational designed as the DNA precursors for aggregates construction.Especially, DNA origami is a technique that uses hundreds of short DNA oligonucleotides(staple strands) to fold a long single-stranded DNA (scaffold strand) into desired shapes of nanostructures with addressable features. [88]Therefore, the responsive sequence, such as cytosine or guanine-rich sequence, would be designed according to the addressable feature of DNA origami to complete intracellular assembly.2. Develop more triggers and rich the trigger form.Various types of biomolecules contents in the confined cellular environment are significantly different from that of the extracellular environment, providing special triggers for intracellular assembly of DNA aggregate.Therefore, in addition to the triggers (pH and mental ions-K + ) already mentioned in the review, triggers-responsive assembly of DNA materials would undergo the "monomer-aggregate" transition when their cross-linked structures are also opened in response to biotic triggers (nucleic acids, enzymes and other biomolecules) or abiotic trigger (light).For example, numerous RNA molecules associated with pathology, such as microRNA (miRNA), [89] small interfering RNA (siRNA), messenger RNA (mRNA), and circular RNA (circRNA), not only could expose DNA sticky ends by strand displacement reaction but also is potential to directly serve as triggers to initiate HCR to form DNA aggregates.As for the impressive potential enzyme-based trigger, it is well known that nuclease enzymes would be able to cleave the phosphodiester bond of DNA to break the DNA strand at specific recognition sites. [21]When the recognition sites are introduced into the edges of DNA precursor, the enzyme can be employed to expose sticky ends of the DNA precursor, followed by the DNA precursor assembly into DNA aggregates through base complementary pairing.
In addition, lots of enzymes exhibited higher enzymatic activities in cancer cells compared with those of normal cells, [90] which provide specific environmental for construction of DNA aggregate in cancer cells.For example, apurinic/apyrimidinic endonuclease 1 (APE1), which is capable of cleaving apurinic/apyrimidinic (AP) site, is overexpressed in cancer cells [91] and could lead to exposing sticky ends of DNA precursors.Telomerase was a ribonucleoprotein reverse transcriptase which catalyzes the addition of hexamer DNA sequence repeats onto the end of telomeres and is greatly expressed in almost all cancer cells. [92]Up to now, telomerase-mediated DNA chain extensions have been as an excellent strategy for exposing specific single strand section of DNA through strand displacement, [93,94] which provides an opportunity for exposing sticky ends of DNA precursors.Besides the endogenic biotic triggers, various abiotic stimuli are potential to be employed as triggers for intracellular DNA aggregates construction. [95]For instance, light has precise spatiotemporal response properties and can be used as a physical stimulus factor to construct stable DNA aggregates. [96]In specific, photosensitive groups such as photoisomerizable molecules, photon-cleavable molecules, photon-caging molecules, or photopolymerizable could be introduced into DNA precursors.When DNA precursor is irradiated by different light, its conformational shift interrupts the DNA precursors, leading to exposing DNA sticky ends of DNA precursors to form DNA aggregates through base complementary pairing.Particularly, it is reassuring that the type, power, and wavelength of light are precisely adjustable and the light stimulation is reversible.Moreover, designing the assembly process mediated by biotic and and abiotic dual triggers would cater more to the needs of precision medicine.To achieve more precise and controllable construction strategies, it is essential to combine more advanced characterization techniques or instruments to visualize and characterize the assembly process and location of DNA aggregates in living cells.3. Enrich the biological function of the DNA aggregates.
DNA aggregates have received widespread attention in various disciplines such as chemistry, biology, and medicine and have resulted in a new research field of nanotechnology.We have tried to construct various exogenous DNA aggregates for organelle interference such as mitochondrial and lysosome interference.In living cells, different organelles have different internal structures among themselves and perform their own functions in intracellular material exchange, metabolic processes and signaling pathways.So, constructing exogenous DNA aggregates for other organelles (Endoplasmic reticulum, Golgi apparatus and cell membrane, etc.) interference can provide a new route for treatment of disease related to the different organelles, which also helps to explore the mechanisms of different diseases at the molecular level.In addition, aptamers, antisense oligonucleotides, and immunostimulatory CpG motifs, can be efficiently attached or integrated into DNA aggregates, which extend the function of the DNA aggregates.Moreover, DNA aggregates would be designed as artificial organelles by investing with specific function of specific organelle, which would be a potential strategy to replace or supplement the diseased organelles. [97]erall, precise construction of DNA aggregates in living cells is an emerging strategy for interfering with the biological processes.However, up to now, the vision of applying DNA aggregates to clinical applications is still in its infancy, and there are some key issues to be solved for the future clinical application of DNA aggregates.On the one hand, the common problems of DNA materials are the short blood circulation time in vivo and poor accumulation in target tissue.On the other hand, for the DNA aggregates, the duration of the DNA aggregates bio-interference is currently uncontrollable, which would bring about occurrence of undesirable side effects; in addition, the pharmacology and toxicology of the DNA aggregates is unclear, and the assembly of DNA nanomaterials into micro-sized aggregates would slow down their metabolism, thus leading to the biotoxicity.Therefore, the future development of this field would direct to the construction of assembly system that controllable in function and adjustable in assembly process, as well as the processes of absorption, distribution, metabolism, and excretion of assembly of DNA aggregates in vivo, and this will be a promising solution for precision and personalized medicine.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

F I G U R E 3
Construction of organelle-like DNA aggregates and cytoskeletal interference in living cells.(A) Design and synthesis of C-monomer by PCR.(B) Illustration of C-monomer into C-nanoparticle mediated by Mg 2+ and formation of DNA aggregates at pH 5.0.(C) Scheme of intracellular acidic microenvironment inducing topological transformation from nanoparticle to organelle-like DNA aggregates in lysosome and endosomal escape of the DNA aggregates.(D and E) Biological effects of organelle-like DNA aggregates on cell cytoskeleton (D) and migration (E).

F I G U R E 4
Construction of artificial peroxisome and regulation of ROS in living cells.(A) Illustration of the construction of DCNC.SE, sticky end.(B) Intracellular lysosomal acidic condition-induced assembly of DCNC into artificial peroxisome.ROS, reactive oxygen species.(C) Flow cytometry of ROS levels treated with a DCFH-DA fluorescent probe.(D) Relative concentration of intracellular GSH.(E) Representative confocal microscopy images of F-actin stained with FITC-phalloidine.(F) Microscopy images of cells migration in a transwell cell test.(G) Fluorescence microscopy images showing living/dead cell assay.

F I G U R E 5
Construction of the assembly of TDNs-G-TPP and mitochondrial interference in living cells.(A) Illustration of the formation of DNA aggregates and interference with mitochondria.(B) Mitochondrial membrane potential measurement with JC-1 Assay Kit.(C) Biological transmission electron microscopy (Bio-TEM) images of MCF-7 cells with different treatments.(D) Intracellular adenosine triphosphate (ATP) levels of MCF-7 cells with different treatments.(E) Schematic illustration of transwell migration assay.(F) Cellular migration of MCF-7 cells with different treatments in transwell migration assay.

F I G U R E 6
Construction of DNA nanocomplex aggregates and lysosome interference in living cells.(A) Proton-driven dynamic assembly of NIPAM-DNA aggregates inside cells couples with the lysosome-mediated endocytosis pathways/lysosomal maturation and lysosome interference.(B) Flow cytometry for analyzing the intracellular retention of NIPAM-DNA aggregates.(C) TRAP staining images of cells treated with NIPAM-DNA aggregates.(D) Gray value statistics of TRAP staining in (C).(E) Fluorescence images of β-actin in cells treated with different formulations.(F) Mean fluorescence intensity statistics of β-actin in (E)

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C K N O W L E D G M E N T S This work was supported in part by National Natural Science Foundation of China (grant numbers: 22225505 and 22105050), China Postdoctoral Science Foundation (grant number: 2021M70079), and the Special Research Assistant Project of Chinese Academy of Sciences (grant number: E2F25111).