Monomeric DNA Research Articles

Targeted Reprogramming of Pathogenic Fibroblast Genes at the 3'-Untranslated Regions by DNA Nanorobots for Periodontitis.

Periodontitis, with its persistent nature, causes significant distress for most sufferers. Current treatments, such as mechanical cleaning and surgery, often fail to fully address the underlying overactivation of fibroblasts that drives this degradation. Targeting the post-transcriptional regulation of fibroblasts, particularly at the 3'-untranslated regions (3'UTR) of pathogenic genes, offers a therapeutic strategy for periodontitis. Herein, we developed a DNA nanorobot for this purpose. This system uses a dynamic DNA nanoframework to incorporate therapeutic microRNAs through molecular recognition and covalent bonds, facilitated by DNA monomers modified with disulfide bonds. The assembled-DNA nanoframework is encapsulated in a cell membrane embedded with a fibroblast-targeting peptide. By analyzing the 3'UTR regions of pathogenic fibroblast genes FOSB and JUND, we identified the therapeutic microRNA as miR-1-3p and integrated it into this system. As expected, the DNA nanorobot delivered the internal components to fibroblasts by the targeting peptide and outer membrane that responsively releases miR-1-3p under intracellular glutathione. It resulted in a precise reduction of mRNA and suppression of protein function in pathogenic genes, effectively reprogramming fibroblast behavior. Our results confirm that this approach not only mitigates the inflammation but also promotes tissue regeneration in periodontal models, offering a promising therapeutic avenue for periodontitis.

Study of different heterocycles showing significant anti-severe acute respiratory syndrome 2 activity in vitro and in vivo.

With the emergence of severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), antiviral drug development has gained increased significance due to the high incidence and potentially severe complications of the resulting coronavirus infection. Heterocycle compounds, acting as antimetabolites of DNA and RNA monomers, rank among the most effective antiviral drugs. These compounds' antiviral effects on various SARS-CoV-2 isolates, as found in existing data collections, form the basis for further research. The aim of this study was to examine the possible antiviral effect of some originally synthesized heterocyclic compounds. The main methods were cell culturing, cytotoxicity assay, qRT-PCR assay, tissue and blood cells analysis, and micro-computed tomography (micro-CT) imaging. In both in vitro and in vivo conditions, the elimination of SARS-Cov-2 occurred significantly earlier after administration of the compounds compared to the control group. In hamsters, the primary symptoms of coronavirus disease disappeared following administration of heterocycle compounds. Using delta and omicron strains of the SARS-CoV-2 virus, newly created heterocycle compound analogs dramatically reduced SARS-CoV-2 multiplication, resulting in a drop in viral RNA load in the supernatant under in vitro conditions. Improvements in pathological manifestations in the blood, bone marrow, and internal organs of hamsters demonstrated that heterocycle compounds inhibited SARS-CoV-2 replication both in vitro and in vivo.

Prediction of DNA origami shape using graph neural network.

Unlike proteins, which have a wealth of validated structural data, experimentally or computationally validated DNA origami datasets are limited. Here we present a graph neural network that can predict the three-dimensional conformation of DNA origami assemblies both rapidly and accurately. We develop a hybrid data-driven and physics-informed approach for model training, designed to minimize not only the data-driven loss but also the physics-informed loss. By employing an ensemble strategy, the model can successfully infer the shape of monomeric DNA origami structures almost in real time. Further refinement of the model in an unsupervised manner enables the analysis of supramolecular assemblies consisting of tens to hundreds of DNA blocks. The proposed model enables an automated inverse design of DNA origami structures for given target shapes. Our approach facilitates the real-time virtual prototyping of DNA origami, broadening its design space.

An acid-responsive DNA hydrogel-mediated cascaded enzymatic nucleic acid amplification system for the sensitive imaging of alkaline phosphatase in living cells.

Alkaline phosphatase (ALP) is a class of hydrolase that catalyzes the dephosphorylation of phosphorylated species in biological tissues, playing an important role in many physiological and pathological processes. Sensitive imaging of ALP activity in living cells is contributory to the research on these processes. Herein, we propose an acid-responsive DNA hydrogel to deliver a cascaded enzymatic nucleic acid amplification system into cells for the sensitive imaging of intracellular ALP activity. The DNA hydrogel is formed by two kinds of Y-shaped DNA monomers and acid-responsive cytosine-rich linkers. The amplification system contained Bst DNA polymerase (Bst DP), Nt.BbvCI endonuclease, a Recognition Probe (RP, containing a DNAzyme sequence, a Nt.BbvCI recognition sequence, and a phosphate group at the 3'-end), and a Signal Probe (SP, containing a cleavage site for DNAzyme, Cy3 and BHQ2 at the two ends). The amplification system was trapped into the DNA hydrogel and taken up by cells, and the cytosine-rich linkers folded into a quadruplex i-motif in the acidic lysosomes, leading to the collapse of the hydrogel and releasing the amplification system. The phosphate groups on RPs were recognized and removed by the target ALP, triggering a polymerization-nicking cycle to produce large numbers of DNAzyme sequences, which then cleaved multiple SPs, restoring Cy3 fluorescence to indicate the ALP activity. This strategy achieved sensitive imaging of ALP in living HeLa, MCF-7, and NCM460 cells, and realized the sensitive detection of ALP in vitro with a detection limit of 2.0 × 10-5 U mL-1, providing a potential tool for the research of ALP-related physiological and pathological processes.

Enzyme-Free Exponential Amplification via Growth and Scission of Crisscross Ribbons from Single-Stranded DNA Components.

The self-assembly of DNA-based monomers into higher-order structures has significant potential for realizing various biomimetic behaviors including algorithmic assembly, ultrasensitive detection, and self-replication. For these behaviors, it is desirable to implement high energetic barriers to undesired spurious nucleation, where such barriers can be bypassed via seed-initiated assembly. Joint-neighbor capture is a mechanism enabling the construction of such barriers while allowing for algorithmic behaviors, such as bit-copying. Cycles of polymerization with division could accordingly be used for implementing exponential growth in self-replicating materials. Previously, we demonstrated crisscross polymerization, a strategy that attains robust seed-dependent self-assembly of single-stranded DNA and DNA-origami monomers via joint-neighbor capture. Here, we expand the crisscross assembly to achieve autonomous, isothermal exponential amplification of ribbons through their concurrent growth and scission via toehold-mediated strand displacement. We demonstrate how this crisscross chain reaction, or 3CR, can be used as a detection strategy through coupling to single- and double-stranded nucleic acid targets and introduce a rule-based stochastic modeling approach for simulating molecular self-assembly behaviors such as crisscross-ribbon scission.

Multifunctional DNA nanosphere platform for real-time monitoring and fluorescence imaging-guided photodynamic therapy

The DNA nanosphere (DNS) is constructed through the assembly of multifunctional Y-scaffold DNA monomers, which encompass aptamers, RNA interferences, and photodynamic therapy agents. The DNS nanoplatform facilitates efficient cellular uptake via aptamer-mediated delivery. The RNA interferences inhibit the microRNA-21 and microRNA-155, thereby enabling precise imaging guidance for photodynamic therapy, ultimately leading to apoptosis in MCF-7 cells. The platform allows for real-time monitoring of the therapeutic process, presenting a more comprehensive approach to cancer treatment. The results of in vitro and in vivo studies suggest the potential of this multifunctional DNS platform as a promising approach for cancer therapy that can be tailored to individual patients based on their specific cancer type.

Self-Assembled DNA Nanospheres: Design and Applications

Self-assembled DNA nanospheres, as versatile and ideal vehicles, have offered new opportunities to create intelligent delivery systems for precise bioimaging and cancer therapy, due to their good biostability and cell permeability, large loading capacity, and programmable self–assembly behaviors. DNA nanospheres can be synthesized by the self–assembly of Y–shaped DNA monomers, ultra–long single-stranded DNA (ssDNA), and even metal–DNA coordination. Interestingly, they are size–controllable by varying some parameters including concentration, reaction time, and mixing ratio. This review summarizes the design of DNA nanospheres and their extensive biomedical applications. First, the characteristics of DNA are briefly introduced, and different DNA nanostructures are mentioned. Then, the design of DNA nanospheres is emphasized and classified into three main categories, including Y–shaped DNA unit self-assembly by Watson–Crick base pairing, liquid crystallization and the dense packaging of ultra–long DNA strands generated via rolling circle amplification (RCA), and metal–DNA coordination–driven hybrids. Meanwhile, the advantages and disadvantages of different self–assembled DNA nanospheres are discussed, respectively. Next, the biomedical applications of DNA nanospheres are mainly focused on. Especially, DNA nanospheres serve as promising nanocarriers to deliver functional nucleic acids and drugs for biosensing, bioimaging, and therapeutics. Finally, the current challenges and perspectives for self-assembled DNA nanospheres in the future are provided.

1H, 13C, and 15N resonance assignments of SarA monomer from Staphylococcus aureus in complex with DNA.

SarA is a global transcription regulator in S. aureus which regulates the expression of over 120 genes related to quorum sensing, biofilm synthesis, drug resistance and many other important physiological processes during host infection. SarA can bind to the promoter region of agr and other target genes to activate or repress the transcription. The crystal structure of SarA uncovered a MarR protein-like conformation with two symmetrical winged helix domains, while its DNA binding mechanism is still unknown. We have constructed a monomeric DNA binding domain of SarA (SarAΔN19) for the study of the interaction between SarA and DNA with NMR spectroscopy. Here, we report the 1H, 13C and 15N NMR assignment of SarAΔN19/DNA complex which is the first step towards further structure and function analysis.

Mid- and far-infrared spectroscopy of nucleobases: molar extinction coefficients, integrated molar absorptivity, and temperature dependence of the main bands

ABSTRACT The nucleobases, namely the purines adenine and guanine and the pyrimidines cytosine, thymine, and uracil, are the key monomers of the information polymers DNA and RNA. All the nucleobases were recently detected in the analysis of certain meteorites known as carbonaceous chondrites. The concentration of the nucleobases found in meteorites is smaller than the concentration of amino acids. However, the nucleobases were successfully synthesized abiotically by ultraviolet irradiation of interstellar ice analogues at 10 K. Consequently, it is possible that the purines and pyrimidines may be present in the interstellar medium or in other space environments and may be detectable by the new orbiting telescopes that indeed are able to cover not only the mid-infrared spectral range but also the far-infrared. This work supplies the reference spectra of the five nucleobases used by the terrestrial biochemistry in the mid-infrared and in the far-infrared with the purpose to facilitate the search and identification of these astrobiological and astrochemical relevant molecules in space. The mid-infrared spectra of all nucleobases were recorded at three different temperatures from −180 °C to nearly ambient temperature and at +250 °C. Furthermore, the molar extinction coefficients (ε) of all mid- and far-infrared bands were determined, as well as the integrated molar absorptivities (ψ). With the reported infrared spectra together with the ε and ψ values, it will be possible to identify qualitatively the nucleobases and estimate their relative abundance in space.

Autonomous and Programmable Reorganization of DNA‐Based Polymers Using Redox Chemistry**

We demonstrate here a strategy that allows the programmable and autonomous reorganization of self-assembled DNA polymers using redox chemistry. We have rationally designed different DNA monomers (tiles) that can co-assemble into tubular structures. The tiles can be orthogonally activated/deactivated with disulfide-linked DNA fuel strands that are degraded over time upon reduction because of the presence of a reducing agent in the system. The concentration of the disulfide fuels determines the activation kinetics of each DNA tile, which controls the degree of order/disorder in the formed co-polymer. The disulfide-reduction pathway can be employed together with enzymatic fuel-degradation pathways providing an additional level of control in the re-organization of DNA structures. Taking advantage of the different pH-sensitivities of disulfide-thiol and enzymatic reactions, we show that we can control the order in DNA-based co-polymers as a function of pH.

Spotlights on Recent JACS Publications, Volume 145, Issue 9.

ADVERTISEMENT RETURN TO ISSUESpotlightsNEXTSpotlights on Recent JACS Publications, Volume 145, Issue 9ACS Contributing CorrespondentsACS Contributing CorrespondentsMore by ACS Contributing CorrespondentsCite this: J. Am. Chem. Soc. 2023, 145, 9, 4901–4902Publication Date (Web):February 28, 2023Publication History Received27 February 2023Published online28 February 2023Published inissue 8 March 2023https://doi.org/10.1021/jacs.3c02138Copyright © 2023 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views792Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (551 KB) Get e-AlertsSUBJECTS:DNA origami,Drug delivery,Gene delivery,Monomers,Peptides and proteins Get e-Alerts

Geometry guided crystallization of anisotropic DNA origami shapes.

Three-dimensional assembly based on DNA origami structures is an ideal method to precisely fabricate nano-scale materials. Additionally, applying an anisotropic assembly unit facilitates constructing complex materials with extraordinary structure. However, it still remains challenging to crystallize anisotropic DNA nano-structures using simple design, because the assembly of low-symmetry monomers often requires harsh auxiliary conditions and more complicated crystallization processes. In this work, we managed to crystallize the anisotropic elongated octahedral DNA origami frames by non-specific connections, and acquired two kinds of highly ordered superlattices purely by conducting multiple annealing processes and increasing the rigidity of the connection parts. In the case where the connection parts were composed of soft DNA sticky ends, we obtained the theoretically inaccessible simple cubic superlattices by this anisotropic DNA origami shape. Through characterization by small-angle X-ray scattering and scanning electron microscopy, we found that the DNA monomers are arbitrarily arranged due to the stress buffering of the soft DNA SEs, while in the stiffer case, simple tetragonal superlattices with translational arrangement of most anisotropic DNA origami shapes were synthesized as expected. This work deepened the understanding of geometry-guided crystallization of DNA origami shapes and provided a new path for constructing three-dimensional functional devices with simple design.

Technology of genomic balancing of chromatin of autologous hematopoietic stem cells for gene therapy of fatal immune-mediated diseases of civilization, extended life expectancy and sudden human death prevention.

A biotechnology for personalized ex vivo gene therapy based on molecular genomic balancing of hematopoietic stem cell (HSC) chromatin with nucleosome monomers of human genomic DNA (hDNAnmr) has been developed and implemented in the clinic to change (to "correct") mutant chromosome loci genomes of dominant HSC clones that form mono- and oligoclonal hematopoiesis during aging and major (oncological, cardiovascular, neurodegenerative and autoimmune) fatal immune-mediated diseases of civilization. A fundamentally new biotechnological approach has been applied to the delivery of genetic material into eukaryotic stem and progenitor cells by establishing an artificial "recombinogenic situation" in them to induce homologous recombination (equivalent replacement) of mutant DNA regions with healthy hDNAnmr. In experimental preclinical trials, the effectiveness of genomic balancing technology has been proven to reduce the risk of sudden death in old animals and to increase the lifespan of outbred mice by 30% and Wistar rats by 57%. The improvement in their quality of life, compared with the control, is explained by an increase in the telomeric regions of the HSCs and HPCs chromosomes by 1.5-2 times. The potential of the technology to slow down the hereditary neurodegenerative diseases on the model of amyotrophic lateral sclerosis is shown. The effectiveness of this technology in clinical practice is presented on the example of a terminal patient with stage 4 neuroendocrine cancer. This technology used in the treatment of a number of oncological, neurodegenerative, autoimmune and hereditary diseases with clonal hematopoiesis is able to arrest the progression of the disease, prevent its recurrence, prolong the active life of a person, increase the average life expectancy and prevent sudden death.

DNA micelle-templated copper nanoclusters for fluorescent imaging of MUC1-positive cancer cells.

DNA micelles formed by hydrophobic, self-assembly of amphiphilic DNA monomers have enormous potential in biological imaging owing to its unique and programmable, three-dimensional nanostructure. Herein, we rationally design double-stranded DNA oligonucleotides with two cholesterols that can spontaneously form the lipid-mediated DNA micelles and generate the high fluorescence signal after the formation of DNA-templated copper nanoclusters (CuNCs). Furthermore, the DNA aptamer specific to MUC1 protein, aberrantly overexpressed on the surface of cancer cells, is attached to lipid-mediated DNA micelles to confer the selectivity towards the target cancer cells. With the well-defined DNA nanostructures, the cell membrane of MUC1-positive cancer cells are stained by CuNCs exhibiting an intense, red fluorescence signal, which are clearly distinguished from MUC1-negative cancer cells. This approach may not only expand the application scope of both DNA micells and CuNCs, especially in the area of cellular imaging, but also provides a basis for developing other types of DNA nanostructures to detect target biomarkers.

Size-Controllable and Self-Assembled DNA Nanosphere for Amplified MicroRNA Imaging through ATP-Fueled Cyclic Dissociation.

Visualizing intracellular microRNA (miRNA) is of great importance for revealing its roles in the development of disease. However, cell membrane barrier, complex intracellular environment and low abundance of target miRNA are three main challenges for efficient imaging of intracellular miRNA. Here, we report a size-controllable and self-assembled DNA nanosphere with ATP-fueled dissociation property for amplified miRNA imaging in live cells and mice. The DNA nanosphere was self-assembled from Y-shaped DNA (Y-DNA) monomers through predesigned base pair hybridization, and the size could be easily controlled by varying the concentration of Y-DNA. Once the nanosphere was internalized into cells, the intracellular specific target miRNA would trigger the cyclic dissociation of the DNA nanosphere driven by ATP, resulting in amplified FRET signal. The programmable DNA nanosphere has been proven to work well for detecting the expression of miRNA in cancer cells and in mice, which demonstrates its fairish cell penetration, stability and sensitivity.

Recognition of DNA Supercoil Handedness during Catenation Catalyzed by Type II Topoisomerases.

Although the presence of catenanes (i.e., intermolecular tangles) in chromosomal DNA stabilizes interactions between daughter chromosomes, a lack of resolution can have serious consequences for genomic stability. In all species, from bacteria to humans, type II topoisomerases are the enzymes primarily responsible for catenating/decatenating DNA. DNA topology has a profound influence on the rate at which these enzymes alter the superhelical state of the double helix. Therefore, the effect of supercoil handedness on the ability of human topoisomerase IIα and topoisomerase IIβ and bacterial topoisomerase IV to catenate DNA was examined. Topoisomerase IIα preferentially catenated negatively supercoiled over positively supercoiled substrates. This is opposite to its preference for relaxing (i.e., removing supercoils from) DNA and may prevent the enzyme from tangling the double helix ahead of replication forks and transcription complexes. The ability of topoisomerase IIα to recognize DNA supercoil handedness during catenation resides in its C-terminal domain. In contrast to topoisomerase IIα, topoisomerase IIβ displayed little ability to distinguish DNA geometry during catenation. Topoisomerase IV from three bacterial species preferentially catenated positively supercoiled substrates. This may not be an issue, as these enzymes work primarily behind replication forks. Finally, topoisomerase IIα and topoisomerase IV maintain lower levels of covalent enzyme-cleaved DNA intermediates with catenated over monomeric DNA. This allows these enzymes to perform their cellular functions in a safer manner, as catenated daughter chromosomes may be subject to stress generated by the mitotic spindle that could lead to irreversible DNA cleavage.

Abstract 1738: DNA-launched nanoparticle vaccines induce cytolytic T cell responses for in vivo tumor control

Abstract Nanotechnology-based approaches have achieved promising results for the development of novel immunotherapeutic cancer vaccines through the decoration of antigens on nanoparticle scaffolds for multivalent display. However, their translation have been limited due to challenges with purification, scalability, and robust induction of cytolytic T cell responses critical to anti-tumor activity. We designed a nanovaccine construct by fusing the HPV E7 immunodominant epitope onto lumazine synthase scaffold. We demonstrate that through electroporation-facilitated DNA delivery encoding the antigen-scaffold construct, this nanovaccine is expressed and self-assembles in vivo into 60-mer nanoparticles. Compared to CpG-adjuvanted peptide and monomeric DNA vaccination, this DNA-launched nanovaccine induced greater CD8+ cellular responses against the E7 epitope and target tumor cells. In both flank and lung models of TC-1 HPV-associated cancers, the nanovaccine achieved significant tumor regression and prolonged survival compared to CpG-adjuvanted peptide. Anti-tumor immunity was retained upon rechallenge >150 days after initial challenge, supporting the induction of long-term immune memory. In addition, we demonstrate that this DNA-launched nanovaccine platform can be used to form hybrid nanoparticles displaying multiple antigens simultaneously. These hybrid nanoparticles exhibit synergistic efficacy, with improved tumor control over administration of separate single-antigen nanovaccines. These promising results, along with facile characterization, scalability, and stability, support that DNA-launched nanovaccines can induce potent anti-tumor cellular immunity and are ideally positioned for further translational development for clinical evaluation. Citation Format: Kevin Liaw, Ziyang Xu, Neethu Chokkalingam, Daniel Kulp, David Weiner. DNA-launched nanoparticle vaccines induce cytolytic T cell responses for in vivo tumor control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1738.

Construction of Branched DNA-based Nanostructures for Diagnosis, Therapeutics and Protein Engineering.

Branched DNA with multibranch-like anisotropic topology serves as a promising and powerful building block in constructing multifunctional-integrated nanomaterials in a programmable and controllable manner. Recently, a series of branched DNA-based functional nanomaterials were developed by elaborate molecular design. In this review, we focused on the construction of branched DNA-based nanostructures for biological and biomedical applications. First, the molecular design and synthesis method of branched DNA monomer were briefly described. Then, the construction strategies of branched DNA-based nanostructures were categorially discussed, including target-triggered polymerization, enzymatic extension and hybrid assembly. Finally, the biological and biomedical applications including diagnosis, therapeutics and protein engineering were summarized. We envision that the review will contribute to the further development of branched DNA-based nanomaterials with great application potential in the field of biomedicine, thus building a new bridge between material chemistry and biomedicine.

Centromere Innovations with a Mouse Species

Mammalian centromeres direct faithful genetic inheritance at cell division and are typically found in regions of highly repetitive DNA, such as a‐satellite in humans and minor satellite in many mouse species. In the house mouse M. musculus, the 120 bp DNA monomers are nearly identical (~95%) to each other, and CENP‐A nucleosomes containing the centromeric histone H3 variant, CENP‐A, are positioned with their center position, the nucleosomal dyad, within the DNA recognition element for the sequence‐specific DNA‐binding protein, CENP‐B. We find that CENP‐B‐/‐ mice have precisely the same positioning of CENP‐A nucleosomes as CENP‐B+/+ mice, indicating a potential role for DNA sequence in the positioning of CENP‐A nucleosomes. M. pahari diverged from M. musculus ~ 4 M years ago and has been reported to have a different general DNA sequence organization at centromeres, so we sought to determine the features of centromeric chromatin that were conserved between these species. We found that, on most M. pahari centromeres, CENP‐B is of very low abundance but is extremely enriched on a single chromosome. Using multiple genomic strategies, we identified the most abundant centromere repeat in M. pahari that we term pi‐satellite, which is ~189 bp long and organized into tandem repeat arrays. Chromosomes harboring pi‐satellite lack CENP‐B boxes entirely. Additionally, we employed a more sophisticated strategy to identify a less abundant centromere satellite that contains the CENP‐B box. This variant exhibits greater homogenization, is 188 bp long, and is found exclusively on the single centromere containing extremely high levels of CENP‐B. This centromere also recruits more constitutive heterochromatin, marked by H3K9me3, consistent with a role for CENP‐B in driving pericentromeric heterochromatin. Together, these findings reveal that a mouse species is undergoing dramatic molecular differences directed by evolutionary innovations in centromere DNA sequence.

Formation of non-base-pairing DNA microgels using directed phase transition of amphiphilic monomers.

Programmability of DNA sequences enables the formation of synthetic DNA nanostructures and their macromolecular assemblies such as DNA hydrogels. The base pair-level interaction of DNA is a foundational and powerful mechanism to build DNA structures at the nanoscale; however, its temperature sensitivity and weak interaction force remain a barrier for the facile and scalable assembly of DNA structures toward higher-order structures. We conducted this study to provide an alternative, non-base-pairing approach to connect nanoscale DNA units to yield micrometer-sized gels based on the sequential phase transition of amphiphilic unit structures. Strong electrostatic interactions between DNA nanostructures and polyelectrolyte spermines led to the formation of giant phase-separated aggregates of monomer units. Gelation could be initiated by the addition of NaCl, which weakened the electrostatic DNA-spermine interaction while attractive interactions between cholesterols created stable networks by crosslinking DNA monomers. In contrast to the conventional DNA gelation techniques, our system used solid aggregates as a precursor for DNA microgels. Therefore, in situ gelation could be achieved by depositing aggregates on the desired substrate and subsequently initiating a phase transition. Our approach can expand the utility and functionality of DNA hydrogels by using more complex nucleic acid assemblies as unit structures and combining the technique with top-down microfabrication methods.