Long and Covalently Conjugated Branched DNA Structures for in Situ Gelation in Vivo

Open AccessCCS ChemistryRESEARCH ARTICLES14 Dec 2022Long and Covalently Conjugated Branched DNA Structures for in Situ Gelation in Vivo Xuehe Lu†, Hong Wang†, Ruofan Chen†, Tiantian Wu, Xiaohui Wu, Yingxu Shang, Yuang Wang, Wantao Tang, Dongsheng Liu, Jianbing Liu and Baoquan Ding Xuehe Lu† CAS Key Laboratory of Nanosystem and Hierarchical Fabrication, National Center for Nanoscience and Technology, Beijing 100190 School of Materials Science and Engineering, Henan Institute of Advanced Technology, Zhengzhou University, Zhengzhou 450001 , Hong Wang† CAS Key Laboratory of Nanosystem and Hierarchical Fabrication, National Center for Nanoscience and Technology, Beijing 100190 , Ruofan Chen† Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Department of Chemistry, Tsinghua University, Beijing 100084 , Tiantian Wu CAS Key Laboratory of Nanosystem and Hierarchical Fabrication, National Center for Nanoscience and Technology, Beijing 100190 , Xiaohui Wu CAS Key Laboratory of Nanosystem and Hierarchical Fabrication, National Center for Nanoscience and Technology, Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 , Yingxu Shang CAS Key Laboratory of Nanosystem and Hierarchical Fabrication, National Center for Nanoscience and Technology, Beijing 100190 , Yuang Wang CAS Key Laboratory of Nanosystem and Hierarchical Fabrication, National Center for Nanoscience and Technology, Beijing 100190 , Wantao Tang CAS Key Laboratory of Nanosystem and Hierarchical Fabrication, National Center for Nanoscience and Technology, Beijing 100190 School of Materials Science and Engineering, Henan Institute of Advanced Technology, Zhengzhou University, Zhengzhou 450001 , Dongsheng Liu Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Department of Chemistry, Tsinghua University, Beijing 100084 , Jianbing Liu *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] CAS Key Laboratory of Nanosystem and Hierarchical Fabrication, National Center for Nanoscience and Technology, Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 and Baoquan Ding *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] CAS Key Laboratory of Nanosystem and Hierarchical Fabrication, National Center for Nanoscience and Technology, Beijing 100190 School of Materials Science and Engineering, Henan Institute of Advanced Technology, Zhengzhou University, Zhengzhou 450001 University of Chinese Academy of Sciences, Beijing 100049 https://doi.org/10.31635/ccschem.022.202202577 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail DNA nanotechnology has been widely employed for biomedical applications. However, most DNA nanomaterials rely on noncovalent complementary base pairing of short single-stranded DNA oligonucleotides. Herein, we describe a general strategy to construct a long and covalently conjugated branched DNA structure for fast and in situ gelation in vivo. In our design, a short and covalently conjugated branched DNA structure can normally be employed as the DNA primer in the terminal deoxynucleotidyl transferase-dependent enzymatic polymerization system. After enzymatic extension, the DNA aptamer-modified branched DNA structures with the sequences of poly T or poly A can immediately coassemble for in situ encapsulation of the target protein and tumor cell. The fast and in situ gelation system can function in a murine model of local tumor recurrence for targeting residual tumor cells to achieve long-term drug release for efficient tumor inhibition in vivo. This rationally developed DNA self-assembly strategy provides a new avenue for the development of multifunctional DNA nanomaterials. Download figure Download PowerPoint Introduction DNA, well known as a genetic information carrier, has also been successfully employed as the building block for various exquisite nanostructures, such as DNA polyhedrons, DNA origamis, and DNA hydrogels.1–10 These precisely assembled DNA nanostructures have been widely developed for biomedical applications, including bioimaging, biosensing, and drug delivery.11–29 Mostly, these assembled DNA nanostructures are constructed by the noncovalent complementary base pairing of rationally designed short DNA sequences. In these assembly systems, DNA junctions with multiple double-stranded DNA-based arms are generated as the connectors.30–35 These rigid and noncovalently assembled DNA junctions restrict the efficient construction of flexible and thermally stable DNA structures. In order to address this issue, many efforts have been developed to add flexible and thermally stable elements to the assembly system, such as unpaired DNA loops and covalently conjugated branched DNA structures.36–49 The unpaired DNA loop can increase the flexibility of the DNA connectors to construct a series of DNA nanostructures with different morphology.36,37 Meanwhile, these single-stranded DNA oligonucleotides modified with flexible spacers in the terminal can be cross-linked by branched organic molecules to construct flexible and thermally stable branched DNA structures.38–44 These branched DNA structures have been rationally designed as the monomer for the construction of nanoparticle (low concentration) or DNA hydrogel (high concentration).45–49 However, due to the short DNA arms, these covalently conjugated branched DNA structures demonstrate a relatively slow coassembly rate with a high concentration of monomers. Thus, we hypothesize that the flexible and covalently conjugated branched DNA structures with extended long DNA arms may result in the fast and in situ formation of highly dense and thermally stable DNA materials. Terminal deoxynucleotidyl transferase (TdT) is a kind of DNA polymerase. Different from the classic polymerase chain reaction system, the TdT-dependent enzymatic polymerization system is free of DNA template (target double-stranded DNA), specific primer sequences (designed forward and reverse primer pairs), and cyclic temperature conditions (different temperatures for denaturation, annealing, and elongation). Simply, a random DNA oligonucleotide with exposed 3′ hydroxyl (as the primer), deoxy-ribonucleoside triphosphate (dNTP) (as the monomer), and TdT (as the DNA polymerase) are enough for the extension of single-stranded DNA under a constant temperature of 37 °C. Due to these inherent advantages, the TdT-dependent enzymatic polymerization system has been widely employed as the chemical modifier for efficient modification of single-stranded DNA in the 3′ terminal.50–52 Representatively, the controllable extension of 3′ overhang of staple strands in the DNA origami has been achieved to form a protective DNA layer for enhanced enzymatic stability.53 Meanwhile, the extension of the noncovalent DNA junctions has been realized for the efficient construction of the DNA hydrogel.54 To the best of our knowledge, the employment of the TdT-dependent enzymatic polymerization system for the extension of flexible and covalently conjugated branched DNA structures has not been previously reported. In this work, we describe a general strategy for the construction of a long and covalently conjugated branched DNA structure for fast and in situ gelation in vivo (Figure 1a–c). The short, branched DNA structures with different numbers of branches (Bn: B2–B6) are synthesized by a copper-free click reaction between azide-modified dipentaerythritol and dibenzocyclooctyne (DBCO)-modified DNA oligonucleotides. The branched DNA structures can generally function as the DNA primers in the TdT-dependent enzymatic polymerization system to obtain the long and covalently conjugated branched DNA structures. After extension with the monomers of thymidine 5′-triphosphate (dTTP) or adenosine 5′-triphosphate (dATP), the length-tunable branched DNA structures with the sequences of poly T or poly A can coassemble together to form the highly dense DNA network (two-dimensional, 2D) or thermally stable DNA hydrogel (three-dimensional, 3D) by adjusting the assembly concentration, respectively. After precise decoration with multiple DNA aptamers, this kind of branched DNA structures can be employed to efficiently recognize the target protein and tumor cell to induce the subsequent successful encapsulation during their fast and in situ coassembly process. Based on this coassembly system, the thrombin, as a representative protein model, can be efficiently encapsulated to induce an obvious anticoagulation effect in the whole blood system. Furthermore, the fast and in situ gelation in a murine model of local tumor recurrence for targeting residual tumor cells to achieve a long-term drug release can be performed for efficient tumor inhibition in vivo. Figure 1 | Design of a long and covalently conjugated branched DNA structure for fast and in situ gelation. (a) Schematic illustration for the construction of long and covalently conjugated branched DNA structures based on TdT-dependent enzymatic polymerization for fast DNA self-assembly. (b) Utilization of the branched DNA structures hybridized with DNA aptamer for efficient protein encapsulation in the complex whole blood system. (c) Fast and in situ gelation in a murine model of local tumor recurrence for targeting residual tumor cells to achieve a long-term drug release for efficient tumor inhibition. Download figure Download PowerPoint Experimental Methods TdT-catalyzed extension of branched DNA structures The TdT-catalyzed extension reaction was performed in a 10 μL reaction system containing DNA primer Bn and dTTP or dATP monomers (ssDNA of Bn: dTTP or dATP monomers = 1:3000), 1×TdT buffer, and 10 units of TdT. The reaction mixture was incubated at 37 °C with different concentrations of DNA primers for different times. The ethylene diamine tetraacetic acid (EDTA) was added to stop the extension. The extended branched DNA structures were then purified by a centrifuge filter and characterized by gel and atomic force microscopy (AFM). Coassembly of extended branched DNA structures B6-Tn (15 μL, 0.25 μM) or B6-An (15 μL, 0.25 μM) was dissolved in a coassembly buffer containing 5 mM MgCl2 and 100 mM NaCl. Then, the B6-Tn (15 μL, 0.25 μM) and B6-An (15 μL, 0.25 μM) were rapidly mixed together and incubated at 25 °C for 1 min. The coassembled products were characterized by gel, AFM, and scanning electron microscopy (SEM). Encapsulation and release of thrombin For encapsulation, the thrombin-Cy5 (2 μL, 0.60 μM) was mixed with 1× phosphate-buffered saline (PBS; 8 μL), thrombin binding aptamer (TBA; 8 μL, 1.50 μM), the DNA junctions J6-TBA-Tn (4 μL, 0.25 μM) followed by J6-TBA-An (4 μL, 0.25 μM), or the branched DNA structures B6-TBA-Tn (4 μL, 0.25 μM) followed by B6-TBA-An (4 μL, 0.25 μM), respectively. After incubation at 25 °C for 30 min, 70 μL of coassembly buffer containing 5 mM MgCl2 and 100 mM NaCl was added and further incubated at 25 °C for 6 h. For release, the B6 DNA network-TBA-treated samples were further added to Triton X-100 (70 μL, 0.15%) or DNase I (70 μL, 2 U/μL) and incubated at 25 °C for 6 h. The final 50 μL of the upper solution was collected for detection of the free thrombin-Cy5 fluorescence intensity by a fluorescence spectrophotometer (Cary Eclipse, Agilent Technologies, California, United States). Cell culture MCF7, MCF7-EGFP, and HEK293 cells were cultured in Dulbecco's modified Eagle's medium complete medium (Hyclone, Thermo Scientific, Massachusetts, United States) supplemented with 10% fetal bovine serum (Hyclone, Thermo Scientific, Massachusetts, United States), 1% penicillin, and streptomycin (Grand Island Biological Company (GIBCO), Invitrogen, California, United States) in an atmosphere of 5% CO2 at 37 °C. Cell encapsulation A total of 1 × 104 MCF7-EGFP cells in 90 μL PBS were collected in a tube. Then, B6-Apt-Tn (5 μL, 2.0 μM) was added. After incubation for 5 min, B6-Apt-An (5 μL, 2.0 μM) was added for coassembly and in situ cell encapsulation. The number of free cells in the supernatant was counted. The encapsulation rate was calculated by the following equation: Encapsulation rate = [(cell added – cell in the supernatant)/cell added] × 100%. After encapsulation for 5 min, the coassembled DNA structure was stained by Gel Red (red). Then, the encapsulated cells (MCF7-EGFP, green) were imaged by fluorescent microscopy (Leica, Wetzlar, Germany). Meanwhile, the 3D stacking of cells was observed using a confocal laser scanning microscope (Zeiss, Oberkochen, Germany). Tumor growth inhibition in vivo When the MCF7 tumor volume was above 200 mm3, the incomplete resection was performed, and the 10% remnant tumors with the same sizes were left to mimic the residual tumor in situ. Then, the mice were randomly divided into 3 groups (n = 5 per group): subcutaneous injection on the surgical bed with (1) 0.9% saline, (2) free doxorubicin (DOX; 1.5 mg/kg), (3) B6-CBA-2'F-Un, after 1 min, the B6-CBA-2'F-An+DOX was also added for in situ formation of 2'F-DNA gel+DOX (gel: 3 mg/kg, DOX: 1.5 mg/kg). During treatment for 12 days, tumor size was measured using a digital vernier caliper across its longest (a) and shortest (b) diameters, and the volume was calculated according to the formula of V = 0.5ab2. The body weight of the mice was measured using an electronic balance. After 12 days of treatment, the mice were sacrificed, and the tumor was imaged and weighed. The tumor tissues were stained with hematoxylin and eosin (H&E) and TdT-mediated dUTP nick end labeling (TUNEL). The organs were stained with H&E for histopathological evaluations. Results and Discussion Construction of long and covalently conjugated branched DNA structures We initially synthesized an azide-modified branched organic cross-linking molecule (Di-PE-6N3) in two steps (the synthetic procedure is provided in Supporting Information Figure S1). The obtained branched molecule was verified by nuclear magnetic resonance (NMR: 1H and 13C) and high-resolution mass spectrometry analyses ( Supporting Information Figures S2–S4). We next employed this branched molecule as the core to cross-link a short DBCO-modified DNA oligonucleotide in the 5′ terminal by an efficient copper-free click reaction (the synthetic procedure and DNA sequence are provided in Figure 2a and Supporting Information Figure S5a and Table S1). The fabricated short and covalently conjugated branched DNA structures (Bn: B2-B6) with different numbers of arms were purified and characterized by 8% native polyacrylamide gel electrophoresis (PAGE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses (Figure 2b and Supporting Information Figure S5b–g). A stair-like gel image and clear mass spectra were observed to confirm the successful fabrication of the short and covalently conjugated branched DNA structures with a definite number of arms. Figure 2 | Construction of long and covalently conjugated branched DNA structures. (a) Synthetic route for branched DNA structures. (b) 8% native PAGE analysis of short and branched DNA structures (Bn) as primers for enzymatic polymerization. M: DNA marker. (c) 1% agarose gel electrophoresis analysis of extended and branched DNA structures based on TdT-catalyzed extension (extension time for B1-Tn and B2-Tn: 10 min, extension time for B3-Tn, B4-Tn, B5-Tn, and B6-Tn: 30 min). (d) Schematic illustration of enzymatic detection of the extended length of arms in the branched DNA structures by restriction endonuclease cleavage (EcoR I), DNA polymerase extension (Bst 2.0), and single-stranded DNA degradation (S1 nuclease). (e) 1% agarose gel electrophoresis analysis of the extended length of B6-Tn (about 735 nt). Download figure Download PowerPoint We subsequently employed these short and branched DNA structures as the primers in the TdT-dependent enzymatic polymerization system to obtain the long and covalently conjugated branched DNA structures. As a proof of concept, we first utilize the linear DNA monomer (B1) of the branched DNA structures to optimize the conditions for TdT-catalyzed extension. In the extension system, two kinds of dNTPs, including dTTP and dATP, were chosen to construct the poly T (Tn) and poly A (An) sequences for the following complementary base pairing. We found that the types of dNTPs, primer concentrations, and extension times were crucial for the extended length of B1-Tn and B1-An ( Supporting Information Figures S6 and S7). To obtain similar extended lengths, we chose the different extension times for B1-Tn (10 min) and B1-An (60 min) under the same primer concentration (0.25 μM). Next, we replaced the linear DNA primer (B1) with these short and branched DNA structures (Bn: B2-B6), respectively. After extension for 10 min, we found that the groups of B3-Tn, B4-Tn, B5-Tn, and B6-Tn did not exhibit a noticeable difference in electrophoretic mobility, indicating insufficient extension ( Supporting Information Figure S8). In order to obtain the fully extended branched DNA structures, we further increased the extension time to 30 min for Tn and 90 min for An. As shown in Figure 2c, a stair-like gel image was observed, similar to the electrophoretic behavior presented in flexible and branched DNA primers (Figure 2b). After successfully constructing the long and covalently conjugated branched DNA structures, we further tried to evaluate their extended arm lengths in the largest branched DNA structure (B6-Tn) by enzymatic strategy (Figure 2d and Supporting Information Figure S9). First, the B6-Tn was hybridized with the single-stranded DNA (CP-B1: the DNA sequence is provided in Supporting Information Table S1) that forms the structure of B6-Tn-CP-B1 with six restriction endonuclease sites of EcoR I in the double-stranded regions. Then, the EcoR I restriction endonuclease was added to obtain the extended single-stranded poly T (Tn). Next, the poly T was transformed into the double-stranded DNA (ds-TnAn) with the same base number of Tn by annealing with DNA primer A40 in the tailored Bst 2.0 DNA polymerase-dependent extension system. Finally, the ds-TnAn was purified by S1 nuclease-induced degradation of other single-stranded DNA by-products. As shown in Figure 2e, a clear band of ds-TnAn was observed with an extended length of about 735 bp by the internal relationship analysis between the number of base pairs and relative electrophoretic mobility ( Supporting Information Figure S9d). In our design, the length of these extended branched DNA structures can be further increased with the lower primer concentration and longer extension time in the TdT-catalyzed extension system. These obtained results demonstrate that the short and covalently conjugated branched DNA structures can generally function as the DNA primers in the TdT-dependent extension system to efficiently construct the long and covalently conjugated branched DNA structures. Coassembly between branched DNA structures After we successfully constructed these branched DNA structures, we further imaged them with the AFM. The long and branched structures with a definite number of arms (3–6 copies) were clearly observed (Figure 3a, Supporting Information Figures S10 and S11). The length of their arms was also consistent with the estimated value (about 250 nm: 735 nt × 0.34 nm/nt). We next optimized the conditions for efficient coassembly between the T–A paired and extended branched DNA structures. In the DNA coassembly system, the concentrations of structural monomers, temperature conditions, and coassembly buffer are usually crucial for coassembly efficiency. We first evaluated their coassembly behavior under a relatively low concentration (0.125 μM: one half of 0.25 μM primer with the mixture ratio of 1∶1) at 25 °C in a simple coassembly buffer containing 5 mM MgCl2 and 100 mM NaCl. After incubation for only 1 min, these coassembled products were directly characterized by agarose gel ( Supporting Information Figure S12). All of these coassembled products were totally trapped in the wells of gel, indicating the formation of large-sized DNA structures with high molecular weights. Figure 3 | Characterization of extended branched DNA structures and their coassembled DNA network. (a) AFM images of extended branched DNA structures: B3-Tn, B4-Tn, B5-Tn, and B6-Tn (white circles and numbers indicate the location of each arm). Scale bar: 200 nm. (b) AFM images of a coassembled DNA network under the concentration of 0.125 μM: B3-Tn+B3-An, B4-Tn+B4-An, B5-Tn+B5-An, and B6-Tn+B6-An. Scale bar: 400 nm. (c) SEM image of coassembled DNA network: B6-Tn+B6-An. Scale bar: 1 μm. (d) Enlarged SEM image of DNA network: B6-Tn+B6-An. Scale bar: 200 nm. (e) Imaging the DNA network based on the coassembly of B6-Tn and B6-An in solution, B6-Tn was stained by SYBR Green II (green), and B6-An was stained by Gel Red (red). Download figure Download PowerPoint We next imaged these coassembled products by AFM (Figure 3b). A loose DNA network was observed in the group of B3-Tn+B3-An coassembled between B3-Tn and B3-An. The more compact DNA network was achieved by increasing the number of arms involved in the branched DNA structures. The group of B6-Tn+B6-An with the highest number of arms demonstrated the densest morphology of the DNA network. The clear network structure of B6-Tn+B6-An was also observed in the SEM image (Figure 3c,d). The dynamic fluorescence imaging strategy was further employed to monitor the efficient coassembly process in the tube. As shown in Figure 3e, the B6-Tn and B6-An were first stained by SYBR Green II (green) and Gel Red (red), respectively. Then, the stained B6-Tn was quickly added to the stained B6-An. Immediately after about only 10 seconds, a merged orange and flocculent DNA network was detected, indicating the fast and efficient coassembly process. Meanwhile, we investigated the coassembly behavior between the branched DNA structures with different numbers of arms (B6-Tn+2×B3-An). A moderately dense DNA network was observed, which was looser than the DNA network based on B6-Tn+B6-An ( Supporting Information Figure S13). After adding the linear B1-An as the blocker in the coassembly system of B6-Tn+B6-An, no obvious large-sized DNA network was observed ( Supporting Information Figure S14). These results demonstrate that the long and branched DNA structures can be successfully employed as building blocks to achieve a fast and efficient DNA coassembly system with different combinations of monomers. Fabrication of DNA hydrogel Furthermore, we increased the concentration of branched DNA structure and tried to fabricate the 3D DNA hydrogel. According to previous reports, the short and branched DNA structures with three arms (about 25 nt for each arm) can form the DNA hydrogel under the concentration of about 300 μM.55–62 In comparison, the B6-Tn possesses six arms (double) and about 735 nt per arm (almost 30 times). To achieve a similar mass concentration of DNA hydrogel, we chose the corresponding concentration of 5 μM for coassembly analysis. Similar to the coassembly condition for the previous DNA network (under 0.125 μM), we mixed the B6-Tn and B6-An together in the same coassembly buffer at 25 °C for 1 min of incubation. A DNA hydrogel was immediately generated and was clamped up with a tweezer (Figure 4a). The dye-labeled DNA hydrogel was also imaged in an inverted tube, reconfirming the formation of DNA hydrogel ( Supporting Information Figure S15a). In addition, a dense morphology of DNA hydrogel was observed by SEM ( Supporting Information Figure S15b). Then, the rheological characterizations of the obtained DNA hydrogel were collected. The conventional rheological tests, including frequency sweep, strain sweep, and time scan, were performed to confirm the hydrogel characteristics (Figure 4b–d). These results indicate that the long and covalently conjugated branched DNA structures efficiently coassembled to form the DNA hydrogel. Figure 4 | Characterization of coassembled DNA hydrogel. (a) Photoimage of coassembled DNA hydrogel under the concentration of 5.0 μM. (b) Frequency sweep test between 0.1 and 10 Hz at a fixed strain of 1% at 25 °C. (c) Strain sweep test between 0.1% and 1000% strains at a fixed frequency of 1 Hz at 25 °C. (d) Time scan test at a fixed frequency (1 Hz) and strain (1%) at 25 °C for 5 min. (e) Temperature-ramp rheological test of DNA hydrogel from 25 to 90 °C at a fixed frequency (1 Hz) and strain (1%). (f) Reversible thermal test of DNA hydrogel at a fixed frequency (1 Hz) and strain (1%) at 25 and 70 °C, respectively, for 3 cycles. (g) The extended DNA junction (J6-Tn) based on the TdT-catalyzed extension of a short noncovalent DNA junction (J6). (h) Reversible thermal test of DNA hydrogel (J6-Tn+J6-An) based on the coassembly of extended noncovalent DNA junctions J6-Tn and J6-An. (i) A summary for thermal stability of DNA hydrogels. Download figure Download PowerPoint We next investigated whether the short and branched DNA primers themselves could form the DNA hydrogel under the same condition (5 μM) or not. We prepared another short and branched DNA structure (AS-B6: with the complementary sequences of B6; the detailed sequences are provided in Supporting Information Table S1) by click reaction ( Supporting Information Figure S16). No large-sized DNA network or DNA hydrogel was observed in the AFM images of coassembled B6+AS-B6 ( Supporting Information Figure S16d). This result suggests that the successfully coassembled DNA hydrogel of B6-Tn+B6-An under the concentration of 5 μM was dependent on the long T–A complementary base pairing. Meanwhile, considering the covalent conjugation property of the branched DNA primers, we performed the temperature-ramp rheological test of this DNA hydrogel from 25 to 90 °C at a fixed frequency (1 Hz) and strain (1%) to evaluate their thermal stability. As shown in Figure 4e, even under the temperature of 90 °C, the G′ (storage modulus) value was still higher than the G″ (loss modulus) value (the typical characteristic of hydrogel). We further investigated their possible reversible thermal response at 25 and 70 °C, respectively (Figure 4f). A good reversible thermal response was recorded for three cycles. Noticeably, a high Tm value of the DNA hydrogel was also previously observed in a DNA hydrogel-based mechanical metamaterial by the rational combination of a rolling circle amplification and a multiprimed chain amplification.63 In our design, the thermal stability of our constructed DNA hydrogel may largely be attributed to the long and covalently conjugated structure of the branched DNA. To confirm our hypothesis, we replaced the covalently conjugated branched DNA primer (B6) with the noncovalent DNA junction (J6) to evaluate their difference in thermal stability (Figure 4g). We first constructed two kinds of DNA junction (J6 and AS-J6 vs B6 and AS-B6) by rational DNA sequence design (the detailed sequences are provided in Supporting Information Table S2). As expected, no DNA hydrogel was observed under the concentration of 5 μM for J6+AS-J6 ( Supporting Information Figure S17). Then, we increased the concentration of these short and branched DNA monomers to 150 μM to form their corresponding DNA hydrogels ( Supporting Information Figure S18). No noticeable thermal stability was detected in these two kinds of DNA hydrogel (covalent B6+AS-B6 and noncovalent J6+AS-J6). Under the temperature of 70 °C, the DNA hydrogels were both transformed into the solution state, indicating that the long T–A complementary base pairing is essential to achieve the super thermal stability. We further employed the DNA junction (J6) as the primer in the TdT-dependent extension system ( Supporting Information Figure S19). Similarly, a long and noncovalent DNA junction (J6-Tn) was constructed ( Supporting Information Figure S19b). After coassembly under 0.125 μM, a relatively looser DNA network (J6-Tn+J6-An), compared with previously described B6-Tn+B6-An, was observed by AFM imaging ( Supporting Information Figure S19c). This difference in the morphology of DNA networks may be determined by the original rigid double-stranded DNA structure of the noncovalent DNA junction. We next increased