Rational Design of DNA nanostructures as TLR9 Agonists

Rational Design of DNA nanostructures as TLR9 Agonists | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 10 February 2025 V1 Latest version Share on Rational Design of DNA nanostructures as TLR9 Agonists Authors : Chunfa Chen , Cheng Tian , Zhuoer Jin , Yuandong Wen , Cheng Zhi Huang , and Hua Zuo [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173916569.98827616/v1 Published Biomacromolecules Version of record Peer review timeline 270 views 111 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract TLR9 agonists have broad applications in vaccines and cancer immunotherapy. Based on the crystal structures of the ligand-TLR9 complexes, we rationally designed DNA nanostructure-based TLR9 agonists by optimizing the spatial orientation of 5'-TCG ligands and leveraging the multivalent effect of the ligand. In these designed DNA structures, the ligands simultaneously bind to the CpG motif binding site and the 5'-xCx DNA binding site, resulting in maximal TLR9 activation. Cite this paper: Chin. J. Chem. 2024 , 42 , XXX—XXX. DOI: 10.1002/cjoc.202400XXX Rational Design of DNA nanostructures as TLR9 Agonists Chunfa Chen a , # , Cheng Tian a , # , Zhuoer Jin a , Yuandong Wen a , Cheng Zhi Huang a , Hua Zuo a ,* a College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China # These authors contributed equally to this work. *Corresponding author. Email: [email protected] TLR9 agonist | CpG | DNA nanomaterials | Spacing | Protein crystal structure | Ligand-receptor | Comprehensive Summary TLR9 agonists have broad applications in vaccines and cancer immunotherapy. Based on the crystal structures of the ligand-TLR9 complexes, we rationally designed DNA nanostructure-based TLR9 agonists by optimizing the spatial orientation of 5’-TCG ligands and leveraging the multivalent effect of the ligand. In these designed DNA structures, the ligands simultaneously bind to the CpG motif binding site and the 5’-xCx DNA binding site, resulting in maximal TLR9 activation. Background and Originality Content Ligand-receptor interactions are essential for numerous physiological and pathological processes in living organisms. They act as critical mediators of communication between cells and tissues, while also regulating cellular behavior, immune responses, drug actions, and other vital biological functions [1] . For instance, early immune responses to pathogen invasion are triggered by the interaction of pathogen-associated molecular patterns (PAMPs) with pattern recognition-receptors (PRRs) [2] such as Toll-like receptors (TLRs), and nucleotide-binding oligomerization domain-containing-like receptors. The Toll-like receptor (TLR) family comprises TLR3, TLR7, TLR8, TLR9, and TLR13, with TLR13 being specific to mice [3] . These receptors are single-pass transmembrane proteins that play a critical role in recognizing nucleic acids. The TLR9 receptor primarily recognizes single-stranded DNAs (ssDNAs) containing the CpG dinucleotide motif (cytosine-phosphate-guanine) and induces the dimerization of its protein structure. This process is followed by the recruitment of MyD88 to the intracellular domain of the receptor, initiating downstream signaling pathways that regulate the inflammatory response and the release of pro-inflammatory cytokines (e.g., TNFα, IL-6), while also promoting adaptive immune responses [4] . CpG oligonucleotides (ODNs) has been extensively studied and clinically applied in areas such as cancer immunotherapy and vaccine development [5-6] . However, the currently developed CpG ODNs face limitations, including the requirement for high-dose administration [7] , which can result in side effects. Efforts to enhance the efficacy of CpG ODN have centered on optimizing its delivery [8-9] and improving its stability [10-11] . Thiophosphorylation of the DNA backbone is a widely used modification [12] that significantly enhances DNA stability and prolongs the half-life of CpG ODN in the bloodstream [13] . However, thiophosphorylated CpG ODN has been found to exhibit cytotoxicity [14] . The prolonged half-life in the bloodstream can lead to unintended immune responses, while excessive accumulation in the liver and kidneys may impose an increased metabolic burden on these organs [15-16] . DNA nanotechnology, a cutting-edge biomaterial technology, harnesses the unique self-assembly properties of DNA molecules to create nanoscale structures and functional devices. This approach offers unparalled precision in regulating receptor-ligand interactions [17-18] . By self-assembling into intricate two- and three-dimensional architectures [19-21] , DNA molecules offer innovative methods to modulate these interactions. Crystal structures of protein-ligand complexes provide high-resolution insights into the three-dimensional architecture of proteins and their ligands, revealing detailed spatial information about receptor-ligand binding mechanisms and the factors that influence them [22-23] . The DNA sequence specificity required for TLR9 receptor activation has been extensively investigated, with CpG ODNs being the most thoroughly studied. Notably, Mojca Bencina et al conducted detailed studies on CpG motifs, examining variations in sequence, length, and species origin, and identified the optimal motifs for activating TLR9 receptors in both mice and humans [24-25] . Toshiyuki Shimizu and colleagues analyzed the crystal structures of ligand-TLR9 complexes and identified a second DNA-binding site on the TLR9 protein, which binds a DNA strand containing 5’-xCx, with a cytosine as the second base at the 5’ end of the strand. The 5’-xCx DNA alone is insufficient to activate the TLR9 protein; however, in the presence of CpG ODN, the 5’-xCx DNA binds to TLR9, thereby enhancing receptor activation [26] . We were inspired to develop a rational approach for designing optimal DNA structures as TLR9 agonists to enhance receptor binding and activation (Scheme 1). This strategy was guided by the spatial distance and relative positioning between the 5’-xCx DNA binding site and the CpG binding site on the TLR9 receptor. By determining this spatial distance and orientation, we engineered DNA structures with a 5’-TCG motif as the ligand at both ends to enhance receptor interaction. These motifs specifically target the 5’-xCx and CpG sites on the receptor. The DNA structure-based agonists consist of two 5’-TCG motifs, a duplex region, a single-stranded linker (sslinker), and a loop. Furthermore, we incorporated multimeric TCG ligands to amplify receptor interaction and activation. This design facilitates precise binding within a defined nanoscale range, optimizing receptor-ligand interactions for enhanced TLR9 activation. Scheme 1. Rational design of DNA agonists to activate TLR9 and induce the release of inflammatory cytokines by macrophage Results and Discussion Design and self-assembly of DNA nanostructure based TLR9 agonists. The TLR9 protein, upon activation by CpG ODN, binds to DNA in a 2:2 stoichiometric ratio [27] , forming a dimeric complex with two CpG ODNs. According to Toshiyuki Shimizu’s study, the mouse TLR9 forms a dimer upon activation by CpG ODN and 5’-xCx DNA, as evidenced by the crystal structure (PDB: 5ZLN) [28] . Using PyMOL software, we measured the distance between the 5’-xCx DNA binding site and the CpG DNA binding site on the TLR9 receptor. We found that the distance is approximately 61 Å, regardless of whether the sites are on the same TLR9 protein or separate TLR9 proteins. This distance corresponds to a length of 18 base pairs (bps) (Scheme 1). Accordingly, we designed a DNA structure with two free 5’-TCG motifs at both ends to function as the ligand for TLR9. The distances between the binding sites provide a basis for the rational design of DNA nanostructure-based TLR9 agonists. These nanostructures incorporate the 5’-TCG motif as a ligand, functioning as both a 5’-xCx agonist for TLR9 and a crucial component of the CpG sequence that activates the receptor [29] . Each agonist comprises a duplex region, a sslinker, and optionally a loop, with two free 5’-TCG motifs located at both ends, designated as Lx-Dx-Tx (Figure 1a). Lx-Dx-Tx is assembled from two single-stranded DNA (ssDNAs). L0-D21-T2 consisted of a 21 bp duplex region with a 2-thymine sslinker, formed by hybridizing L0-D21-T2a and L0-D21-T2b, and did not contain a loop. Similarly, L0-D21-T4 included a 21 bp duplex region with a 4-thymine sslinker, formed by hybridizing L0-D21-T4a and L0-D21-T4b. L0-D16-T4 featured a 16 bp duplex region with a 4-thymine linker. To enhance structural flexibility, a 5-thymine loop was incorporated into the center of the 21 bp duplex, creating the L5-D21-T4 structure, which comprised a 21 bp duplex, a 4-thymine linker, and a 5-thymine loop. These DNA nanostructures were assembled by annealing two individual ssDNAs at a 1:1 molar ratio, followed by characterization using native polyacrylamide gel electrophoresis (nPAGE). The assembled double-stranded DNA (dsDNA) structures appeared as sharp, single bands and migrated more slowly in the gel (Figure 1b). Figure 1 Design of DNA nanostructure based TLR9 agonists. (a) Detailed design of TLR9 agonists. (b) Characterization of the self-assembled DNA nanostructures by 6% nPAGE at 25 ℃. Activation of TLR9 Receptor by DNA Nanostructure based Agonists. In this study, we selected RAW 264.7 macrophages as the cellular model, as these cells express high levels of TLR9 and are commonly used in CpG-functionalized nanomaterial research [9, 30] . To determine whether these the designed DNA nanostructure could enter RAW 264.7 cells, we incubated Cy5-labeled DNA structures with RAW 264.7 cells and analyzed intracellular fluorescence using flow cytometry and laser confocal microscopy. To prevent false-positive uptake signals, the cells were treated with DNase I to remove medium- or membrane-bound DNA. Subsequently, intracellular fluorescence signals were detected by laser confocal microscopy after 2 h of co-incubation with DNA nanostructures (Figure 2a). From 2 to 8 h, the fluorescence intensity progressively increased, indicating enhanced DNA uptake into the cytoplasm (Figure 2b). It has been demonstrated that the interaction between TLR9 receptors and DNA ligands occurs in the lysosome [31] , an acidic environment with a pH of 5.5. Accordingly, we evaluated the stability of the nanostructures under acidic conditions. The experimental results confirmed that the designed DNA nanostructures remained stable for at least 6 h under both acidic conditions and 10% serum conditions (Figures S1-4). After 24 h of incubation, the supernatants of the cells were collected and analyzed for TNFα levels using enzyme-linked immunosorbent assay (ELISA), with K1826 CpG ODN as a positive control [6] . As shown in Figure 2c, the single-stranded 5’-TCG DNA (L0-D16-T4a) did not induce TNFα release, whereas all duplex-containing structures effectively stimulated TNFα production. When the duplex length was fixed at 21 bp, the structure with a 4-thymidine sslinker (L0-D21-T4) induced higher TNFα secretion compared to the structure with a 2-thymidine sslinker (L0-D21-T2). The increased flexibility provided by longer linkers may enhance the binding affinity of the DNA ligand (5’-TCG) to the TLR9 receptor. Furthermore, when the sslinker consisted of 4 thymidines, reducing the duplex length revealed that the 16 bp duplex-containing structure (L0-D16-T4) induced higher TNFα secretion compared to the 21 bp duplex-containing structure (L0-D21-T4). According to TLR9 receptor crystallographic data, the 16 bp duplex length more closely aligns with the distance between the two DNA-binding sites on the TLR9 receptor, suggesting that the spatial arrangement of the two ligand motifs influences TLR9 activation. Notably, by incorporating a loop into L0-D21-T4, we created L5-D21-T4, which led to a twofold increase in the release of inflammatory cytokines from RAW 264.7 cells compared to the loop-free structure (L0-D21-T4). The addition of the loop enhanced the flexibility [32] of the ligands at both ends, enabling adjustments in their spatial arrangement to better align with the receptor’s binding sites. These findings suggest that the spatial distance of the DNA ligand (5’-TCG) at the ends of the DNA nanostructure, along with the flexibility introduced by the loop, significantly influence the binding of the DNA ligand to the TLR9 receptor, thereby impacting the release of inflammatory cytokines. Figure 2 Activation of TLR9 receptors by DNA based agonists following uptake by RAW 264.7 cells. (a) Confocal microscopy images of RAW264.7 cells following 2 h of co-incubation with DNA agonists (200 nM). Nucleius were stained with Hoechst 33342, and TLR9 agonists were labeled with Cy5. Scale bars: 20 μm. (b) Flow cytometry analysis of Cy5-TLR9 agonists (200 nM) uptake by cells at different time points. (c) ELISA analysis of TNFα expression levels in RAW264.7 cells treated with different TLR9 agonists (2.5 μM). Unmodified CpG ODN K1826 was used as a positive control. Data are presented as the mean ± SEM (n = 3). P values were calculated using Student’s t-test and one-way ANOVA. ***p < 0.001, ****p < 0.0001 compared to the L0-D21-T2 group; ###p ≤ 0.001 compared to the L5-D21-T4 group. DNA Nanostructures Simultaneously Interact with the 5’-xCx and CpG Binding Sites on TLR9 Receptor. To demonstrate that the DNA ligands in our designed DNA nanostructures bind simultaneously to both the 5’-xCx and CpG binding sites on the TLR9 receptor, we included two control groups: (1) Control Group 1, where the 5’-TCG ligand in the DNA nanostructures was replaced with 5’-TCT, 5’-TCC, or 5’-TTG (Figure 3a). These three motifs (5’-TCT, 5’-TCC, and 5’-TTG) lacked CpG functionality. ELISA results showed that the levels of TNFα release induced by these motifs were not significantly different from the control group (Figure 3b). This indicates that the loss of CpG motif functionality rendered the DNA structures incapable of activating the TLR9 receptor or inducing TNFα production. (2) Control Group 2: the 5’-TCG ligand motif in the DNA nanostructures was replaced with 5’-pTCG (phosphorylated at the first base) or 5’-TTCG, both of which do not conform to the 5’-xCx DNA pattern (Figure 3c). Therefore, these motifs cannot bind to the 5’-xCx DNA site on the TLR9 receptor, although they retain the functionality of the CpG motif. The results showed that, in the absence of 5’-xCx DNA, these motifs could still stimulate TNFα release from RAW 264.7 cells, but the level of TNFα secretion was reduced by more than half (Figure 3d). These findings are consistent with the results of Toshiyuki Shimizu et al., which demonstrated that 5’-xCx DNA alone does not activate the TLR9 receptor; however, in the presence of the CpG motif, 5’-xCx DNA can enhance the activity of the CpG motif. It also confirms that the two 5’-TCG ligands in our designed DNA structures simultaneously interact with the 5’-xCx and CpG sites on the TLR9 receptor, synergistically enhancing TLR9 activation. Thus, the spatial orientation of the ligands, precisely controlled by the designed DNA scaffolds, enables optimal binding to the two distinct sites, resulting in optimal TLR9 receptor activation. Figure 3 Evaluation of the effects of different DNA ligands on TNFα secretion by RAW 264.7 cells. (a) Control Group 1, where the 5’-TCG ligand was replaced with 5’-TCT, 5’-TCC, or 5’-TTG (b) ELISA analysis of TNFα release by RAW 264.7 cells following the uptake of the designed ligands, 5’-TCT, 5’-TCC, or 5’-TTG (1.5 μM) after co-incubation for 24 h. (c) Control Group 2: The 5’-TCG ligand motif was replaced with 5’-pTCG or 5’-TTCG. (d) ELISA analysis of TNFα release by RAW 264.7 cells following the uptake of the designed ligands, 5’-pTCG or 5’-TTCG (1.5 μM) after co-incubation for 24 h. Data are presented as the mean ± SEM (n = 3). P values were calculated using one-way ANOVA. ***p < 0.001; ns indicates non-significant. Optimization of DNA Nanostructure-based TLR9 Agonists. To maximize TLR9 receptor activation by DNA-based agonists, we further optimized the DNA nanostructures based on the L5-D21-T4 structure. We fixed the duplex length at 21 bp and adjusted the lengths of the sslinker or the loop to increase structural flexibility, potentially affecting the binding of the 5’-TCG motifs at both ends to TLR9. The sslinker length was increased from four thymidine bases (T4) in L5-D21-T4 to six (T6) and eight (T8) thymidine bases in L5-D21-T6 and L5-D21-T8, respectively (Figure 4a). These DNA nanostructures were incubated with RAW 264.7 cells, and TNFα levels were measured using ELISA. The results revealed that the T6 group exhibited the highest TNFα levels. In contrast, shorter (T4) or longer (T8) sslinkers resulted in lower TNFα levels, likely due to suboptimal ligand orientation for binding to TLR9 (Figure 4b). We then fixed the sslinker length at two thymidine bases (T2) and varied the loop length, designing L3-D21-T2, L4-D21-T2 and L5-D21-T2, which included three (T3), four (T4), and five (T5) thymidine bases in the loop, respectively (Figure 4a). As shown in Figure 4b, the TNFα release followed the order: T5 < T3 < T4. Insufficient flexibility (T3) or excessive flexibility (T5) resulted in reduced binding and lower TNFα levels. These findings indicate that both the spatial distance between the two ligands and the flexibility of the structure influence the activity of the DNA based agonist. Rationally designed DNA nanostructures enable optimal ligand organization, promoting precise binding to TLR9 and maximizing receptor activation. This demonstrates that, guided by protein-ligand crystallography data, ligands can be rationally designed to better fit receptor-binding sites by simply adjusting their spatial orientation, thereby achieving optimal regulation of receptor-ligand binding. Figure 4 Effect of the flexibility of DNA nanostructures on TNFα level. (a) Design of DNA nanostructure based agonists by varing the length of sslinker or loop. (b) TNFα levels released by RAW 264.7 cells measured by ELISA after after 24 h of incubation with DNAs (1.5 μM). Data are presented as the mean ± SEM (n = 3). P values were calculated using Student’s t-test and one-way ANOVA. ***p < 0.001 and ****p < 0.0001 compared to the L5-D21-T6 group; #p < 0.05 and ##p < 0.01 compared to the L4-D21-T2 group; ns indicates non-significant. Exploring the Enhancement of TLR9 Receptor Activation by Dimeric DNA Nanostructure Based Agonists. Multivalency plays a crucial role in regulating molecular interactions [33] . For instance, some proteins form multimeric complexes with other molecules through multivalent interaction, thereby modulating their function or enhancing their interactions [34] . To achieve maximal TLR9 activation, we designed dimeric DNA nanostructure-based TLR9 agonists, guided by the rules regarding the spatial distance between the two ligands and the structural flexibility. We rationally designed P-L5-D21-T6 and P-L5-D21-T4, where P denotes the palindromic sequence at the 3’ end (Figure 5a). The DNA strands were annealed in TAE/Mg buffer and characterized by nPAGE. All single strands (P-L5-D21-T6a, P-L5-D21-T6b, P-L5-D21-T4a, and P-L5-D21-T4b) exhibited a trailing state in nPAGE due to the presence of palindromic sequences (Figure 5b). After self-assembly, both P-L5-D21-T6 and P-L5-D21-T4 predominantly migrated at approximately 125 bp, corresponding to their dimeric structures. P-L5-D21-T6 was formed by hybridization of P-L5-D21-T6a and P-L5-D21-T6b, with P-L5-D21-T6 further forming a self-dimer through the self-complementarity of the palindromic sequence (Figure 5c). Similarly, P-L5-D21-T4 was assembled from P-L5-D21-T4a and P-L5-D21-T4b, with P-L5-D21-T4 also forming a self-dimer via the self-complementary palindromic sequence (Figure S5). We hypothesized that incorporating a loop into the duplex region might cause the structure to bend into a closed shape, rather than forming a polymer. The self-dimer structure remains stable for over 6 hours (Figure S6) in acidic environments and retains its stable conformation for 4 hours in 10% serum (Figure S7). After 24 h of co-incubation with RAW 264.7 cells, the TNFα levels were analyzed using ELISA. The results demonstrated that all dimeric DNA structures (P-L5-D21-T6 and P-L5-D21-T4) enhanced TLR9 receptor activation by 2–3 fold compared to structures without the palindromic sequence (L5-D21-T6 and L5-D21-T4) (Figure 5d). Furthermore, the TNFα levels were equivalent to those of the positive control, thiophosphorylated CpG ODN K1826 (K1826-ps). Interestingly, P-L5-D21-T6 and P-L5-D21-T4 exhibited similar TLR9 activation capabilities, as evidenced by comparable TNFα levels, despite having a two-base difference in the length of their sslinker. Typically, high doses of unmodified CpG-ODN are required to stimulate immune cells due to its instability. To address this, thiophosphorylated CpG ODN K1826 was developed to enhance stability; however, its adverse effects, such as toxicity, remain a subject of ongoing debate [35-36] . In our study, we employed precisely designed unmodified dimeric DNA nanostructures, each containing dimeric 5’-TCG ligands, which achieved comparable efficacy in stimulating immune cells to release inflammatory cytokines at levels similar to those induced by thiophosphorylated CpG ODN K1826. This suggests that receptor-ligand interactions can be effectively modulated to enhance immune cell activation through precise organization of the ligand’s spatial orientation. Confirmation of the Interaction Between DNA Nanostructures and TLR9 by Using Chloroquine. TLR9 recognizes and interacts with DNA, and its activation relies on acidic conditions. To confirm that our designed DNA nanostructures target and activate TLR9, we used chloroquine [37] , a commonly used inhibitor of lysosomal acidification, to block this interaction. RAW 264.7 cells pretreated with 15 µM chloroquine for 1 h and subsequently incubated with 1.5 µM DNA nanostructures for 24 h failed to secrete high levels of TNFα, although the cells proliferated normally (Figures 6a and S8-S9). In contrast, RAW 264.7 cells not pretreated with chloroquine but treated with DNA nanostructures released high levels of TNFα. We also measured the levels of the inflammatory cytokine IL-6, with results similar to those observed for TNFα (Figure 6b). In the absence of chloroquine pretreatment, the DNA nanostructures effectively activated TLR9 and stimulated IL-6 release. However, following chloroquine pretreatment, the DNA nanostructures failed to induce high levels of IL-6 release (Figure 6b). These findings suggest that blocking TLR9 function in lysosomes prior to stimulation prevents DNA agonists from triggering TNFα release. The DNA agonist we designed functions by binding to TLR9 and activating downstream signaling pathways. Figure 5 Optimization of TLR9 agonists using dimeric DNA structures. (a) Design of dimeric DNA structures. A 16-nt palindromic sequence is introduced at the 3’ end of the DNA strand. (b) Characterization of the structure by nPAGE. (c) AFM images of P-L5-D21-T6. Scale bar: 200 nm. (d) TNFα levels measured by ELISA after co-incubation of RAW cells with DNAs (1.5 μM) for 24 h. Data are presented as the mean ± SEM (n = 3). P values were calculated using one-way ANOVA. *p < 0.05, **p < 0.01, ns indicates non-significant. Figure 6 Confirmation of the interaction between DNA nanostructruture and TLR9 by using chloroquine. (a) The level of TNFα released by RAW 264.7 cells measured using ELISA. After pre-treatment with chloroquine (15 μM) for 1 h, cells were co-incubated with DNA (1.5 μM) for 24 h, and TNFα levels were subsequently measured. (b) The level of IL-6 measured by ELISA. Conclusions In our study, we aimed to investigate the optimal binding mechanism for maximal activation of the TLR9 receptor by precisely organizing the spatial orientation of the ligand. Based on crystallographic data, the TLR9 receptor is activated by both the CpG motif and 5’-xCx DNA. Utilizing the programmability of DNA, we rationally designed DNA nanostructures to regulate the spatial orientation of the two 5’-TCG ligands, enabling simultaneous binding to the CpG motif binding site and the 5’-xCx DNA binding site. We further designed dimeric DNA nanostructures by incorporating a palindromic sequence into the structure. This multivalent effect resulted in enhanced TLR9 activation. These findings suggest that by precisely controlling the spatial orientation of ligands and engineering multivalent ligands, TLR9 activation can be maximized. This implies that in future applications, enhancing receptor-ligand binding efficiency could reduce the required drug concentration for immune activation. Experimental Stains-all (C 30 H 27 BrN 2 S 2 ) and Hoechest33342 were purchased from Sigma Aldrich (USA). Tris base, EDTA.Na 2 .H 2 O, magnesium acetate, acrylamide, and bis-acrylamide were purchased from Sangon Biotech Co., Ltd (Shanghai, China). N , N , N’ , N’ -Tetramethyl ethylenediamine (TEMED) and ammonium persulfate were purchased from Beijing Dingguo Changsheng Biotech Co., Ltd (Beijing, China). Acetic acid was purchased from Chengdu Kelong Chemical Co., Ltd (Chengdu, China). Polylysine was purchased from Ted Pella, Inc. (USA). Phosphate buffer saline (PBS) and cell counting kit-8 (CCK8) were purchased from Biosharp Bio-Technology Co., Ltd (Anhui, China). DMEM was obtained from Thermo Fisher Scientific Inc (USA). Chloroquine was purchased in Aladdin Biochemical Technology Co. LTD (Shanghai China). Ultra-PAGE-purified DNA oligonucleotides and cy5 labelled DNA were purchased from Sangon Biotech Co., Ltd (Shanghai, China). ELISA Flex: mouse IL-6 (HRP) and mouse TNFα (HRP) were purchased from Mab Tech Inc (USA). Self-assembly of DNA nanostructures. the DNA nanostructures were self-assembled in the TAE/Mg buffer（40 mM Tris base, 2 mM EDTA, 20 mM acetic acid and 12.5 mM MgCl 2 , pH 7.4）and annealed at 95 ℃ for 5 min, 65 ℃ for 30 min, 55℃ for 30 min, 37 ℃ for 30 min, 25 ℃ for 30 min and 4 ℃ for 60 min . Native polyacrylamide gel electrophoresis (nPAGE): 6% nPAGE gel was prepared with 19:1 acrylamide/ bisacrylamide solution and TAE/Mg 2+ buffer. The gel was run at 25 ℃ at 220V in the TAE/Mg 2+ buffer. Then stained with Stains-All (Sigma) and scanned by an HP scanner (CanoScan LiDe400). Atomic force microscopy (AFM) imaging: polylysine (10 μg/mL, 20 μL) was deposited on freshly cleaved mica surface (Ted Pella, Inc.) for 10 min and blew away by nitrogen. 20 μL H 2 O was added to wash the mica surface and blew away immediately by nitrogen. Then 10 μL of diluted DNA (final concentration: 100 nM) was deposited onto the polylysine-coated mica surface and incubated for 3 min. Finally, 20 μL of TAE/Mg 2+ buffer was deposited on the mica surface and scanned in ScanAsyst in Fluid mode on Multimode 8 AFM (Bruker) with ScanAsyst Fluid+ probes (Bruker). Cell viability analyzed by cell counting kit-8 (CCK8): RAW264.7 cells were grown in 96-well plates at a density of 30,000 per well, incubated overnight in an incubator containing 5% CO 2 at 37 ℃. DNA nanostructures (final concentration: 2.5 µM) or chloroquine (final concentration: 15 μM) were added, and after 24 h of co-incubation with the cells, CCK8 reagent (CCK8: DMEM = 1:10) was added, and after 1 h of reaction in the incubator, the absorbance value was measured at 450 nm with a microplate reader SynergyH1 (Bio Tek). The data is processed on the GraphPad Prism software. Assay of inflammatory cytokines by ELISA: TNFα level assay: RAW264.7 cells were cultured in antibiotic-free high-glucose medium (DMEM) with 20% fetal bovine serum (FBS). Cells were incubated overnight in 96-well plates at a density of 30,000 cells per well. DNA nanostructures were then added to each well at a final concentration of 1.5 µM. After 24 hours of co-incubation, the plates were centrifuged at 1000 rpm for 5 min to collect the cell supernatant. TNFα was measured and quantified with a standard curve using the commercial kit Mab Tech ELISA mouse TNFα (cat. 3511-1H-20) according to manufacturer’s instruction. TNFα concentrations were expressed in pg/ml. IL-6 level assay: RAW 264.7 cells were seeded in 24-well plates at a density of 200,000 cells per well and cultured overnight. DNA nanostructures were then added to each well at a final concentration of 1.5 µM. After 24 hours of co-incubation with the DNA agonists, cell supernatants were collected, and IL-6 levels were measured using an ELISA kit (cat. 3361-1H-20) according to the manufacturer’s instructions. IL-6 concentrations were expressed in pg/ml. Cell uptake analysized by laser confocal scanning microscope: RAW 264.7 cells were grown at a density of 200,000 per well in glass-bottom confocal dishes. Cy5-labelled DNA was added to the cells at a final concentration of 200 nM, incubated for 2 h, and then stained with 1 μg/ml hoechest33342 for 10 min. Samples were imaged using the confocal microscope SpinSR (Olympus). Excitation wavelengths of 405 nm and 640 nm were used for Hoechst 33342 and Cy5-DNA, respectively. Image analysis was performed with the software OlyVIA. Cell uptake by flow cytometry: RAW 264.7 cells were seeded in 24-well plates at a density of 200,000 cells per well and incubated overnight at 37 ℃ with 5% CO₂. Cy5-labeled DNA was added to the cells at a final concentration of 200 nM, and cells were collected after incubation for various durations. To eliminate interference from extracellular DNA, the cells were treated with 70 U/ml DNase I at 37 ℃ for 30 min. Following this, the cells were washed with PBS and resuspended in PBS containing 4% paraformaldehyde. Samples were analyzed in FACSVerse11000 flow cytometer (BD), and data processing was done using FACS Diva (BD) and FlowJo (Tree Star). Statistical analysis: Results are presented as mean values ± standard deviation of three independent experiments, with three technical triplicates each. Statistical analyses were determined by two-tailed Student’s t-test or the one-way ANOVA test using GraphPad Prism software (*p ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). Supporting Information The supporting information for this article is available on the WWW under https://doi.org/10.1002/cjoc.202400xxx. Acknowledgement This work was financially supported by National Natural Science Foundation of China (22274135 and 22134005), and Natural Science Foundation of Chongqing, China (CSTB2024TIAD-CYKJCXX0039 and CSTB2024NSCQ-MSX0907). We are grateful for the assistance of Dr. Yan Li (Analytical and Testing Center, Southwest University) for the assistance in confocal observations. References 1. Manz, B. N.; Groves, J. T., Spatial organization and signal transduction at intercellular junctions. Nat. Rev. Mol. Cell Biol. 2010, 11 (5), 342-352. 2. 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M., Therapeutic potential of Toll-like receptor 9 activation. Nat. Rev. Drug Discov. 2006, 5 (6), 471-484. 37. Uno, S.; Nishikawa, M.; Mohri, K.; Umeki, Y.; Matsuzaki, N.; Takahashi, Y.; Fujita, H.; Kadowaki, N.; Takakura, Y., Efficient delivery of immunostimulatory DNA to mouse and human immune cells through the construction of polypod-like structured DNA. Nanomedicine 2014, 10 (4), 765-774. Manuscript received: XXXX, 2024 Manuscript revised: XXXX, 2024 Manuscript accepted: XXXX, 2024 Version of record online: XXXX, 2024 Entry for the Table of Contents Rational Design of DNA nanostructures as TLR9 Agonists Chunfa Chen a, # , Cheng Tian a, # , Zhuoer Jin a , Yuandong Wen a , Cheng Zhi Huang a , Hua Zuo a,* Chin. J. Chem. 2024 , 42 , XXX—XXX. DOI: 10.1002/cjoc.202400XXX Based on the two DNA binding sites on the TLR9 receptor, a DNA-ligand-functionalized nanostructure was designed to regulate the spatial steric hindrance between DNA ligands at the nanoscale. This design enhances the nanostructure’s ability to bind to the TLR9 receptor and induce downstream inflammatory responses. Information & Authors Information Version history V1 Version 1 10 February 2025 Peer review timeline Published Biomacromolecules Version of Record 8 Oct 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords cpg dna nanomaterials ligand-receptor protein crystal structure spacing tlr9 agonist Authors Affiliations Chunfa Chen Southwest University College of Pharmaceutical Sciences View all articles by this author Cheng Tian Southwest University College of Pharmaceutical Sciences View all articles by this author Zhuoer Jin Southwest University College of Pharmaceutical Sciences View all articles by this author Yuandong Wen Southwest University College of Pharmaceutical Sciences View all articles by this author Cheng Zhi Huang Southwest University College of Pharmaceutical Sciences View all articles by this author Hua Zuo [email protected] Southwest University College of Pharmaceutical Sciences View all articles by this author Metrics & Citations Metrics Article Usage 270 views 111 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Chunfa Chen, Cheng Tian, Zhuoer Jin, et al. Rational Design of DNA nanostructures as TLR9 Agonists. 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