Identification of Molecular Subtype and Prognostic Signature for Prostate Adenocarcinoma based on Neutrophil Extracellular Traps.

Background: Prostate adenocarcinoma (PRAD) is one of the most common cancers in male. Increasing evidences pointed out that Neutrophil Extracellular Traps (NETs) play an important role in tumor angiogenesis, tumor metastasis and drug resistance. However, limited systematic studies regarding the role of NETs in PRAD have been performed. Identification of biomarkers based on NETs might facilitate risk stratification which help optimizing the clinical strategies. Methods: NETs-related genes with differential expressions were identified between PRAD and adjacent normal tissues in TCGA-PRAD dataset. Consensus cluster analysis was performed to determine the PRAD subtypes based on the different-expressed NETs-related genes. The difference of pathway enrichment, infiltrating immune cell and genomic mutation were also evaluated between subtypes. LASSO cox regression analysis was conducted to construct a NETs-related prognostic signature. Result: We identified 19 NETs related genes with differential expressions between PRAD and adjacent normal tissue in TCGA-PRAD dataset. Two significant subtypes were identified based on these 19 genes by consensus cluster analysis, namely subtype 1 and subtype 2. Significant differences in prognosis, immune infiltration and tumor mutation burden were observed in subtypes. LASSO Cox regression analysis identified a NETs-associated prognostic signature including 13 genes, and this signature had a good performance in predicting the progression-free survival of PRAD patients. Further integrated analysis indicated that MMP9 mostly expressed in Mono/Macrophage cells might play a role in regulating NETs formation via neutrophil activation in PRAD. Conclusion: To sum up, the current study identified two NETs-related molecular subtypes and based on which constructed a prognostic signature for PRAD.

Purines enrich root-associated Pseudomonas and improve wild soybean growth under salt stress.

The root-associated microbiota plays an important role in the response to environmental stress. However, the underlying mechanisms controlling the interaction between salt-stressed plants and microbiota are poorly understood. Here, by focusing on a salt-tolerant plant wild soybean (Glycine soja), we demonstrate that highly conserved microbes dominated by Pseudomonas are enriched in the root and rhizosphere microbiota of salt-stressed plant. Two corresponding Pseudomonas isolates are confirmed to enhance the salt tolerance of wild soybean. Shotgun metagenomic and metatranscriptomic sequencing reveal that motility-associated genes, mainly chemotaxis and flagellar assembly, are significantly enriched and expressed in salt-treated samples. We further find that roots of salt stressed plants secreted purines, especially xanthine, which induce motility of the Pseudomonas isolates. Moreover, exogenous application for xanthine to non-stressed plants results in Pseudomonas enrichment, reproducing the microbiota shift in salt-stressed root. Finally, Pseudomonas mutant analysis shows that the motility related gene cheW is required for chemotaxis toward xanthine and for enhancing plant salt tolerance. Our study proposes that wild soybean recruits beneficial Pseudomonas species by exudating key metabolites (i.e., purine) against salt stress.

Engineering Modular DNA Reaction Networks for Signal Processing.

Diversified molecular information-processing methods have significant implications for nanoscale manipulation and control, monitoring and disease diagnosis of organisms, and direct intervention in biological activities. However, as an effective approach for implementing multifunctional molecular information processing, DNA reaction networks (DRNs) with numerous functionally specialized molecular structures have challenged them on scale and modular design, leading to increased network complexity, further causing problems such as signal leakage, attenuation, and cross-talk in network reactions. Our study developed a strategy for performing various signal-processing tasks through engineering modular DRNs composed of simple molecular structures. This strategy is based on a universal core unit with signal selection capability, and a timeadjustable signal self-resetting module is achieved by combing the core unit and self-resetting unit, which improves the time controllability of modular DRNs. In addition, multi-input and -output signal crosscatalytic and continuously adjustable signal delay modules were realized by combining core and threshold units, providing a flexible, precise method for modular DRNs to process the signal. The strategy simplifies the design of DRNs, helps generate design ideas for largescale integrated DRNs with multiple functions, and provides prospects in biocomputing, gene regulation, and biosensing.

DNA-QLC: an efficient and reliable image encoding scheme for DNA storage.

BACKGROUND: DNA storage has the advantages of large capacity, long-term stability, and low power consumption relative to other storage mediums, making it a promising new storage medium for multimedia information such as images. However, DNA storage has a low coding density and weak error correction ability. RESULTS: To achieve more efficient DNA storage image reconstruction, we propose DNA-QLC (QRes-VAE and Levenshtein code (LC)), which uses the quantized ResNet VAE (QRes-VAE) model and LC for image compression and DNA sequence error correction, thus improving both the coding density and error correction ability. Experimental results show that the DNA-QLC encoding method can not only obtain DNA sequences that meet the combinatorial constraints, but also have a net information density that is 2.4 times higher than DNA Fountain. Furthermore, at a higher error rate (2%), DNA-QLC achieved image reconstruction with an SSIM value of 0.917. CONCLUSIONS: The results indicate that the DNA-QLC encoding scheme guarantees the efficiency and reliability of the DNA storage system and improves the application potential of DNA storage for multimedia information such as images.

Efficient data reconstruction: The bottleneck of large-scale application of DNA storage.

Over the past decade, the rapid development of DNA synthesis and sequencing technologies has enabled preliminary use of DNA molecules for digital data storage, overcoming the capacity and persistence bottlenecks of silicon-based storage media. DNA storage has now been fully accomplished in the laboratory through existing biotechnology, which again demonstrates the viability of carbon-based storage media. However, the high cost and latency of data reconstruction pose challenges that hinder the practical implementation of DNA storage beyond the laboratory. In this article, we review existing advanced DNA storage methods, analyze the characteristics and performance of biotechnological approaches at various stages of data writing and reading, and discuss potential factors influencing DNA storage from the perspective of data reconstruction.

Storing Images in DNA via base128 Encoding.

Current DNA storage schemes lack flexibility and consistency in processing highly redundant and correlated image data, resulting in low sequence stability and image reconstruction rates. Therefore, according to the characteristics of image storage, this paper proposes storing images in DNA via base128 encoding (DNA-base128). In the data writing stage, data segmentation and probability statistics are carried out, and then, the data block frequency and constraint encoding set are associated with achieving encoding. When the image needs to be recovered, DNA-base128 completes internal error correction by threshold setting and drift comparison. Compared with representative work, the DNA-base128 encoding results show that the undesired motifs were reduced by 71.2-90.7% and that the local guanine-cytosine content variance was reduced by 3 times, indicating that DNA-base128 can store images more stably. In addition, the structural similarity index (SSIM) and multiscale structural similarity (MS-SSIM) of image reconstruction using DNA-base128 were improved by 19-102 and 6.6-20.3%, respectively. In summary, DNA-base128 provides image encoding with internal error correction and provides a potential solution for DNA image storage. The data and code are available at the GitHub repository: https://github.com/123456wk/DNA_base128.

Transcriptome analysis reveals how cadmium promotes root development and accumulates in Apocynum venetum, a promising plant for greening cadmium-contaminated soil.

Cadmium (Cd) contamination poses a substantial threat the environment, necessitating effective remediation strategies. Phytoremediation emerges as a cost-efficient and eco-friendly approach for reducing Cd levels in the soil. In this study, the suitability of A. venetum for ameliorating Cd-contaminated soils was evaluated. Mild Cd stress promoted seedling and root growth, with the root being identified as the primary tissue for Cd accumulation. The Cd content of roots ranged from 0.35 to 0.55 mg/g under treatment with 10-50 µM CdCl2·2.5 H2O, and the bioaccumulation factor ranged from 28.78 to 84.43. Transcriptome sequencing revealed 20,292 unigenes, and 7507 nonredundant differentially expressed genes (DEGs) were identified across five comparison groups. DEGs belonging to the "MAPK signaling pathway-plant," "monoterpenoid biosynthesis," and "flavonoid biosynthesis pathway" exhibited higher expression levels in roots compared to stems and leaves. In addition, cytokinin-related DEGs, ROS scavenger genes, such as P450, glutathione-S-transferase (GST), and superoxide dismutase (SOD), and the cell wall biosynthesis-related genes, CSLG and D-GRL, were also upregulated in the root tissue, suggesting that Cd promotes root development. Conversely, certain ABC transporter genes, (e.g, NRAMP5), and some vacuolar iron transporters, predominantly expressed in the roots, displayed a strong correlation with Cd content, revealing the mechanism underlying the compartmentalized storage of Cd in the roots. KEGG enrichment analysis of DEGs showed that the pathways associated with the biosynthesis of flavonoids, lignin, and some terpenoids were significantly enriched in the roots under Cd stress, underscoring the pivotal role of these pathways in Cd detoxification. Our study suggests A. venetum as a potential Cd-contaminated phytoremediation plant and provides insights into the molecular-level mechanisms of root development promotion and accumulation mechanism in response to Cd stress.

Transcriptomics integrated with metabolomics reveal the competitive relationship between co-cultured Trichoderma asperellum HG1 and Bacillus subtilis Tpb55.

Microbial co-culture has proven to be an effective way to improve the ability of microorganisms to biocontrol. However, the interactive mechanisms of co-cultural microbes, especially between fungi and bacteria, have rarely been studied. By comparative analysis of morphology, transcriptomics and metabolomics, the interactive mechanisms of a sequential co-culture system of Trichoderma asperellum HG1 and Bacillus subtilis Tpb55 was explored in this study. The results revealed that co- culture has no significant effect on the growth and cell morphology of the two strains, but lead to mycelium wrinkling of HG1. RNA-seq analysis showed that co-culture significantly upregulated the HG1 genes concerning amino acid degradation and metabolism, proteolysis, resisting environmental stress, cell homeostasis, glycolysis, the glyoxylate cycle, and the citric acid (TCA) cycle, while Tpb55 genes related to cell homeostasis, spore formation and membrane fluidization were significantly upregulated, but genes associating to TCA, glycolytic cycles and fatty acid ß-oxidation were significantly downregulated. Metabolomic results revealed that some amino acids related to energy metabolism were significantly altered in HG1, whereas palmitic acid, which is related to cell membrane functions, was upregulated in Tpb55. These results indicated that HG1 could interfere with carbon metabolism and cell membrane fluidity, but accelerate spore formation of Tpb55. Biophysical assays further convinced that co-culture could decrease ATP content and inhibit ATPase activity in HG1, and could promote spore formation and reduce the cell membrane fluidity of Tpb55. In addition, co-culture also accelerated the production of intracellular anti-oomycete compound octhilinone. The above results indicate that HG1 and Tpb55 are mainly in a competitive relationship in the co culture system. These findings provide new insights for understanding the interaction mechanism between co cultured microbes.

Ensifer sp. GMS14 enhances soybean salt tolerance for potential application in saline soil reclamation.

Rhizosphere microbiomes play an important role in enhancing plant salt tolerance and are also commonly employed as bio-inoculants in soil remediation processes. Cultivated soybean (Glycine max) is one of the major oilseed crops with moderate salt tolerance. However, the response of rhizosphere microbes me to salt stress in soybean, as well as their potential application in saline soil reclamation, has been rarely reported. In this study, we first investigated the microbial communities of salt-treated and non-salt-treated soybean by 16S rRNA gene amplicon sequencing. Then, the potential mechanism of rhizosphere microbes in enhancing the salt tolerance of soybean was explored based on physiological analyses and transcriptomic sequencing. Our results suggested that Ensifer and Novosphingobium were biomarkers in salt-stressed soybean. One corresponding strain, Ensifer sp. GMS14, showed remarkable growth promoting characteristics. Pot experiments showed that GMS14 significantly improved the growth performance of soybean in saline soils. Strain GMS14 alleviated sodium ions (Na+) toxicity by maintaining low a Na+/K+ ratio and promoted nitrogen (N) and phosphorus (P) uptake by soybean in nutrient-deficient saline soils. Transcriptome analyses indicated that GMS14 improved plant salt tolerance mainly by ameliorating salt stress-mediated oxidative stress. Interestingly, GMS14 was evidenced to specifically suppress hydrogen peroxide (H2O2) production to maintain reactive oxygen species (ROS) homeostasis in plants under salt stress. Field experiments with GMS14 applications showed its great potential in saline soil reclamation, as evidenced by the increased biomass and nodulation capacity of GMS14-inoculated soybean. Overall, our findings provided valuable insights into the mechanisms underlying plant-microbes interactions, and highlighted the importance of microorganisms recruited by salt-stressed plant in the saline soil reclamation.

CSTF2 mediated mRNA N6-methyladenosine modification drives pancreatic ductal adenocarcinoma m6A subtypes.

N6-methyladenosine (m6A) modification of gene transcripts plays critical roles in cancer. Here we report transcriptomic m6A profiling in 98 tissue samples from 65 individuals with pancreatic ductal adenocarcinoma (PDAC). We identify 17,996 m6A peaks with 195 hyper-methylated and 93 hypo-methylated in PDAC compared with adjacent normal tissues. The differential m6A modifications distinguish two PDAC subtypes with different prognosis outcomes. The formation of the two subtypes is driven by a newly identified m6A regulator CSTF2 that co-transcriptionally regulates m6A installation through slowing the RNA Pol II elongation rate during gene transcription. We find that most of the CSTF2-regulated m6As have positive effects on the RNA level of host genes, and CSTF2-regulated m6As are mainly recognized by IGF2BP2, an m6A reader that stabilizes mRNAs. These results provide a promising PDAC subtyping strategy and potential therapeutic targets for precision medicine of PDAC.

LncRNA BCAN-AS1 stabilizes c-Myc via N6-methyladenosine-mediated binding with SNIP1 to promote pancreatic cancer.

C-Myc overexpression contributes to multiple hallmarks of human cancer but directly targeting c-Myc is challenging. Identification of key factors involved in c-Myc dysregulation is of great significance to develop potential indirect targets for c-Myc. Herein, a collection of long non-coding RNAs (lncRNAs) interacted with c-Myc is detected in pancreatic ductal adenocarcinoma (PDAC) cells. Among them, lncRNA BCAN-AS1 is identified as the one with highest c-Myc binding enrichment. BCAN-AS1 was abnormally elevated in PDAC tumors and high BCAN-AS1 level was significantly associated with poor prognosis. Mechanistically, Smad nuclear-interacting protein 1 (SNIP1) was characterized as a new N6-methyladenosine (m6A) mediator binding to BCAN-AS1 via recognizing its m6A modification. m6A-modified BCAN-AS1 acts as a scaffold to facilitate the formation of a ternary complex together with c-Myc and SNIP1, thereby blocking S phase kinase-associated protein 2 (SKP2)-mediated c-Myc ubiquitination and degradation. Biologically, BCAN-AS1 promotes malignant phenotypes of PDAC in vitro and in vivo. Treatment of metastasis xenograft and patient-derived xenograft mouse models with in vivo-optimized antisense oligonucleotide of BCAN-AS1 effectively represses tumor growth and metastasis. These findings shed light on the pro-tumorigenic role of BCAN-AS1 and provide an innovant insight into c-Myc-interacted lncRNA in PDAC.

A moderately thermophilic origin of a novel family of marine group II euryarchaeota from deep ocean.

Marine group II (MGII) is the most abundant planktonic heterotrophic archaea in the ocean. The evolutionary history of MGII archaea is elusive. In this study, 13 new MGII metagenome-assembled genomes were recovered from surface to the hadal zone in Challenger Deep of the Mariana Trench; four of them from the deep ocean represent a novel group. The optimal growth temperature (OGT) of the common ancestor of MGII has been estimated to be at about 60°C and OGTs of MGIIc, MGIIb, and MGIIa at 47°C-50ºC, 37°C-44ºC, and 30°C-37ºC, respectively, suggesting the adaptation of these species to different temperatures during evolution. The estimated OGT range of MGIIc was supported by experimental measurements of cloned ß-galactosidase that showed optimal enzyme activity around 50°C. These results indicate that MGIIc may have originated from a common ancestor that lived in warm or even hot marine environment, such as hydrothermal vents.

Biomolecule-Driven Two-Factor Authentication Strategy for Access Control of Molecular Devices.

The rise of DNA nanotechnology is promoting the development of molecular security devices and marking an essential change in information security technology, to one that can resist the threats resulting from the increase in computing power, brute force attempts, and quantum computing. However, developing a secure and reliable access control strategy to guarantee the confidentiality of molecular security devices is still a challenge. Here, a biomolecule-driven two-factor authentication strategy for access control of molecular devices is developed. Importantly, the two-factor is realized by applying the specificity and nicking properties of the nicking enzyme and the programmable design of the DNA sequence, endowing it with the characteristic of a one-time password. To demonstrate the feasibility of this strategy, an access control module is designed and integrated to further construct a role-based molecular access control device. By constructing a command library composed of three commands (Ca, Cb, Ca and Cb), the authorized access of three roles in the molecular device is realized, in which the command Ca corresponds to the authorization of role A, Cb corresponds to the authorization of role B, and Ca and Cb corresponds to the authorization of role C. In this way, when users access the device, they not only need the correct factor but also need to apply for role authorization in advance to obtain secret information. This strategy provides a highly robust method for the research on access control of molecular devices and lays the foundation for research on the next generation of information security.

Effect of planting salt-tolerant legumes on coastal saline soil nutrient availability and microbial communities.

Soil salinization is a serious global environmental problem affecting sustainable development of agriculture. Legumes are excellent candidates for the phytoremediation of saline soils; however, how soil microbes mediate the amelioration of coastal saline ecosystems is unknown. In this study, two salt-tolerant legumes, Glycine soja and Sesbania cannabina were planted in coastal saline soil for three years. Soil nutrient availability and microbiota structure (including bacteria, fungi, and diazotrophs) were compared between the phytoremediated soils and control soil (barren land). Planting legumes reduced soil salinity, and increased total carbon, total nitrogen, and NO3--N contents. Among the soil microbiota, some nitrogen-fixing bacteria (e.g., Azotobacter) were enriched in legumes, which were probably responsible for soil nitrogen accumulation. The complexity of the bacterial, fungal, and diazotrophic networks increased significantly from the control to the phytoremediated soils, suggesting that the soil microbial community formed closer ecological interactions during remediation. Furthermore, the dominant microbial functions were chemoheterotrophy (24.75%) and aerobic chemoheterotrophy (21.97%) involved in the carbon cycle, followed by nitrification (13.68%) and aerobic ammonia oxidation (13.34%) involved in the nitrogen cycle. Overall, our findings suggested that G. soja and S. cannabina legumes were suitable for ameliorating saline soils as they decreased soil salinity and increased soil nutrient content, with microorganisms especially nitrogen-fixing bacteria, playing an important role in this remediation process.

Multi-functional nanogel with cascade catalytic performance for treatment of diabetic oral mucosa ulcer.

Introduction: Diabetic oral mucosa ulcers face challenges of hypoxia, hyperglycemia and high oxidative stress, which result in delayed healing process. Oxygen is regarded as an important substance in cell proliferation, differentiation and migration, which is beneficial to ulcer recovery. Methods: This study developed a multi-functional GOx-CAT nanogel (GCN) system for the treatment of diabetic oral mucosa ulcers. The catalytic activity, ROS scavenge and oxygen supply ability of GCN was validated. The therapeutic effect of GCN was verified in the diabetic gingival ulcer model. Results: The results showed that the nanoscale GCN was capable of significantly eliminating intracellular ROS, increasing intracellular oxygen concentration and accelerating cell migration of human gingival fibroblasts, which could promote diabetic oral gingival ulcer healing in vivo by alleviating inflammation and promoting angiogenesis. Discussion: This multifunctional GCN with ROS depletion, continuous oxygen supply and good biocompatibility, which might provide a novel therapeutic strategy for effective treatment of diabetic oral mucosa ulcers.

Potential noninvasive biomarkers for the malignant transformation of oral leukoplakia: A systematic review and meta-analysis.

BACKGROUND: The rising cancer incidence in patients with oral leukoplakia (OL) highlights the importance of identifying potential biomarkers for high-risk individuals and lesions because these biomarkers are useful in developing personalized management strategies for OL patients. This study systematically searched and analyzed the literature on potential saliva and serum biomarkers for OL malignant transformation. METHODS: PubMed and Scopus were searched for studies published up to April 2022. The primary outcome of this study was the difference in biomarker concentrations in saliva or serum samples from healthy control (HC), OL and oral cancer (OC) populations. Cohen's d with 95% credible interval was calculated and pooled using the inverse variance heterogeneity method. RESULTS: A total of seven saliva biomarkers were analyzed in this paper, including interleukin-1alpha, interleukin-6 (IL-6), interleukin-6-8, tumor necrosis factor alpha (TNF-α), copper, zinc, and lactate dehydrogenase. IL-6 and TNF-α exhibited statistically significant deviations in comparisons between HC versus OL and OL versus OC. A total of 13 serum biomarkers were analyzed, including IL-6, TNF-α, C-reactive protein, total cholesterol, triglycerides, high-density lipoproteins, low-density lipoproteins, albumin, protein, ß2-microglobulin, fucose, lipid-bound sialic acid (LSA), and total sialic acid (TSA). LSA and TSA exhibited statistically significant deviations in comparisons between HC versus OL and OL versus OC. CONCLUSION: IL-6 and TNF-α in saliva have strong predictive values for OL deterioration, and LSA and TSA concentration levels in serum also have the potential to serve as biomarkers for OL deterioration.

High Net Information Density DNA Data Storage by the MOPE Encoding Algorithm.

DNA has recently been recognized as an attractive storage medium due to its high reliability, capacity, and durability. However, encoding algorithms that simply map binary data to DNA sequences have the disadvantages of low net information density and high synthesis cost. Therefore, this paper proposes an efficient, feasible, and highly robust encoding algorithm called MOPE (Modified Barnacles Mating Optimizer and Payload Encoding). The Modified Barnacles Mating Optimizer (MBMO) algorithm is used to construct the non-payload coding set, and the Payload Encoding (PE) algorithm is used to encode the payload. The results show that the lower bound of the non-payload coding set constructed by the MBMO algorithm is 3%-18% higher than the optimal result of previous work, and theoretical analysis shows that the designed PE algorithm has a net information density of 1.90 bits/nt, which is close to the ideal information capacity of 2 bits per nucleotide. The proposed MOPE encoding algorithm with high net information density and satisfying constraints can not only effectively reduce the cost of DNA synthesis and sequencing but also reduce the occurrence of errors during DNA storage.

Ulva prolifera polysaccharide enhances the root colonisation by Bacillus amyloliquefaciens strain Cas02.

The application of biocontrol agent is important for the sustainable development of agriculture. Unsuccessful or limited colonisation by plant growth-promoting rhizobacteria (PGPR) has become an important constraint factor for their commercial application. Here, we report that Ulva prolifera polysaccharide (UPP) promotes root colonisation by Bacillus amyloliquefaciens strain Cas02. UPP serves as an environmental signal for bacterial biofilm formation and its glucose residue is used as a carbon source for the synthesis of the exopolysaccharides and poly-gamma-glutamate present in biofilm matrix. Greenhouse experiments demonstrated that UPP could effectively enhance the root colonisation by Cas02 in both the bacterial population and survival time under natural semiarid soil conditions. Furthermore, the microbiome analysis also indicated the promoted colonisation by Cas02, as well as the improved bacterial rhizosphere community structure, after combined treatment of UPP and Cas02. This study provides a practical approach to improve the biocontrol agent with seaweed polysaccharides.

FMG: An observable DNA storage coding method based on frequency matrix game graphs.

Using complex biomolecules for storage is a new carbon-based storage method. For example, DNA has the potential to be a good method for archival long-term data storage. Reasonable and efficient coding is the first and most important step in DNA storage. However, current coding methods, such as altruism algorithm, have the problem of low coding efficiency and high complexity, and coding constraints and sets make it difficult to see the coding results visually. In this study, a new DNA storage coding method based on frequency matrix game graph (FMG) is proposed to generate DNA storage coding satisfying combinatorial constraints. Compared with the randomness of the heuristic algorithm that satisfies the constraints, the coding method based on the FMG is deterministic and can clearly explain the coding process. In addition, the constraints and coding results have observable characteristics and are better than the previously published results for the size of the coding set. For example, when length of the code n = 10, hamming distance d = 4, the results obtained by proposed approach combining chaos game and graph are 24% better than the previous results. The proposed coding scheme successfully constructs high-quality coding sets with less complexity, which effectively promotes the development of carbon-based storage coding.

TripDesign: A DNA Triplex Design Approach Based on Interaction Forces.

A DNA triplex has the advantages of improved nanostructure stability and pH environment responsiveness compared with single-stranded and double-stranded nucleic acids. However, sequence stability and low design efficiency hinder the application of DNA triplexes. Therefore, a DNA triplex design approach (TripDesign) based on interaction forces is proposed. First, we present the stacking force constraint, torsional stress constraint, and G-quadruplex motif constraint and then use an improved memetic algorithm to design triplex sequences under combinatorial constraints. Finally, to quantify the process of triplex formation, we also explore the minimum length of the triplex-forming oligos (TFOs) required to form the triplex and the factors that produce depletion in cyclic pH-jump experiments. The experimental results show that the sequences produced by TripDesign have high stability and reversibility, and the proposed approach achieves efficient and automatic sequence design. In addition, this study characterizes multiple basic parameters of DNA triplex formation and promotes the wider application of DNA triplexes in nanotechnology.