Multifunctional DNA Research Articles

A multifunctional DNA repair enzyme and magnetic dual-triggered theranostic nanosystem for intelligent drug delivery

Due to the high specificity and efficiency of enzymes, enzyme-responsive drug delivery systems are important in cancer therapy and diagnosis. Human apurinic/apyrimidinic endonuclease (APE1) is a representative DNA repair enzyme overexpressed in various cancer cells, which is also proven to be a promising stimuli in our previous studies. In this work, to realize targeted chemotherapy, an APE1 and magnetic dual-responsive nanocarrier was designed. The carrier is composed of Fe3O4@SiO2 nanoparticles and specially designed AP-DNA strand that can be effectively hydrolyzed by APE1. The chemotherapeutic drug doxorubicin (DOX) was loaded onto the AP-DNA to form the theranostic nanosystem (FS@DNA-DOX). As the AP-DNA was parted under the hydrolysis of APE1 in cancer cells, DOX was released to inhibit tumor growth. In vitro experiment results indicated that the nanosystem possesses a high specificity to APE1 and effectively release DOX in MCF-7 cells. In vivo experiments demonstrated that the targeted delivery of the nanosystem to the tumor site was successfully achieved and that strong tumor suppressor effects were observed. Overall, the FS@DNA-DOX is a multifunctional and theranostic nanosystem for the chemotherapy of cancer by increasing the therapeutic effect as well as reducing the toxicity of drugs.

Up-regulated PIF1 predicts poor clinical outcomes and correlates with low immune infiltrates in clear cell renal cell carcinoma.

Background: Petite Integration Factor 1 (PIF1) is a multifunctional helicase and DNA processing enzyme that plays an important role in the process of several cancer types. However, the relationship between clear cell renal cell carcinoma (ccRCC) and PIF1 remains unclear. This study aims to explore the role of PIF1 in ccRCC tumorigenesis and prognosis. Methods: Based on The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) database, we retrieved and verified the expression of PIF1 in ccRCC tissues as well as normal tissues. To assess the protein expression of PIF1 by using the Human Protein Atlas and the Clinical Proteomic Tumor Analysis Consortium (CPTAC). We also performed receiver operating characteristic (ROC) curve analysis to differentiate the effectiveness of PIF1 in ccRCC and adjacent normal tissues. To evaluate the value of PIF1 on clinical outcomes in ccRCC patients by using multivariate methods and Kaplan‒Meier survival curves. Protein‒protein interaction (PPI) networks were made with STRING. We determined the relationship between the expression of PIF1 and immune cell infiltration with single-sample gene set enrichment analysis (ssGSEA). Results: Compared with normal tissues, the expression of PIF1 was significantly elevated in ccRCC. The mRNA expression of PIF1 is correlated with high TNM stage and high pathologic stage. The receiver operating characteristic (ROC) curve analysis showed that PIF1 was related to an area under the curve (AUC) value of 0.928 to distinguish between ccRCC tissues and normal tissues. Kaplan‒Meier survival analysis showed that the overall survival (OS) of ccRCC patients with a high level of PIF1 was significantly shorter than that of those with a low level of PIF1. PIF1 may play an important role in the occurrence of tumors. Correlation analysis showed that PIF1-mediated carcinogenesis may participate in the process of tumor immune escape in ccRCC. Conclusion: PIF1 could be a reference biomarker to identify ccRCC patients with poor prognosis. PIF1 may play a distinct role in the microenvironment of ccRCC by regulating tumor infiltration of immune cells, which is a new therapeutic target to affect the growth of the tumor.

Multifunctional DNA nanoprobe for tumor-targeted synergistic therapy by integrating chemodynamic therapy with gene silencing.

Due to the high complexity, diversity and heterogeneity of tumor occurrence and development, multi-mode synergistic therapy is more effective than single treatment modes to improve the antitumor efficacy. Also, multifunctional probes are crucial to realize synergistic therapy. Herein, a multifunctional DNA tetrahedron nanoprobe was ingeniously designed to simultaneously achieve chemodynamic therapy (CDT) and gene silencing for synergistic antitumor. The multifunctional DNA tetrahedron nanoprobe, DNA tetrahedron-silver nanocluster-antagomir-21 (D-sgc8-DTNS-AgNCs-Anta-21), integrated a CDT reagent (DNA-AgNCs) and miRNA-21 inhibitor (Anta-21) with a specific recognition probe (aptamer). After targeted entry in cancer cells, D-sgc8-DTNS-AgNCs-Anta-21 silenced endogenous miRNA-21 by Anta-21 and produced highly toxic ˙OH by reacting with H2O2, which induced apoptosis in the tumor cells. The targeted recognition of aptamers led to the concentration-dependent death of HeLa cells. On the contrary, the cell survival rate of normal cells was basically unaffected with an increase in the concentration of D-sgc8-DTNS-AgNCs-Anta-21. Therefore, the diverse functions, biocompatibility and programmability of DNA provide a useful and easy way to assemble multifunctional probes for synergistic therapy.

Potential mechanism of the AgNCs-hydrogel in promoting the regeneration of diabetic infectious wounds.

Diabetic infectious wound treatment is challenging due to insistent wound infections. To treat such complicated pathological diabetic infectious wounds, multifunctional materials need to be developed, and their mechanisms need to be understood. Here, we developed a material termed AgNCs-hydrogel, which is a multifunctional DNA hydrogel used as dressings by integrating it with antibacterial silver nanoclusters. The AgNCs-hydrogel was applied to promote the regeneration of diabetic infectious wounds in mice because it exhibited superior antibacterial activity and effective ROS-scavenging properties. Based on skin proteomics, we explored the potential mechanism of the AgNCs-hydrogel in treating mouse skin wounds. We found that the AgNCs-hydrogel can regulate some key proteins located primarily in the extracellular exosomes, involved in the negative regulation of the apoptotic process, and perform ATP binding to accelerate diabetic infected wound closure. Therefore, this study provided a multifunctional AgNCs-hydrogel and revealed its potential mechanism in promoting the regeneration of diabetic infectious wounds.

NONO enhances mRNA processing of super-enhancer-associated GATA2 and HAND2 genes in neuroblastoma.

High-risk neuroblastoma patients have poor survival rates and require better therapeutic options. High expression of a multifunctional DNA and RNA-binding protein, NONO, in neuroblastoma is associated with poor patient outcome; however, there is little understanding of the mechanism of NONO-dependent oncogenic gene regulatory activity in neuroblastoma. Here, we used cell imaging, biochemical and genome-wide molecular analysis to reveal complex NONO-dependent regulation of gene expression. NONO forms RNA- and DNA-tethered condensates throughout the nucleus and undergoes phase separation invitro, modulated by nucleic acid binding. CLIP analyses show that NONO mainly binds to the 5' end of pre-mRNAs and modulates pre-mRNA processing, dependent on its RNA-binding activity. NONO regulates super-enhancer-associated genes, including HAND2 and GATA2. Abrogating NONO RNA binding, or phase separation activity, results in decreased expression of HAND2 and GATA2. Thus, future development of agents that target RNA-binding activity of NONO may have therapeutic potential in this cancer context.

267 Generation and Characterization of a CRISPR-Cas9-Mediated XPC gene Knockout in Human Skin Cells

Xeroderma pigmentosum group C (XPC) is a multifunctional DNA damage recognition protein involved in the global genome nucleotide excision repair (GG-NER) pathway. Mammalian cells employ this pathway to eliminate bulky DNA lesions that induce double helix distortion caused by mutagens such as UV radiation or cisplatin. Loss-of-function mutations in the XPC gene lead to various malignancies, including skin cancers. Photosensitivity and the buildup of DNA damage can both define the phenotype of XPC patients’ cells.

Biomineralization of DNA Nanoframeworks for Intracellular Delivery, On-Demand Diagnosis, and Synergistic Cancer Treatments.

DNA nanoframeworks, with great biological information and controlled framework structures, exhibit great potentials in biological applications. Their applications are normally limited by unstable structures susceptible to hydrolysis, depurination, depyrimidination, oxidation, alkylation, or nuclease degradations. Herein, to ensure the mechanical and chemical stabilities of DNA nanoframeworks for intracellular applications, biomineralization of multifunctional DNA nanoframeworks with a tetrahedral skeleton is employed. Via silicification, the S-S bond is simultaneously introduced to obtain the silica-armored DNA nanoframeworks (Si-DNA nanoframeworks), mechanically and chemically stabilized for efficient intracellular deliveries. This successfully prevents degradations and leakages of reagents loaded on Si-DNA nanoframeworks, including biomolecular siRNA and small DOX drugs. Furthermore, the nucleic acid strands of the nanoframeworks are labeled with FAM and the quencher, facilitating miRNA detection upon "turn-on" signals from hybridizations. Therefore, the nanoframeworks collapse via double responses of the silica coating (silica acidic dissolution and S-S reduction by GSH) in cancer cells, realizing on-demand reagent release for miRNA detection and synergistic treatments (by siRNA and DOX). Demonstrated by both in vivo and in vitro experiments, the biomineralization has stabilized DNA nanomaterials for biological applications.

In situ imaging miRNAs using multifunctional linear DNA nanostructure

The microRNAs (miRNAs) play a critical role in many biological processes and are essential biomarkers for diagnosing disease. However, the sensitive and specific quantification of microRNAs (miRNAs) expression in living cells still faces a huge challenge. Our study designed a multifunctional linear DNA nanostructure (MLN) as a carrier of molecular beacons (MB-21) for detecting and intracellular imaging miRNA-21. The MLN-MB consists of three parts: aptamer, MLN, and MB-21. The aptamer (AS1411) could media MLN-MB enter live cells without additional transfection reagents. Once inside the cells, the intracellular miRNA-21 could hybridize the MB-21s, resulting in significantly enhanced fluorescence signals. The whole process was enzyme-free, autonomous, and continuous, which avoided the necessity of adding external fuel strands or enzymes. We demonstrated that the MLN-MB could be used to screen the miRNA-21 with a detection limit of 320 pM in a short time (10 min) and show high specificity toward miRNA-21 against other miRNAs. Moreover, the proposed MLN-MB could detect the miRNA-21 in complex matrixes stably. With its outstanding stability, dual recognition, and biocompatibility, MLN-MB is capable of delivering into living cells to identify specific cancer cells. Therefore, our sensing approach, with high sensitivity, specificity, and simplicity advantages, holds great potential for early cancer diagnosis.

Elucidation of the role of nucleolin as a cell surface receptor for nucleic acid-based adjuvants

Nucleic acid-based adjuvants such as CpG oligonucleotides (CpG ODNs) and poly(I:C) are potential vaccine adjuvants for infectious diseases and cancers. However, the mechanism by which their cell surface receptors promote their uptake into dendritic cells (DCs) and shuttle them to intracellular Toll-like receptors remains to be further investigated. Here, we demonstrated a role for nucleolin, a multifunctional DNA- and RNA-binding protein and a major constituent of the nucleolus, as one of the cell-surface receptors for nucleic acid-based adjuvants. Nucleolin on mouse DC surface bound directly to A-type CpG ODN, B-type CpG ODN, and poly(I:C) and promoted their internalization into cells following DC maturation in vitro. In human DCs, nucleolin also contributed to the binding and internalization of both types of CpG ODNs and subsequent cytokine production. Furthermore, nucleolin played a crucial role in cytokine production and activating antigen-specific antibodies and T cell responses induced by B-type CpG ODN in vivo in mice. Our findings provide valuable information that can help improve the efficacy and safety of these adjuvants.

A pleiotropic variant in DNAJB4 is associated with multiple myeloma risk.

Pleiotropy, which consists of a single gene or allelic variant affecting multiple unrelated traits, is common across cancers, with evidence for genome-wide significant loci shared across cancer and noncancer traits. This feature is particularly relevant in multiple myeloma (MM) because several susceptibility loci that have been identified to date are pleiotropic. Therefore, the aim of this study was to identify novel pleiotropic variants involved in MM risk using 28 684 independent single nucleotide polymorphisms (SNPs) from GWAS Catalog that reached a significant association (P < 5× 10-8 ) with their respective trait. The selected SNPs were analyzed in 2434 MM cases and 3446 controls from the International Lymphoma Epidemiology Consortium (InterLymph). The 10 SNPs showing the strongest associations with MM risk in InterLymph were selected for replication in an independent set of 1955 MM cases and 1549 controls from the International Multiple Myeloma rESEarch (IMMEnSE) consortium and 418 MM cases and 147 282 controls from the FinnGen project. The combined analysis of the three studies identified an association between DNAJB4-rs34517439-A and an increased risk of developing MM (OR=1.22, 95%CI 1.13-1.32, P=4.81× 10-7 ). rs34517439-A is associated with a modified expression of the FUBP1 gene, which encodes a multifunctional DNA and RNA-binding protein that it was observed to influence the regulation of various genes involved in cell cycle regulation, among which various oncogenes and oncosuppressors. In conclusion, with a pleiotropic scan approach we identified DNAJB4-rs34517439 as a potentially novel MM risk locus.

Multifunctional DNA Hydrogel Enhances Stemness of Adipose‐Derived Stem Cells to Activate Immune Pathways for Guidance Burn Wound Regeneration

AbstractA grade 3 burn is a nonstatic fatal injury, which can lead to complete damage to the skin structure, accompanied by a series of symptoms such as persistent inflammation, pain, pruritus, ulcer, and peripheral neuropathy. Although the primary clinical burn treatment is skin grafting, it cannot comprehensively solve burn symptoms. Here, a multifunctional DNA hydrogel integrated system is conveniently obtained through dynamic cross‐linking of the DNA unit, polyacrylamide, and l‐ascorbate 2‐phosphate (l‐A2P) formation of dense hydrogen bonds. The DNA hydrogel is doped with borneol for pain and itch relief. The obtained DNA hydrogel provides a hotbed similar to the extracellular matrix structure in vivo for the growth and development of stem cells, which can regulate cell proliferation, maintain cell viability, and achieve perfect release in a suitable environment. Additionally, the pharmacological wound dressing shell features excellent mechanical behavior, tissue adhesion, and antibacterial properties. Beyond that, the multifunctional DNA hydrogel integrated system can promote macrophage transformation, angiogenesis, and neurogenesis. Notably, the system activates the phosphatidylinositol 3′‐kinase (PI3K)‐Akt signaling pathway, which helps to promote tissue regeneration. Therefore, the DNA hydrogel integrated system opens the cascade mode of effective integrated treatment of burn wounds, further driving the in‐depth study of clinical transformation mechanisms in the future.

A Facile, Label-free and Versatile Fluorescence Sensing Nanoplatform Based on Titanium Carbide Nanosheets for the Detection of Various Targets.

The construction of a universal nanoplatform for sensitive detection of multiple targets of interest is of great importance in different research fields. Herein, by ingeniously integrating the target recognition sequences and G-rich sequences into a single-stranded multifunctional DNA probe and adopting Ti3C2 nanosheets as an efficient fluorescence quencher, a simple, low-cost and easy operation fluorescence sensing nanoplatform was proposed. Without an analytical target, the hydrogen bond and metal chelate interaction between the target recognition region of the DNA probe and Ti3C2 nanosheet induce the selective self-assembly of highly fluorescent thioflavin T (ThT)-intercalated DNA probe onto the surface of Ti3C2 nanosheets, resulting in dramatic decrease of fluorescence emitted by ThT-G-quadruplex. In the presence of a target, the target recognition region will selectively bind with the target and the constrained DNA probe is released from the Ti3C2 nanosheets surface, leading to enhanced fluorescence recovery of ThT-G-quadruplex. As a proof of concept, the sensitive and selective detection of p53 gene, Hg2+, and adenosine with the assistance of Ti3C2 nanosheets-based fluorescence sensing nanoplatform were successfully realized. Moreover, it is also applicable for the evaluation the level of these analytical targets in real samples. By simply switching the recognition sequences of DNA probe, the universal sensing strategy could also be applied for detecting many other types of targets. The simple and universal sensing nanoplatform is expected to promote wide applications in environment monitoring and bioanalysis.

Ultrasensitive Fluorescence Detection and Accurate Colocalization Visualization of Dual-miRNAs in Cancer Cells Based on the Conjugated Chain Reaction of Multifunctional Pentagon DNA Nanostructures.

Herein, a multifunctional pentagon DNA nanostructure (MPDN) was assembled by the hybridization of a circular DNA scaffold containing five different fragments with five diverse DNA oligonucleotides for simultaneous sensitive detection and accurate colocalization imaging of dual-miRNAs in cancer cells. Exactly, the MPDN could specifically and efficiently internalize into folate (FA) receptor-overexpressed cells via specific binding of FA and the FA receptor to distinguish cancer cells from normal cells and transform trace amounts of targets miRNA-21 and miRNA-155 into substantial FAM and Cy5-labeled DNA polymers as the signal probe to generate two remarkable fluorescence emissions, realizing simultaneously sensitive detection of dual-miRNAs. Impressively, compared with traditional small fragment DNA probes with high fluidity, the DNA copolymers with extremely low diffusivity kept it in the originally generated position to achieve the colocalization imaging of dual-miRNAs more accurately for revealing the spatial expression information of dual-miRNAs in tissues and cells. This strategy provided programmable tool to simultaneously detect and accurately colocate dual-miRNAs for understanding normal physiology and the tumor mechanism.

Recent Advances in Self-Assembled DNA Nanostructures for Bioimaging.

DNA nanotechnology has been proven to be a powerful platform to assist the development of imaging probes for biomedical research. The attractive features of DNA nanostructures, such as nanometer precision, controllable size, programmable functions, and biocompatibility, have enabled researchers to design and customize DNA nanoprobes for bioimaging applications. However, DNA probes with low molecular weights (e.g., 10-100 nt) generally suffer from low stability in physiological buffer environments. To improve the stability of DNA nanoprobes in such environments, DNA nanostructures can be designed with relatively larger sizes and defined shapes. In addition, the established modification methods for DNA nanostructures are also essential in enhancing their properties and performances in a physiological environment. In this review, we begin with a brief recap of the development of DNA nanostructures including DNA tiles, DNA origami, and multifunctional DNA nanostructures with modifications. Then we highlight the recent advances of DNA nanostructures for bioimaging, emphasizing the latest developments in probe modifications and DNA-PAINT imaging. Multiple imaging modules for intracellular biomolecular imaging and cell membrane biomarkers recognition are also summarized. In the end, we discuss the advantages and challenges of applying DNA nanostructures in bioimaging research and speculate on its future developments.

Empowering Electrochemical Biosensing through Nanostructured or Multifunctional Nucleic Acid or Peptide Biomaterials

AbstractElectrochemical biosensors continue to evolve at an astonishing pace, consolidating as competitive tools for determining a wide range of targets and relentlessly strengthening their attributes in terms of sensitivity, selectivity, simplicity, response time, and antifouling ability, making them suitable for getting a foothold in real‐world applications. The design and exploitation of nanostructured or multifunctional nucleic acid or peptide biomaterials are playing a determinant role in these achievements. With the aim of highlighting the potential and opportunities of these biomaterials, this perspective article critically discusses and overviews the electrochemical biosensors reported since 2019 involving nanostructured and multifunctional DNA biomaterials, multifunctional aptamers, modern peptides, and CRISPR/Cas systems. The use of these biomaterials as recognition elements, electrode modifiers (acting as linkers or creating scaffolds with antifouling properties), enzyme substrates, and labeling/carrier agents for signal amplification is discussed through rationally and strategically selected examples, concluding with a personal perspective about the challenges to be faced and future lines of action.

Ultrasensitive MERS-CoV Biosensor Composed of Multi-functional DNA 3 Way-Junction Probe based on Electrochemical-Surface Enhanced Raman Spectroscopy (EC-SERS) Dual Detection System

Professor Taek Lee, associate professor, Biomedical Engineering Lab, Department of Chemical Engineering, Kwangwoon University, South Korea, is working on the development of biosensors and in vitro diagnostic devices, nucleic acid nanoengineering-based therapy and biomolecular-based bioelectronics for computation. Middle East respiratory syndrome coronavirus (MERS-CoV) is the novel coronavirus that causes Middle East respiratory syndrome (MERS). Lee is investigating the potential of aptamers to be used in the detection of viruses such as MERS-CoV. This could assist with diagnosis and treatment and protect peoples' health. In a world first, Lee and the team developed a MERS aptamer using the SELEX technique and then created the world's first MERS-nanovesicle biosensor composed of multi-functional DNA aptamer/graphene-MoS2 nanocomposite based on electrochemical and surface-enhanced Raman spectroscopy. The biosensor can detect the virus in a buffer solution and in saliva and the researchers believe it could be applicable to the detection of other viruses such as COVID-19. So far, the team has developed 30 types of aptamer that have potential to be used in biosensors and the researchers hope their work to establish a rapid virus detection system using fast SELEX and biosensor fabrication techniques will benefit health and wellbeing.

Versatile electrochemiluminescence sensor for dual-potential “off” and “on” detection of double targets based on a novel terbium organic gel and multifunctional DNA network probes

In this work, a multifunctional signal probe with DNA network-loaded Ru(phen)32+ and Ag nanocluster (NCs) was assembled, and combined with a novel terbium organic gel to design a versatile electrochemiluminescence (ECL) biosensor for dual-potential “on” “off” assays of Cd2+ and Mg2+. Firstly, target Cd2+-driven DNA walker-amplification process produced a large amount of output DNA, which was used to link the multifunctional DNA network (DNA Net) structure probes with both Ru(phen)32+ and C-rich base to the terbium organic gel (TOG)-modified electrode. Thus the positive potential ECL signal of Ru(phen)32+ was generated, and the negative-potential ECL signal of TOG was quenched by Ag NCs via resonance energy transfer and co-reactant-competition simultaneously, achieving dual signal “on” and “off” assay of Cd2+. Furthermore, the present Mg2+ (second-target) specifically sheared the substrate of DNA enzyme on the electrode, releasing the DNA Net probes with Ru(phen)32+ and Ag NCs to realize “off” and “on” detection of Mg2+ by dual-potential signals. This strategy developed a new versatile biosensor by using dual-potential ECL probes and novel DNA network structure, and opened up a new way for multi-target detection by dual “on” “off” signals, which shows great potential in practical detection. The multifunctional sensing platform was also applied to Cd2+ assay in rice.

RelA Is an Essential Target for Enhancing Cellular Responses to the DNA Repair/Ref-1 Redox Signaling Protein and Restoring Perturbated Cellular Redox Homeostasis in Mouse PDAC Cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a poor response to current treatment regimens. The multifunctional DNA repair-redox signaling protein Ref-1 has a redox signaling function that activates several transcriptional factors (TFs) including NF-κB (RelA), STAT3, AP-1. These have been implicated in signaling in PDAC and associated with cancer progression and therapy resistance. Numerous studies have shown a role for RelA in PDAC inflammatory responses and therapy resistance, little is known as to how these inflammatory responses are modulated through Ref-1 redox signaling pathways during pancreatic pathogenesis. RelA and STAT3 are two major targets of Ref-1 and are important in PDAC pathogenesis. To decipher the mechanistic role of RelA in response to Ref-1 inhibition, we used PDAC cells (KC3590) from a genetically engineered Kras G12D-driven mouse model that also is functionally deficient for RelA (Parent/Vector) or KC3590 cells with fully functional RelA added back (clone 13; C13). We demonstrated that RelA deficient cells are more resistant to Ref-1 redox inhibitors APX3330, APX2009, and APX2014, and their sensitivity is restored in the RelA proficient cells. Knockdown of STAT3 did not change cellular sensitivity to Ref-1 redox inhibitors in either cell type. Gene expression analysis demonstrated that Ref-1 inhibitors significantly decreased IL-8, FOSB, and c-Jun when functional RelA is present. We also demonstrated that PRDX1, a known Ref-1 redox modulator, contributes to Ref-1 inhibitor cellular response. Knockdown of PRDX1 when functional RelA is present resulted in dramatically increased PDAC killing in response to Ref-1 inhibitors. The enhanced cell killing was not due to increased intracellular ROS production. Although Ref-1 inhibition decreased the NADP/NADPH ratio in the cells, the addition of PRDX1 knockdown did not further this redox imbalance. This data suggests that the mechanism of cell killing following Ref-1 inhibition is at least partially mediated through RelA and not STAT3. Further imbalancing of the redox signaling through disruption of the PRDX1-Ref-1 interaction may have therapeutic implications. Our data further support a pivotal role of RelA in mediating Ref-1 redox signaling in PDAC cells with the Kras G12D genotype and provide novel therapeutic strategies to combat PDAC drug resistance.

The human AP-endonuclease 1 (APE1) is a DNA G-quadruplex structure binding protein and regulates KRAS expression in pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive types of cancer, is characterized by aberrant activity of oncogenic KRAS. A nuclease-hypersensitive GC-rich region in KRAS promoter can fold into a four-stranded DNA secondary structure called G-quadruplex (G4), known to regulate KRAS expression. However, the factors that regulate stable G4 formation in the genome and KRAS expression in PDAC are largely unknown. Here, we show that APE1 (apurinic/apyrimidinic endonuclease 1), a multifunctional DNA repair enzyme, is a G4-binding protein, and loss of APE1 abrogates the formation of stable G4 structures in cells. Recombinant APE1 binds to KRAS promoter G4 structure with high affinity and promotes G4 folding in vitro. Knockdown of APE1 reduces MAZ transcription factor loading onto the KRAS promoter, thus reducing KRAS expression in PDAC cells. Moreover, downregulation of APE1 sensitizes PDAC cells to chemotherapeutic drugs in vitro and in vivo. We also demonstrate that PDAC patients’ tissue samples have elevated levels of both APE1 and G4 DNA. Our findings unravel a critical role of APE1 in regulating stable G4 formation and KRAS expression in PDAC and highlight G4 structures as genomic features with potential application as a novel prognostic marker and therapeutic target in PDAC.

Swelling of Serum-Stable DNA Nanoparticles upon Target-Induced Conformational Rearrangement of Sensing Probes for the Signal-On Detection of Cancer-Related Genes.

Nuclease-resistant assay probes are of significant importance for biochemical analysis and disease diagnosis. In this contribution, a reconfigurable lipidic moiety-attached DNA nanoparticle (LDN) is constructed from a cholesterol-conjugated multifunctional hairpin-type DNA probe (Chol-DP) by hydrophobicity-mediated self-assembly. The LDN holds high serum stability and displays a low false-positive signal even in a complex biological milieu. The hydrophobic cholesterol moiety enables the hydrophobicity-mediated assembly, while hydrophilic DNA sequence serves as a recognition element and a polymerization template. The initiator-activated strand displacement amplification (SDA) reaction can convert the hairpin-shaped probe into rigid double-stranded DNA (dsDNA), causing the conformational rearrangement-based LDN swelling that can be used to reliably and fluorescently signal the cancer-related p53 gene. The size increase and structural reconfiguration are confirmed by dynamic light scattering (DLS) analysis and confocal microscopy imaging, respectively. Target p53 is specifically detected down to 10 pM. The whole assay process involved only several simple mixing steps. Recovery test and blind test further confirm the feasibility of the use of the LDN for the detection of target DNA in a complex biological milieu, indicating a promising nanotool for biomedical applications.