DNA Hybridization Research Articles

Ascites exosomal lncRNA PLADE enhances platinum sensitivity by inducing R-loops in ovarian cancer.

Cisplatin resistance is a major cause of therapeutic failure in patients with high-grade serous ovarian cancer (HGSOC). Long noncoding RNAs (lncRNAs) have emerged as key regulators of human cancers; however, their modes of action in HGSOC remain largely unknown. Here, we provide evidence to demonstrate that lncRNA Platinum sensitivity-related LncRNA from Ascites-Derived Exosomes (PLADE) transmitted by ascites exosomes enhance platinum sensitivity in HGSOC. PLADE exhibited significantly decreased expression in ascites exosomes and tumor tissues, as well as in the corresponding metastatic tumors from patients with HGSOC cisplatin-resistance. Moreover, HGSOC patients with higher PLADE expression levels exhibited longer progression-free survival. Gain- and loss-of-function studies have revealed that PLADE promotes cisplatin sensitivity by suppressing cell proliferation, migration and invasion, and enhancing apoptosis in vitro and in vivo. Furthermore, the functions of PLADE in increasing cisplatin sensitivity were proven to be transferred by exosomes to the cultured recipient cells and to the adjacent tumor tissues in mouse models. Mechanistically, PLADE binds to and downregulates heterogeneous nuclear ribonucleoprotein D (HNRNPD) by VHL-mediated ubiquitination, thus inducing an increased amount of RNA: DNA hybrids (R-loop) and DNA damage, consequently promoting cisplatin sensitivity in HGSOC. Collectively, these results shed light on the understanding of the vital roles of long noncoding RNAs in cancers.

Construction of test strips for lung cancer detection based on aptamers

Lung cancer is the most common malignancy worldwide. Early diagnosis helps to reduce mortality and improve survival. Aptamers are widely used in cancer screening because of their high specificity, good stability and low cost. In this study, using the specific aptamer of lung cancer serum, the sandwich method colloidal gold test strip was prepared by isothermal amplification technique and the principle of nucleic acid hybridisation for the early diagnosis of lung cancer. The results showed that the test strip was positive in 8 patients with lung cancer, which was consistent with the actual cases. The test strip can accurately identify lung cancer patients. The concentration range of nucleic acid detection is 1 × 10−4 - 7 × 10−4 mol/L, and the detection limit is 0.67 mM. The test strip detection method has low cost and simple operation, and provides a reference for the development of home portable tumor early detection.

Crop Evolution of Foxtail Millet.

Studies on the domestication, genetic differentiation, and crop evolution of foxtail millet are reviewed in this paper. Several genetic studies were carried out to elucidate the genetic relationships among foxtail millet accessions originating mainly from Eurasia based on intraspecific hybrid pollen semi-sterility, isozymes, DNA markers, and single-nucleotide polymorphisms. Most studies suggest that China is the center of diversity of foxtail millet, and landraces were categorized into geographical groups. These results indicate that this millet was domesticated in China and spread over Eurasia, but independent origin in other regions cannot be ruled out. Furthermore, the evolution of genes was reviewed (i.e., the Waxy gene conferring amylose content in the endosperm, the Si7PPO gene controlling polyphenol oxidase, the HD1 and SiPRR37 genes controlling heading time, the Sh1 and SvLes1 genes involved in grain shattering, and the C gene controlling leaf sheath pigmentation), and the variation and distribution of these genes suggested complex patterns of evolution under human and/or natural selection.

DNA hybrid florescent chrominance sensor: Drug sensing through direct gradient florescent color transformation of hybrid DNA crystals loaded lanthanide (Eu3+/Tb3+) complexes

In this study, a highly sensitive anticancer drug sensor was synthesized using DNA crystals loaded with luminescent Ln3+(Eu3+/Tb3+) complexes. The sensors exhibited a pronounced color transformation property due to the differential quenching of Eu3+ and Tb3+ emissions upon drug interactions. These luminescent hybrid DNA crystals present a significant advancement in sensing anticancer drugs like curcumin, transitioning from monitoring fluorescence intensity changes to a visually discernable color change. This new approach eliminates the need for specialized instrumentation. Owing to the intrinsic biocompatibility of materials composed of DNA, this hybrid sensor has the potential to sense drugs within the human body. Hybrid crystals loaded with a single complex will lose their fluorescence properties due to drug quenching; however, crystals loaded with mixed complexes emit distinct fluorescent colors, such as yellow-green and orange-red, at different drug concentrations. This color transformation allows for the direct determination of drug concentration based on crystal color, making the sensing process more simple and easily observable.

Force-induced unzipping of DNA in the presence of solvent molecules

The melting of double-stranded DNA (dsDNA) in the presence of solvent molecules is a fundamental process with significant implications for understanding the thermal and mechanical behavior of DNA and its interactions with the surrounding environment. The solvents play an essential role in the structural transformation of DNA subjected to a pulling force. In this study, we simulate the thermal and force induced denaturation of dsDNA and elucidate the solvent dependent melting behavior, identifying key factors that influence the stability of DNA melting in presence of solvent molecules. Using a statistical model, we first find the melting profile of short heterogeneous DNA molecules in the presence of solvent molecules in Force ensemble. We also investigate the effect of solvent's strengths on the melting profile of DNA. In the force ensemble, we consider two homogeneous DNA chains and apply the force on different locations along the chain in the presence of solvent molecules. Different pathways manifest the melting of the molecule in both ensembles, and we found several interesting features of melting DNA in a constant force ensemble, such as lower critical force when the chain is pulled from the base pair close to a solvent molecule. The results provide new insights into the force-induced unzipping of DNA and could be used to develop new methods for controlling the unzipping process. By providing a better understanding of melting and unzipping of dsDNA in the presence of solvent molecules, this study provides valuable guidelines for predicting DNA thermodynamic quantities and for designing DNA nanostructures.

Temperature-Dependent Twist of Double-Stranded RNA Probed by Magnetic Tweezer Experiments and Molecular Dynamics Simulations.

RNA plays critical roles in the transmission and regulation of genetic information and is increasingly used in biomedical and biotechnological applications. Functional RNAs contain extended double-stranded regions, and the structure of double-stranded RNA (dsRNA) has been revealed at high resolution. However, the dependence of the properties of the RNA double helix on environmental effects, notably temperature, is still poorly understood. Here, we use single-molecule magnetic tweezer measurements to determine the dependence of the dsRNA twist on temperature. We find that dsRNA unwinds with increasing temperature, even more than DNA, with ΔTwRNA = -14.4 ± 0.7°/(°C·kbp), compared to ΔTwDNA = -11.0 ± 1.2°/(°C·kbp). All-atom molecular dynamics (MD) simulations using a range of nucleic acid force fields, ion parameters, and water models correctly predict that dsRNA unwinds with rising temperature but significantly underestimate the magnitude of the effect. These MD data, together with additional MD simulations involving DNA and DNA-RNA hybrid duplexes, reveal a linear correlation between the twist temperature decrease and the helical rise, in line with DNA but at variance with RNA experimental data. We speculate that this discrepancy might be caused by some unknown bias in the RNA force fields tested or by as yet undiscovered transient alternative structures in the RNA duplex. Our results provide a baseline to model more complex RNA assemblies and to test and develop new parametrizations for RNA simulations. They may also inspire physical models of the temperature-dependent dsRNA structure.

Yolk-shell nanospheres based silane-functionalized carbon dots: Designed synthesis and application in highly selective determination of aflatoxin biosynthesis-related genes nor-1 in cereal crops

This study presented advanced monitoring approaches for the aflatoxin gene cluster in Aspergillus flavus and Aspergillus parasiticus, which are effective tools for controlling aflatoxin proliferation in the food chain. A novel approach involved a ratiometric fluorescence probe based on yolk-shell nanospheres (bCDs@SiO2@gQDs). The probe's fluorescence was activated by DNA hybridization with the modified bCDs@SiO2@gQDs and gold nanoparticles (AuNPs), functioning through donor–acceptor pairs. The exceptional properties of the gQDs and the high FRET efficiency of this system established a functionalized platform for DNA labeling. When the nor-1 gene was present, FRET between the gQDs and AuNPs triggered fluorescence quenching, enabling nor-1 gene detection. The yolk-shell nanostructure of the fluorescent nanoprobes provided an internal reference, reducing experimental errors due to background noise, instrumentation factors, and probe concentration. This allowed for accurate and highly sensitive quantitative determination of the nor-1 gene. The ratiometric fluorescence assay demonstrated a broad linear range (0.1–100 nM) and a low detection limit (12.83 pM) for the nor-1 gene. Tests on non-contaminated maize samples spiked with various concentrations of the nor-1 gene achieved recovery rates between 95.43 % and 101.46 %. This biosensor represents a significant advancement in food safety, offering a simple, rapid, and sensitive method for monitoring agricultural products.

Low-temperature photothermal-chemotherapy enhancing tumor immunotherapy by tetrahedral framework nucleic acids nanogels based drug delivery system

To achieve a balance between antitumor efficacy in photothermal therapy (PTT) and high-temperature (>50℃) induced side effects, a novel tetrahedral framework nucleic acid (FNA) nanogel (FNANG) coated with polydopamine (PDA) (abbreviation: PP-FNANG) was designed to achieve siRNA-mediated low-temperature PTT. First, siRNA targeted heat shock protein 70 (HSP70) was used as crosslinker to guide FNA through nucleic acid hybridization to obtain FNANG, and simultaneously loaded doxorubicin (DOX). The obtained FNANG was coated with PEGylated PDA, which not only protected the FNANG from enzymatic degradation, but also made the FNANG have excellent photothermal conversion ability under near infrared (NIR) irradiation. FNANG realized the effective delivery of siHSP70 and realized the low-temperature photothermal therapy mediated by siRNA. Notably, PP-FNANG combined with its loaded DOX showed excellent synergistic effect, and could effectively induce the immunogenic cell death (ICD), promote the activation and infiltration of T lymphocyte, promote the maturation of dendritic cells and the secretion of related cytokines, and effectively inhibit melanoma. This study provided a simple and effective exploration strategy for new vector design based on FNA and collaborative therapy.

Fluorescent aptasensor for highly sensitive detection of Staphylococcus aureus based on dual-amplification strategy by integrating DNA walking and hybridization chain reaction

Food-borne diseases caused by bacteria threaten human health. Herein, we presented a new fluorescent aptasensor by coupling DNA walking and hybridization chain reaction (HCR) for convenient and sensitive quantification of bacteria. Staphylococcus aureus (S. aureus) was selected as target. When there was target in the system, the binding of S. aureus with its aptamer caused the disintegration of aptamer/DNA walker on the surface of AuNPs and released DNA walker. With the help of Nt.BsmAI, DNA walker moved along the surface of AuNPs and trigger probe was detached from AuNPs. The trigger probe could initiate hybridization chain reaction (HCR) and opened the stems of H1@AuNPs probe and H2@AuNPs probe. After the addition of nicking endonuclease, the adjacent upconversion nanoparticles (UCNPs, NaYF4:Yb3+, Er3+) were further away from the quenchers (AuNPs) of H1 and H2. Therefore, the fluorescence intensity of UCNPs could be restored via fluorescence resonance energy transfer (FRET). Bacteria were thus detected by recording the fluorescence intensity of UCNPs. This method is simple, rapid and sensitive. It can directly detect bacteria in a low background signal. The limit of detection (LOD) was 10 CFU/mL, detection time was less than 3 h. Recovery rates in simulated milk, honey and human serum samples ranged from 93.6 % to 105.8 %. The strategy opens up new paths for early diagnosis of diseases and food monitoring.

Label-free detection of biomolecules using inductively coupled plasma mass spectrometry (ICP-MS).

Bioassays using inductively coupled plasma mass spectrometry (ICP-MS) have gained increasing attention because of the high sensitivity of ICP-MS and the various strategies of labeling biomolecules with detectable metal tags. The classic strategy to tag the target biomolecules is through direct antibody-antigen interaction and DNA hybridization, and requires the separation of the bound from the unbound tags. Label-free ICP-MS techniques for biomolecular assays do not require direct labeling: they generate detectable metal ions indirectly from specific biomolecular reactions, such as enzymatic cleavage. Here, we highlight the development of three main strategies of label-free ICP-MS assays for biomolecules: (1) enzymatic cleavage of metal-labeled substrates, (2) release of immobilized metal ions from the DNA backbone, and (3) nucleic acid amplification-assisted aggregation and release of metal tags to achieve amplified detection. We briefly describe the fundamental basis of these label-free ICP-MS assays and discuss the benefits and drawbacks of various designs. Future research is needed to reduce non-specific adsorption and minimize background and interference. Analytical innovations are also required to confront challenges faced by in vivo applications.

Multimodal Golden DNA Superstructures (GDSs) for Highly Efficient Photothermal Immunotherapy.

DNA-templated metallization has emerged as an efficient strategy for creating nanoscale-metal DNA hybrid structures with a desirable conformation and function. Despite the potential of DNA-metal hybrids, their use as combinatory therapeutic agents has rarely been examined. Herein, we present a simple approach for fabricating a multipurpose DNA superstructure that serves as an efficient photoimmunotherapy agent. Specifically, we adsorb and locally concentrate Au ions onto DNA superstructures through induced local reduction, resulting in the formation of Au nanoclusters. The mechanical and optical properties of these metallic nanoclusters can be rationally controlled by their conformations and metal ions. The resulting golden DNA superstructures (GDSs) exhibit significant photothermal effects that induce cancer cell apoptosis. When sequence-specific immunostimulatory effects of DNA are combined, GDSs provide a synergistic effect to eradicate cancer and inhibit metastasis, demonstrating potential as a combinatory therapeutic agent for tumor treatment. Altogether, the DNA superstructure-templated metal casting system offers promising materials for future biomedical applications.

Label-Free DNA Hybridization Detection Using a Highly Sensitive Fiber Microcavity Biosensor.

A novel label-free optical fiber biosensor, based on a microcavity fiber Mach-Zehnder interferometer, was developed and practically demonstrated for DNA detection. The biosensor was fabricated using offset splicing standard communication single-mode fibers (SMFs). The light path of the sensor was influenced by the liquid sample in the offset open cavity. In the experiment, a high sensitivity of -17,905 nm/RIU was achieved in the refractive index (RI) measurement. On this basis, the probe DNA (pDNA) was immobilized onto the sensor's surface using APTES, enabling real-time monitoring of captured complementary DNA (cDNA) samples. The experimental results demonstrate that the biosensor exhibited a high sensitivity of 0.32 nm/fM and a limit of detection of 48.9 aM. Meanwhile, the sensor has highly repeatable and specific performance. This work reports an easy-to-manufacture, ultrasensitive, and label-free DNA biosensor, which has significant potential applications in medical diagnostics, bioengineering, gene identification, environmental science, and other biological fields.

Detection of 13 foodborne pathogens in aquatic products using visual chromogenic chips based on asymmetric multiplex polymerase chain reaction and nucleic acid hybridization

Detection of 13 foodborne pathogens in aquatic products using visual chromogenic chips based on asymmetric multiplex polymerase chain reaction and nucleic acid hybridization

Scalable lipid droplet microarray fabrication, validation, and screening.

High throughput screening of small molecules and natural products is costly, requiring significant amounts of time, reagents, and operating space. Although microarrays have proven effective in the miniaturization of screening for certain biochemical assays, such as nucleic acid hybridization or antibody binding, they are not widely used for drug discovery in cell culture due to the need for cells to internalize lipophilic drug candidates. Lipid droplet microarrays are a promising solution to this problem as they are capable of delivering lipophilic drugs to cells at dosages comparable to solution delivery. However, the scalablility of the array fabrication, assay validation, and screening steps has limited the utility of this approach. Here we take several new steps to scale up the process for lipid droplet array fabrication, assay validation in cell culture, and drug screening. A nanointaglio printing process has been adapted for use with a printing press. The arrays are stabilized for immersion into aqueous solution using a vapor coating process. In addition to delivery of lipophilic compounds, we found that we are also able to encapsulate and deliver a water-soluble compound in this way. The arrays can be functionalized by extracellular matrix proteins such as collagen prior to cell culture as the mechanism for uptake is based on direct contact with the lipid delivery vehicles rather than diffusion of the drug out of the microarray spots. We demonstrate this method for delivery to 3 different cell types and the screening of 92 natural product extracts on a microarray covering an area of less than 0.1 cm2. The arrays are suitable for miniaturized screening, for instance in high biosafety level facilities where space is limited and for applications where cell numbers are limited, such as in functional precision medicine.

Diagnosis of Mycobacterium tuberculosis using palladium-platinum bimetallic nanoparticles combined with paper-based analytical devices.

In this study, we demonstrate that palladium-platinum bimetallic nanoparticles (Pd@Pt NPs) as the nanozyme, combined with a multi-layer paper-based analytical device and DNA hybridization, can successfully detect Mycobacterium tuberculosis. This nanozyme has peroxidase-like properties, which can increase the oxidation rate of the substrate. Compared with horseradish peroxidase, which is widely used in traditional detection, the Michaelis constants of Pd@Pt NPs are fourteen and seventeen times lower than those for 3,3',5,5'-tetramethylbenzidine and H2O2, respectively. To verify the catalytic efficiency of Pd@Pt NPs, this study will execute molecular diagnosis of Mycobacterium tuberculosis. We chose the IS6110 fragment as the target DNA and divided the complementary sequences into the capture DNA and reporter DNA. They were modified on paper and Pd@Pt NPs, respectively, to detect Mycobacterium tuberculosis on a paper-based analytical device. With the above-mentioned method, we can detect target DNA within 15 minutes with a linear range between 0.75 and 10 nM, and a detection limit of 0.216 nM. These results demonstrate that the proposed platform (a DNA-nanozyme integrated paper-based analytical device, dnPAD) can provide sensitive and on-site infection prognosis in areas with insufficient medical resources.

Variable gain DNA nanostructure charge amplifiers for biosensing.

Electronic measurements of engineered nanostructures comprised solely of DNA (DNA origami) enable new signal conditioning modalities for use in biosensing. DNA origami, designed to take on arbitrary shapes and allow programmable motion triggered by conjugated biomolecules, have sufficient mass and charge to generate a large electrochemical signal. Here, we demonstrate the ability to electrostatically control the DNA origami conformation, and thereby the resulting signal amplification, when the structure binds a nucleic acid analyte. Critically, unlike previous studies that employ DNA origami to amplify an electrical signal, we show that the conformation changes under an applied field are reversible. This applied field also simultaneously accelerates structural transitions above the rate determined by thermal motion. We tuned this property of the structures to achieve a response that was ≈2 × 104 times greater (i.e., a gain or amplification) than the value from DNA hybridization under similar conditions. Because this signal amplification is independent of DNA origami-analyte interactions, our approach is agnostic of the end application. Furthermore, since large signal changes are only triggered in response to desirable interactions, we minimize the deleterious effects of non-specific binding. The above benefits of self-assembled DNA origami make them ideally suited for multiplexed biosensing when paired with highly parallel electronic readout.

Rapid and quantitative detection of DNA hybridization using a simplified Fabry-Perot interferometric biosensor.

This study introduces a miniaturized fiber-optic Fabry-Perot (FP) interferometric biosensor, distinctively engineered for cost-effective, rapid, and quantitative DNA sequence detection. By leveraging the interference patterns generated within a Fabry-Perot microcavity, our sensor precisely monitors DNA hybridization events in real-time. We have verified the sensor's biofunctionalization via fluorescent labeling and have extensively validated its performance through numerous hybridization and regeneration cycles with 1 μM single-stranded DNA (ssDNA) solutions. Demonstrating remarkable repeatability and reusability, the sensor effectively discerns ssDNA sequences exhibiting varying degrees of mismatches. Its ability to accurately distinguish between sequences with 2 and 7 mismatches underscores its potential as a valuable asset for swift DNA analysis. Characterized by its rapid response time-typically yielding results within 6 minutes-and its adeptness at mismatch identification, our biosensor stands as a potent tool for facilitating accelerated DNA diagnostics and research.

CRISPR/Cas13a-assisted amplification-free miRNA biosensor via dark-field imaging and magnetic gold nanoparticles.

MicroRNAs (miRNAs) are short (about 18-24 nucleotides) non-coding RNAs and have emerged as potential biomarkers for various diseases, including cancers. Due to their short lengths, the specificity often becomes an issue in conventional amplification-based methods. Next-generation sequencing techniques could be an alternative, but the long analysis time and expensive costs make them less suitable for routine clinical diagnosis. Therefore, it is essential to develop a rapid, selective, and accurate miRNA detection assay using a simple, affordable system. In this work, we report a CRISPR/Cas13a-based miRNA biosensing using point-of-care dark-field (DF) imaging. We utilized magnetic-gold nanoparticle (MGNPs) complexes as signal probes, which consist of 200 nm-sized magnetic beads and 60 nm-sized gold nanoparticles (AuNPs) linked by DNA hybridization. Once the CRISPR/Cas13a system recognized the target miRNAs (miR-21-5p), the activated Cas13a cleaved the bridge linker containing RNA sequences, releasing 60 nm-AuNPs detected and quantified by a portable DF imaging system. The combination of CRISPR/Cas13a, MGNPs, and DF imaging demonstrated amplification-free detection of miR-21-5p within 30 min at a detection limit of 500 attomoles (25 pM) and with single-base specificity. The CRISPR/Cas13a-assisted MGNP-DF assay achieved rapid, selective, and accurate detection of miRNAs with simple equipment, thus providing a potential application for cancer diagnosis.

A simple and rapid colorimetric detection of Staphylococcus aureus relied on the distance-dependent optical properties of silver nanoparticles.

The quick and accurate diagnosis of pathogens has appeared as a pressing issue in clinical diagnostics, environmental monitoring, and food safety. The available assays are suffering from limited capacities in simple, fast, low-cost, and on-site detection to increase prevention and proper treatment. Herein, we address these challenges by developing a simple, speedy, affordable, and ultrasensitive nanoplasmonic biosensor for colorimetric detection of cDNA from staphylococcal RNA relying on the distance-dependent optical features of silver nanostructures for the measurement of color variations and spectral shifts owing to the plasmon coupling generated by the cross-linking accumulation of AgNPs. The method described utilizes silver nanoparticles (AgNPs) immobilized with two different single-stranded oligonucleotides (ssDNA1 and ssDNA2) that specifically recognize the target DNA. Sandwich hybridization of target DNA with ssDNA1 and ssDNA2 induced color variations and spectral shifts of AgNPs, whereas test samples without the target DNA remained yellow as the initial color of colloidal silver. The designed nanoplasmonic biosensor demonstrated high specificity with the detection limit (LOD) of ∼1.8 amol target DNA (∼106 molecules per test) in the broad linear dynamic range from 0.01 to 100 nM, and LOD down to a few cells was attained for amplified bacterial nucleic acids and a linear range from 102 CFU mL-1 to 107 CFU mL-1. The sensing approach showed great potential for the timely diagnosis of pathogens in low-density samples, and it has considerable merits over traditional culture approaches and qPCR techniques.

Large bubble drives circular DNA melting.

We investigate the melting transition of non-supercoiled circular DNA of different lengths, employing Brownian dynamics simulations. In the absence of supercoiling, we find that melting of circular DNA is driven by a large bubble, which agrees with the previous predictions of circular DNA melting in the presence of supercoiling. By analyzing sector-wise changes in average base-pair distance, our study reveals that the melting behavior of circular DNA closely resembles that of linear DNA. Additionally, we find a marked difference in the thermal stability of circular DNA over linear DNA at very short length scales, an effect that diminishes as the length of circular DNA increases. The stability of smaller circular DNA is linked to the occurrence of transient small bubbles, characterized by a lower probability of growth.