Double-stranded Nucleic Acids Research Articles

Liposomes-Encapsulating Double-Stranded Nucleic Acid (Poly I:C) for Head and Neck Cancer Treatment.

Polyriboinosinic acid-polyribocytidylic acid (Poly I:C) serves as a synthetic mimic of viral double-stranded dsRNA, capable of inducing apoptosis in numerous cancer cells. Despite its potential, therapeutic benefits, the application of Poly I:C has been hindered by concerns regarding toxicity, stability, enzymatic degradation, and undue immune stimulation, leading to autoimmune disorders. To address these challenges, encapsulation of antitumor drugs within delivery systems such as cationic liposomes is often employed to enhance their efficacy while minimizing dosages. In this study, we investigated the potential of cationic liposomes to deliver Poly I:C into the Head and Neck 12 (HN12) cell line to induce apoptosis in the carcinoma cells and tumor model. Cationic liposomes made by the hydrodynamic focusing method surpass traditional methods by offering a continuous flow-based approach for encapsulating genes, which is ideal for efficient tumor delivery. DOTAP liposomes efficiently bind Poly I:C, confirmed by transmission electron microscopy images displaying their spherical morphology. Liposomes are easily endocytosed in HN12 cells, suggesting their potential for therapeutic gene and drug delivery in head and neck squamous carcinoma cells. Activation of apoptotic pathways involving MDA5, RIG-I, and TLR3 is evidenced by upregulated caspase-3, caspase-8, and IRF3 genes upon endocytosis of Poly(I:C)-encapsulated liposomes. Therapeutic evaluations revealed significant inhibition of tumor growth with Poly I:C liposomes, indicating the possibility of MDA5, RIG-I, and TLR3-induced apoptosis pathways via Poly I:C liposomes in HN12 xenografts in J:NU mouse models. Comparative histological analysis underscores enhanced cell death with Poly I:C liposomes, warranting further investigation into the precise mechanisms of apoptosis and inflammatory cytokine response in murine models for future research.

Enzyme-Free Exponential Amplification via Growth and Scission of Crisscross Ribbons from Single-Stranded DNA Components.

The self-assembly of DNA-based monomers into higher-order structures has significant potential for realizing various biomimetic behaviors including algorithmic assembly, ultrasensitive detection, and self-replication. For these behaviors, it is desirable to implement high energetic barriers to undesired spurious nucleation, where such barriers can be bypassed via seed-initiated assembly. Joint-neighbor capture is a mechanism enabling the construction of such barriers while allowing for algorithmic behaviors, such as bit-copying. Cycles of polymerization with division could accordingly be used for implementing exponential growth in self-replicating materials. Previously, we demonstrated crisscross polymerization, a strategy that attains robust seed-dependent self-assembly of single-stranded DNA and DNA-origami monomers via joint-neighbor capture. Here, we expand the crisscross assembly to achieve autonomous, isothermal exponential amplification of ribbons through their concurrent growth and scission via toehold-mediated strand displacement. We demonstrate how this crisscross chain reaction, or 3CR, can be used as a detection strategy through coupling to single- and double-stranded nucleic acid targets and introduce a rule-based stochastic modeling approach for simulating molecular self-assembly behaviors such as crisscross-ribbon scission.

GRASSERIE - A BACULOVIRUS DISEASE OF THE SILKWORM BOMBYX MORI L.

Grasserie is a major infectious disease of silkworms caused by the Bombyx mori nuclear polyhedrosis virus. The virus belongs to the family Baculoviridae and is composed of a polyhedron with a single molecule of circular supercoiled double-stranded deoxyribose nucleic acid. The disease causes massive mortality (more than 60% of the total crop loss caused by different diseases), causing significant great crop loss to the sericulturists by reducing the cocoon crop and silk produce. There has been no effective solution for preventing and treating the disease. In the present review, the authors explore the subject in detail and inspire the researchers to search for molecular mechanisms of gene action and promote disease-resistant silkworm breeding.. KEYWORDS :Bombyx mori,,Baculoviridae, Disease resistance, Nuclear polyhedrosis virus, Polyhedron, Silkworm

Zα domain proteins mediate the immune response.

The Zα domain has a compact α/β architecture containing a three-helix bundle flanked on one side by a twisted antiparallel β sheet. This domain displays a specific affinity for double-stranded nucleic acids that adopt a left-handed helical conformation. Currently, only three Zα-domain proteins have been identified in eukaryotes, specifically ADAR1, ZBP1, and PKZ. ADAR1 is a double-stranded RNA (dsRNA) binding protein that catalyzes the conversion of adenosine residues to inosine, resulting in changes in RNA structure, function, and expression. In addition to its editing function, ADAR1 has been shown to play a role in antiviral defense, gene regulation, and cellular differentiation. Dysregulation of ADAR1 expression and activity has been associated with various disease states, including cancer, autoimmune disorders, and neurological disorders. As a sensing molecule, ZBP1 exhibits the ability to recognize nucleic acids with a left-handed conformation. ZBP1 harbors a RIP homotypic interaction motif (RHIM), composed of a highly charged surface region and a leucine-rich hydrophobic core, enabling the formation of homotypic interactions between proteins with similar structure. Upon activation, ZBP1 initiates a downstream signaling cascade leading to programmed cell death, a process mediated by RIPK3 via the RHIM motif. PKZ was identified in fish, and contains two Zα domains at the N-terminus. PKZ is essential for normal growth and development and may contribute to the regulation of immune system function in fish. Interestingly, some pathogenic microorganisms also encode Zα domain proteins, such as, Vaccinia virus and Cyprinid Herpesvirus. Zα domain proteins derived from pathogenic microorganisms have been demonstrated to be pivotal contributors in impeding the host immune response and promoting virus replication and spread. This review focuses on the mammalian Zα domain proteins: ADAR1 and ZBP1, and thoroughly elucidates their functions in the immune response.

DNA mimic foldamers affect chromatin composition and disturb cell cycle progression.

The use of synthetic chemicals to selectively interfere with chromatin and the chromatin-bound proteome represents a great opportunity for pharmacological intervention. Recently, synthetic foldamers that mimic the charge surface of double-stranded DNA have been shown to interfere with selected protein-DNA interactions. However, to better understand their pharmacological potential and to improve their specificity and selectivity, the effect of these molecules on complex chromatin needs to be investigated. We therefore systematically studied the influence of the DNA mimic foldamers on the chromatin-bound proteome using an in vitro chromatin assembly extract. Our studies show that the foldamer efficiently interferes with the chromatin-association of the origin recognition complex in vitro and in vivo, which leads to a disturbance of cell cycle in cells treated with foldamers. This effect is mediated by a strong direct interaction between the foldamers and the origin recognition complex and results in a failure of the complex to organise chromatin around replication origins. Foldamers that mimic double-stranded nucleic acids thus emerge as a powerful tool with designable features to alter chromatin assembly and selectively interfere with biological mechanisms.

Long noncoding RNA MALAT1 is dynamically regulated in leader cells during collective cancer invasion

Cancer cells collectively invade using a leader-follower organization, but the regulation of leader cells during this dynamic process is poorly understood. Using a dual double-stranded locked nucleic acid (LNA) nanobiosensor that tracks long noncoding RNA (lncRNA) dynamics in live single cells, we monitored the spatiotemporal distribution of lncRNA during collective cancer invasion. We show that the lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is dynamically regulated in the invading fronts of cancer cells and patient-derived spheroids. MALAT1 transcripts exhibit distinct abundance, diffusivity, and distribution between leader and follower cells. MALAT1 expression increases when a cancer cell becomes a leader and decreases when the collective migration process stops. Transient knockdown of MALAT1 prevents the formation of leader cells and abolishes the invasion of cancer cells. Taken together, our single-cell analysis suggests that MALAT1 is dynamically regulated in leader cells during collective cancer invasion.

Noncovalent Adsorption of Single-Stranded and Double-Stranded DNA on the Surface of Gold Nanoparticles

Understanding the patterns of noncovalent adsorption of double-stranded nucleic acids (dsDNA) on gold nanoparticles (GNPs) was the aim of this study. It was found that the high-affinity motifs in DNA can and do act as an “anchor” for the fixation of the whole molecule on the GNP (up to 98 ± 2 single-stranded (ss)DNA molecules per particle with diameter of 13 ± 2 nm). At the same time, the involvement of an “anchor” in the intramolecular DNA interaction can negatively affect the efficiency of the formation of ss(ds)DNA–GNP structures. It has been shown that the interaction of GNP with DNA duplexes is accompanied by their dissociation and competitive adsorption of ssDNAs on GNP, wherein the crucial factor of DNA adsorption efficiency is the intrinsic affinity of ssDNA to GNP. We propose a detailed scheme for the interaction of dsDNA with GNPs, which should be taken into account in studies of this type. Researchers focused on this field should accept the complicated nature of such objects and take into account the many competing processes, including the processes of adsorption and desorption of DNA on gold as well as the formation of secondary structures by individual DNA strands.

Enhancement of the thermal stability of G-quadruplex structures by urea

The solute urea has been used extensively as a denaturant in protein folding studies; double-stranded nucleic acid structures are also destabilized by urea, but comparatively less than proteins. In previous research, the solute has been shown to strongly destabilize folded G-quadruplex DNA structures. This contribution demonstrates the stabilizing effect of urea on the G-quadruplex formed by the oligodeoxyribonucleotide (ODN), G3T (d[5′-GGGTGGGTGGGTGGG-3′]), and related sequences in the presence of sodium or potassium cations. Stabilization is observed up to 7 M urea, which was the highest concentration we investigated. The folded structure of G3T has three G-tetrads and three loops that consist of single thymine residues. ODNs related to G3T, in which the thymine residues in the loop are substituted by adenosine residues, also exhibit enhanced stability in the presence of molar concentrations of urea. The circular dichroism (CD) spectra of these ODNs in the presence of urea are consistent with that of a G-quadruplex. As the urea concentration increases, the spectral intensities of the peaks and troughs change, while their positions change very little. The heat-induced transition from the folded to unfolded state, Tm, was measured by monitoring the change in the UV absorption as a function of temperature. G-quadruplex structures with loops containing single bases exhibited large increases in Tm with increasing urea concentrations. These data imply that the loop region play a significant role in the thermal stability of tetra-helical DNA structures in the presence of the solute urea.

Specific Adsorption of Magnetic Fe3O4@hydroxyapatite Nanocomposites to a Hybridized Duplex for Immobilization and Label-Free Electrochemical Biosensing of MicroRNA

The unique discriminating ability of hydroxyapatite (HAP) to single- and double-stranded nucleic acid implies its potential as a biosensing material for nucleic acid analysis. Herein, a novel immobilization- and label-free electrochemical strategy has been developed for miRNA let-7a detection based on the specific adsorption of magnetic Fe3O4@hydroxyapatite (Fe3O4@HAP) to a duplex structure. Typically, the highly efficient homogeneous reaction between probe DNA (pDNA) and target miRNA let-7a produces the nucleic acid duplex, which is specifically adsorbed on the surface of Fe3O4@HAP. Thereafter, the electroactive intercalator Nile blue (NB) is introduced as the label-free signal molecule and inserts between the stacked base pairs of the pDNA–miRNA duplex. Thus, the Fe3O4@HAP bearing pDNA–miRNA and numerous intercalated NBs are adsorbed by the magnetic glassy carbon electrode, giving rise to an intense sensing signal without the need of nuclease-assisted cyclic amplification. With this advantage, the sensing system shows a wide linear range from 1.0 fM to 100 nM and a low detection limit of 0.051 fM for miRNA let-7a analysis. What is more, the synergy of the base-pairing-driven hybridization, the specific intercalation of NB to the duplex structure, and the selective adsorption of Fe3O4@HAP contribute to a high sensing specificity of the biosensing strategy. The sensing method provides a state-of-the-art strategy for the sensitive and facile bioanalysis and clinical diagnosis of miRNA.

Integrated analysis of circulating cell free nucleic acids for cancer genotyping and immune phenotyping of tumor microenvironment.

The circulating cell-free nucleic acids (ccfNAs) consist of a heterogenous cocktail of both single (ssNA) and double-stranded (dsNA) nucleic acids. These ccfNAs are secreted into the blood circulation by both healthy and malignant cells via various mechanisms including apoptosis, necrosis, and active secretion. The major source of ccfNAs are the cells of hematopoietic system under healthy conditions. These ccfNAs include fragmented circulating cell free DNA (ccfDNA), coding or messenger RNA (mRNA), long non-coding RNA (lncRNA), microRNA (miRNA), and mitochondrial DNA/RNA (mtDNA and mtRNA), that serve as prospective biomarkers in assessment of various clinical conditions. For, e.g., free fetal DNA and RNA migrate into the maternal plasma, whereas circulating tumor DNA (ctDNA) has clinical relevance in diagnostic, prognostic, therapeutic targeting, and disease progression monitoring to improve precision medicine in cancer. The epigenetic modifications of ccfDNA as well as circulating cell-free RNA (ccfRNA) such as miRNA and lncRNA show disease-related variations and hold potential as epigenetic biomarkers. The messenger RNA present in the circulation or the circulating cell free mRNA (ccf-mRNA) and long non-coding RNA (ccf-lncRNA) have gradually become substantial in liquid biopsy by acting as effective biomarkers to assess various aspects of disease diagnosis and prognosis. Conversely, the simultaneous characterization of coding and non-coding RNAs in human biofluids still poses a significant hurdle. Moreover, a comprehensive assessment of ccfRNA that may reflect the tumor microenvironment is being explored. In this review, we focus on the novel approaches for exploring ccfDNA and ccfRNAs, specifically ccf-mRNA as biomarkers in clinical diagnosis and prognosis of cancer. Integrating the detection of circulating tumor DNA (ctDNA) for cancer genotyping in conjunction with ccfRNA both quantitatively and qualitatively, may potentially hold immense promise towards precision medicine. The current challenges and future directions in deciphering the complexity of cancer networks based on the dynamic state of ccfNAs will be discussed.

A Case Series of Amicrobial Pustulosis of Folds: An Eye-opener for the Diagnosis of Autoimmune Disorders

Amicrobial Pustulosis of Folds (APF) is a rare, chronic, relapsing cutaneous disease seen exclusively in younger women with a history of autoimmune disease, most commonly Systemic Lupus Erythematosus (SLE), or who simply have circulating autoantibodies. This case series highlights the occurrence of an unusual manifestation of APF as a marker of autoimmune disorders and emphasises the importance of double-stranded Deoxyribose Nucleic Acid (dsDNA) positivity in such cases. A total of five female patients with APF were included in this case series. All five cases presented with pustular lesions lasting 10-12 weeks, involving conchal bowls, eyes, perineal, and perianal regions, either as an initial presentation or as a flare-up of pre-existing autoimmune disease. All patients underwent screening for autoimmune disorders, which revealed dsDNA positivity and met the diagnostic criteria for APF. Therefore, a diagnosis of APF with SLE was made. All patients showed significant improvement with oral steroids. Although SLE is a complex multisystem disorder where patients may not always present with malar rash, photosensitivity, arthritis, and arthralgia, APF could serve as an eye-opener for diagnosing underlying autoimmune diseases.

Force-dependent elasticity of nucleic acids.

The functioning of double-stranded (ds) nucleic acids (NAs) in cellular processes is strongly mediated by their elastic response. These processes involve proteins that interact with dsDNA or dsRNA and distort their structures. The perturbation of the elasticity of NAs arising from these deformations is not properly considered by most theoretical frameworks. In this work, we introduce a novel method to assess the impact of mechanical stress on the elastic response of dsDNA and dsRNA through the analysis of the fluctuations of the double helix. Application of this approach to atomistic simulations reveals qualitative differences in the force dependence of the mechanical properties of dsDNA with respect to those of dsRNA, which we relate to structural features of these molecules by means of physically-sound minimalistic models.

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.

Kinetic Characterization of SARS-CoV-2 nsp13 ATPase Activity and Discovery of Small-Molecule Inhibitors.

SARS-CoV-2 non-structural protein 13 (nsp13) is a highly conserved helicase and RNA 5′-triphosphatase. It uses the energy derived from the hydrolysis of nucleoside triphosphates for directional movement along the nucleic acids and promotes the unwinding of double-stranded nucleic acids. Nsp13 is essential for replication and propagation of all human and non-human coronaviruses. Combined with its defined nucleotide binding site and druggability, nsp13 is one of the most promising candidates for the development of pan-coronavirus therapeutics. Here, we report the development and optimization of bioluminescence assays for kinetic characterization of nsp13 ATPase activity in the presence and absence of single-stranded DNA. Screening of a library of 5000 small molecules in the presence of single-stranded DNA resulted in the discovery of six nsp13 small-molecule inhibitors with IC50 values ranging from 6 ± 0.5 to 50 ± 6 μM. In addition to providing validated methods for high-throughput screening of nsp13 in drug discovery campaigns, the reproducible screening hits we present here could potentially be chemistry starting points toward the development of more potent and selective nsp13 inhibitors, enabling the discovery of antiviral therapeutics.

Biosensor for a Rapid and Sensitive Detection and Quantification of Nuclease Activity

Nucleases are family of hydrolytic enzymes which cleave the phosphodiesteric bonds between the nucleotides producing 3'-phospho or 5'-phospho end products. These enzymes can be classified as endo- and exo-nucleases, cleaving inside of a polynucleotide chain or at the ends respectively, however, some enzymes may have both activities. Typically, some nucleases prefer double-stranded nucleic acid (e.g. DNase I), while other cleave single-stranded (e.g. Nuclease P1) or RNA-DNA hybrid (RNase H) polynucleotides. Deoxyribonucleases (DNases) are important objects in biotechnology presenting both as tools for manipulations with DNA1 as well as contaminants where nuclease-like activity is undesired.2 In addition, data emerges indicating the importance of DNase activity detection for medical applications, i.e., DNases may be related with various diseases and could be used to monitor the progress of various cancers3 , 4 as well as could be a rapid and fast biomarker of bacterial infections.5 Typically, DNases are being detected and quantified using classical approach: fluorescence detection or gel electrophoresis techniques. Though those methods are highly sensitive and reliable, they are costly, time-consuming as well as require toxic markers such as ethidium bromide or radiolabeled molecules. As alternative methods novel detection techniques for DNase detection were reported, mostly based on fluorescence or chemiluminescence detection incorporating nanomaterials for signal enhancement.6 In addition, electrochemical biosensors, provide simple and inexpensive platform for biomolecule detection while maintaining a high degree of accuracy and sensitivity. However, to this day the absolute majority of electrochemical biosensors are being developed from small biomolecules, e.g., glucose, glycerol, lactate while the development of large marcomolecules, i.e., proteins or enzymes is still lagging. In a case of DNase detection, a few biosensors have been reported. Sato et al. reported electrochemical biosensor for DNase I detection based on ferrocene-modified DNA immobilized on the electrode surface with the detection limit (LOD) up to 10–2 U mL–1. Ding and Qin have reported a potentiometric biosensor for the detection of DNAses based on polycation-sensitive membrane, and for DNase I the LOD was 0.45 U mL–1.7 Despite advances, the developed electrochemical biosensors for DNase detection still lack sensitivities comparable to fluorescence- or radiolabeling-based detection methods and reliability required for the analysis of real samples.In this work we present a rapid and sensitive bioelectrochemical device for the determination of nuclease activity in various fluids. The device system consists of a sensor electrode, a special design DNA target to maximize the nuclease cleavage rate, signal analysis algorithm and the supporting electronics. The developed sensor allows to determine DNase activity in range 0.0003–0.03 U mL–1 with the limit of detection up to 0.00034 U mL–1 over 15 min measurement (calibrated against DNase I standard) as well as has a high storage stability. The system was also implemented using inexpensive single-used electrodes. Moreover, the sensor was tested measuring nuclease activity in real unmodified human saliva samples and demonstrated high accuracy compared to the industry standard DNaseAlert™ QC System. The developed technology could significantly improve nuclease quality control processes in pharma/biotech industry and give new insights into nuclease importance for medical applications. References (1) Pan, Y.; Xiao, L.; Li, A. S. S.; Zhang, X.; Sirois, P.; Zhang, J.; Li, K. Mol Biotechnol 2013, 55 (1), 54–62.(2) Senavirathne, G.; Liu, J.; Lopez, M. A.; Hanne, J.; Martin-Lopez, J.; Lee, J.-B.; Yoder, K. E.; Fishel, R. Nat Methods 2015, 12 (10), 901–902.(3) Patel, P. S.; Patel, B. P.; Rawal, R. M.; Raval, G. N.; Patel, M. M.; Patel, J. B.; Jha, F. P.; Patel, D. D. Tumor Biol 2000, 21(2), 82–89.(4) Balian, A.; Hernandez, F. J. Biomark Res 2021, 9 (1), 86.(5) Flenker, K. S.; Burghardt, E. L.; Dutta, N.; Burns, W. J.; Grover, J. M.; Kenkel, E. J.; Weaver, T. M.; Mills, J.; Kim, H.; Huang, L.; Owczarzy, R.; Musselman, C. A.; Behlke, M. A.; Ford, B.; McNamara, J. O. Molecular Therapy 2017, 25 (6), 1353–1362.(6) Mozioğlu, E.; Akgoz, M.; Kocagöz, T.; Tamerler, C. Anal. Methods 2016, 8 (20), 4017–4021.(7) Ding, J.; Qin, W. Biosensors and Bioelectronics 2013, 47, 559–565. Figure. The general operation scheme of nuclease biosensor. (A) In a case a sample does not contain nuclease the Target nucleotide is not cleaved and its hybridization rate is high. (B) In a case sample contains nucleases the Target nucleotide is cleaved and its hybridization rate is lower and relates to the nuclease activity. Figure 1

Signal-On and Highly Sensitive Electrochemiluminescence Biosensor for Hydrogen Sulfide in Joint Fluid Based on Silver-Ion-Mediated Base Pairs and Hybridization Chain Reaction

Hydrogen sulfide (H2S) in joint fluid acts as a signal molecule to regulate joint inflammation. Direct detection of H2S in joint fluid is of great significance for the diagnosis and treatment of arthritis. However, due to the low volume of joint fluid and low H2S concentration, existing methods face the problem of the insufficient limit of detection. In this study, a highly sensitive biosensor was proposed by designing a primer probe and combining it with hybrid chain reaction (HCR) under the strong interaction between metal ions and H2S to achieve H2S detection. The primer probe containing multiple cytosine (C) sequences was fixed on a gold electrode, and the C–Ag–C hairpin structure was formed under the action of Ag+. In the presence of H2S, it can combine with Ag+ in the hairpin structure to form Ag2S, which leads to the opening of the hairpin structure and triggers the hybridization chain reaction (HCR) with another two hairpin structures (H1 and H2). A large number of double-stranded nucleic acid structures can be obtained on the electrode surface. Finally, Ru(phen)32+ can be embedded into the double chain structure to generate the electrochemiluminescence (ECL) signal. The linear response of the H2S biosensor ranged from 0.1000 to 1500 nM, and the limit of detection concentration of H2S was 0.0398 nM. The developed biosensor was successfully used to determine H2S in joint fluid.

Probing Notch1-Dll4 signaling in regulating osteogenic differentiation of human mesenchymal stem cells using single cell nanobiosensor

Human mesenchymal stem cells (hMSCs) have great potential in cell-based therapies for tissue engineering and regenerative medicine due to their self-renewal and multipotent properties. Recent studies indicate that Notch1-Dll4 signaling is an important pathway in regulating osteogenic differentiation of hMSCs. However, the fundamental mechanisms that govern osteogenic differentiation are poorly understood due to a lack of effective tools to detect gene expression at single cell level. Here, we established a double-stranded locked nucleic acid (LNA)/DNA (LNA/DNA) nanobiosensor for gene expression analysis in single hMSC in both 2D and 3D microenvironments. We first characterized this LNA/DNA nanobiosensor and demonstrated the Dll4 mRNA expression dynamics in hMSCs during osteogenic differentiation. By incorporating this nanobiosensor with live hMSCs imaging during osteogenic induction, we performed dynamic tracking of hMSCs differentiation and Dll4 mRNA gene expression profiles of individual hMSC during osteogenic induction. Our results showed the dynamic expression profile of Dll4 during osteogenesis, indicating the heterogeneity of hMSCs during this dynamic process. We further investigated the role of Notch1-Dll4 signaling in regulating hMSCs during osteogenic differentiation. Pharmacological perturbation is applied to disrupt Notch1-Dll4 signaling to investigate the molecular mechanisms that govern osteogenic differentiation. In addition, the effects of Notch1-Dll4 signaling on hMSCs spheroids differentiation were also investigated. Our results provide convincing evidence supporting that Notch1-Dll4 signaling is involved in regulating hMSCs osteogenic differentiation. Specifically, Notch1-Dll4 signaling is active during osteogenic differentiation. Our results also showed that Dll4 is a molecular signature of differentiated hMSCs during osteogenic induction. Notch inhibition mediated osteogenic differentiation with reduced Alkaline Phosphatase (ALP) activity. Lastly, we elucidated the role of Notch1-Dll4 signaling during osteogenic differentiation in a 3D spheroid model. Our results showed that Notch1-Dll4 signaling is required and activated during osteogenic differentiation in hMSCs spheroids. Inhibition of Notch1-Dll4 signaling mediated osteogenic differentiation and enhanced hMSCs proliferation, with increased spheroid sizes. Taken together, the capability of LNA/DNA nanobiosensor to probe gene expression dynamics during osteogenesis, combined with the engineered 2D/3D microenvironment, enables us to study in detail the role of Notch1-Dll4 signaling in regulating osteogenesis in 2D and 3D microenvironment. These findings will provide new insights to improve cell-based therapies and organ repair techniques.

Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA.

The discovery that certain diseases have specific miRNA signatures which correspond to disease progression opens a new biomarker category. The detection of these small non-coding RNAs is performed routinely using body fluids or tissues with real-time PCR, next-generation sequencing, or amplification-based miRNA assays. Antibody-based detection systems allow an easy onset handling compared to PCR or sequencing and can be considered as alternative methods to support miRNA diagnostic in the future. In this study, we describe the generation of a camelid heavy-chain-only antibody specifically recognizing miRNAs to establish an antibody-based detection method. The generation of nucleic acid-specific binders is a challenge. We selected camelid binders via phage display, expressed them as VHH as well as full-length antibodies, and characterized the binding to several miRNAs from a signature specific for dilated cardiomyopathy. The described workflow can be used to create miRNA-specific binders and establish antibody-based detection methods to provide an additional way to analyze disease-specific miRNA signatures.

Particle detection and tracking with DNA

We present the first proof-of-concept simulations of detectors using biomaterials to detect particle interactions. The essential idea behind a “DNA detector” involves the attachment of a forest of precisely-sequenced single or double-stranded nucleic acids from a thin holding layer made of a high-density material. Incoming particles break a series of strands along a roughly co-linear chain of interaction sites and the severed segments then fall to a collection area. Since the sequences of base pairs in nucleic acid molecules can be precisely amplified and measured using polymerase chain reaction (PCR), the original spatial position of each broken strand inside the detector can be reconstructed with nm precision. Motivated by the potential use as a low-energy directional particle tracker, we perform the first Monte Carlo simulations of particle interactions inside a DNA detector. We compare the track topology as a function of incoming direction, energy, and particle type for a range of ionising particles. While particle identification and energy reconstruction might be challenging without a significant scale-up, the excellent potential angular and spatial resolution (lesssim 25^circ axial resolution for keV-scale particles and nm-scale track segments) are clear advantages of this concept. We conclude that a DNA detector could be a cost-effective, portable, and powerful new particle detection technology. We outline the outstanding experimental challenges, and suggest directions for future laboratory tests.

Ultrasensitive Electrochemical Detection of Mutated Viral RNAs with Single-Nucleotide Resolution Using a Nanoporous Electrode Array (NPEA).

The detection of nucleic acids and their mutation derivatives is vital for biomedical science and applications. Although many nucleic acid biosensors have been developed, they often require pretreatment processes, such as target amplification and tagging probes to nucleic acids. Moreover, current biosensors typically cannot detect sequence-specific mutations in the targeted nucleic acids. To address the above problems, herein, we developed an electrochemical nanobiosensing system using a phenomenon comprising metal ion intercalation into the targeted mismatched double-stranded nucleic acids and a homogeneous Au nanoporous electrode array (Au NPEA) to obtain (i) sensitive detection of viral RNA without conventional tagging and amplifying processes, (ii) determination of viral mutation occurrence in a simple detection manner, and (iii) multiplexed detection of several RNA targets simultaneously. As a proof-of-concept demonstration, a SARS-CoV-2 viral RNA and its mutation derivative were used in this study. Our developed nanobiosensor exhibited highly sensitive detection of SARS-CoV-2 RNA (∼1 fM detection limit) without tagging and amplifying steps. In addition, a single point mutation of SARS-CoV-2 RNA was detected in a one-step analysis. Furthermore, multiplexed detection of several SARS-CoV-2 RNAs was successfully demonstrated using a single chip with four combinatorial NPEAs generated by a 3D printing technique. Collectively, our developed nanobiosensor provides a promising platform technology capable of detecting various nucleic acids and their mutation derivatives in highly sensitive, simple, and time-effective manners for point-of-care biosensing.