Invention of circRNA promoting RNA to specifically promote circRNA production.

CircRNA, an essential RNA molecule involved in various biological functions and diseases, often exhibits decreased expression in tumor tissues, playing a role as a tumor suppressor, and suggesting therapeutic potential for cancer. However, current methods for promoting circRNA production are limited. This study introduces a novel approach for enhancing circRNA biogenesis, termed circRNA promoting RNA (cpRNA). CpRNA is designed to complement the flanking sequences of reverse complementary matches (RCMs) within pre-mRNA, thereby facilitating circRNA formation through improved exon circularization. Using a split-GFP reporter system, we demonstrated that cpRNA significantly enhance circGFP production. Optimization identified the best conditions for cpRNA to promote circRNA biogenesis, and these cpRNAs were then used to augment the production of endogenous circRNAs. These results indicate that cpRNAs can specifically increase the production of endogenous circRNAs with RCMs, such as circZKSCAN1 and circSMARCA5 in cancer cells, thereby inhibiting cell proliferation and migration by modulating circRNA-related pathways, showcasing the therapeutic potential of cpRNAs. Mechanistic studies have also shown that cpRNA promotes circRNA biogenesis, in part, by antagonizing the unwinding function of DHX9. Overall, these findings suggest that cpRNA represents a promising strategy for circRNA overexpression, offering a potential treatment for diseases marked by low circRNA levels.

Delivery of 5-fluorouracil for cancer therapy using aptamer-based nonlinear hybridization chain reaction.

5-Fluorouracil (5-FU) is a conventional nucleotide analogue used for cancer treatment. However, its clinical application faces challenges such as low stability and non-specific toxicity. With the remarkable advancements in DNA nanotechnology, DNA-based self-assembled nanocarriers have emerged as powerful tools for delivering nucleotide drugs. In this study, we have designed a non-linear hybrid chain reaction involving a fuel strand with AS1411 aptamer sequence to construct a dendritic structure capable of carrying 5-FU. This structure specifically targets cancer cells with overexpressed nucleolin on their surface, allowing the 5-FU to exert its anticancer effects and achieve therapeutic outcomes. Furthermore, we have also investigated the mechanistic action of this drug delivery system, aiming to establish a novel therapeutic platform for 5-FU treatment.

Quantitative measurement of acute myocardial infarction cardiac biomarkers by "All-in-One" immune microfluidic chip for early diagnosis of myocardial infarction.

Acute myocardial infarction (AMI) is a life-threatening condition with a narrow treatment window, necessitating rapid and accurate diagnostic methods. We present an "all-in-one" convenient and rapid immunoassay system that combines microfluidic technology with a colloidal gold immunoassay. A degassing-driven chip replaces a bulky external pump, resulting in a user-friendly and easy-to-operate immunoassay system. The chip comprises four units: an inlet reservoir, an immunoreaction channel, a waste pool, and an immunocomplex collection chamber, allowing single-channel flow for rapid and accurate AMI biomarker detection. In this study, we focused on cardiac troponin I (cTnI). With a minimal sample of just 4 µL and a total detection time of under 3 min, the chip enabled a quantitative visual analysis of cTnI concentration within a range of 0.5 âˆ¼ 60.0 ng mL-1. This all-in-one integrated microfluidic chip with colloidal gold immunoassay offers a promising solution for rapid AMI diagnosis. The system's portability, small sample requirement, and quantitative visual detection capabilities make it a valuable tool for AMI diagnostics.

New advances in signal amplification strategies for DNA methylation detection in vitro.

5-methylcytosine (5 mC) DNA methylation is a prominent epigenetic modification ubiquitous in the genome. It plays a critical role in the regulation of gene expression, maintenance of genome stability, and disease control. The potential of 5 mC DNA methylation for disease detection, prognostic information, and prediction of response to therapy is enormous. However, the quantification of DNA methylation from clinical samples remains a considerable challenge due to its low abundance (only 1% of total bases). To overcome this challenge, scientists have recently developed various signal amplification strategies to enhance the sensitivity of DNA methylation biosensors. These strategies include isothermal nucleic acid amplification and enzyme-assisted target cycling amplification, among others. This review summarizes the applications, advantages, and limitations of these signal amplification strategies over the past six years (2018-2023). Our goal is to provide new insights into the selection and establishment of DNA methylation analysis. We hope that this review will offer valuable insights to researchers in the field and facilitate further advancements in this area.

CPSF3 Promotes Pre-mRNA Splicing and Prevents CircRNA Cyclization in Hepatocellular Carcinoma.

CircRNAs are crucial in tumorigenesis and metastasis, and are comprehensively downregulated in hepatocellular carcinoma (HCC). Previous studies demonstrated that the back-splicing of circRNAs was closely related to 3'-end splicing. As a core executor of 3'-end cleavage, we hypothesized that CPSF3 modulated circRNA circularization. Clinical data were analyzed to establish the prognostic correlations. Cytological experiments were performed to determine the role of CPSF3 in HCC. A fluorescent reporter was employed to explore the back-splicing mechanism. The circRNAs regulated by CPSF3 were screened by RNA-seq and validated by PCR, and changes in downstream pathways were explored by molecular experiments. Finally, the safety and efficacy of the CPSF3 inhibitor JTE-607 were verified both in vitro and in vivo. The results showed that CPSF3 was highly expressed in HCC cells, promoting their proliferation and migration, and that a high CPSF3 level was predictive of a poor prognosis. A mechanistic study revealed that CPSF3 enhanced RNA cleavage, thereby reducing circRNAs, and increasing linear mRNAs. Furthermore, inhibition of CPSF3 by JET-607 suppressed the proliferation of HCC cells. Our findings indicate that the increase of CPSF3 in HCC promotes the shift of pre-mRNA from circRNA to linear mRNA, leading to uncontrolled cell proliferation. JTE-607 exerted a therapeutic effect on HCC by blocking CPSF3.

Application of intelligent responsive DNA self-assembling nanomaterials in drug delivery.

Smart nanomaterials are nano-scaled materials that respond in a controllable and reversible way to external physical or chemical stimuli. DNA self-assembly is an effective way to construct smart nanomaterials with precise structure, diverse functions and wide applications. Among them, static structures such as DNA polyhedron, DNA nanocages and DNA hydrogels, as well as dynamic reactions such as catalytic hairpin reaction, hybridization chain reaction and rolling circle amplification, can serve as the basis for building smart nanomaterials. Due to the advantages of DNA, such as good biocompatibility, simple synthesis, rational design, and good stability, these materials have attracted increasing attention in the fields of pharmaceuticals and biology. Based on their specific response design, DNA self-assembled smart nanomaterials can deliver a variety of drugs, including small molecules, nucleic acids, proteins and other drugs; and they play important roles in enhancing cellular uptake, resisting enzymatic degradation, controlling drug release, and so on. This review focuses on different assembly methods of DNA self-assembled smart nanomaterials, therapeutic strategies based on various intelligent responses, and their applications in drug delivery. Finally, the opportunities and challenges of smart nanomaterials based on DNA self-assembly are summarized.

Circular RNAs: Emerging roles and new insights in human cancers.

Circular RNAs (circRNAs) are single-stranded, covalently closed RNA molecules formed by mRNA exon back-splicing. Although the circRNA functions remain largely unknown, their currently known biological activities include: acting as competing endogenous RNA (ceRNA) to adsorb microRNA (miRNA), binding proteins, regulating transcription or splicing, and ability to be translated into proteins or peptides. A growing number of studies have found that many circRNAs are abnormally expressed in various cancers, and their dysregulation is highly correlated with tumor progression. Therefore, diagnosis and treatment using circRNAs as biomarkers and therapeutic targets, respectively, has gradually become an attractive research topic. In this review, we introduced the canonical biogenesis pathways and degradation mechanisms of circRNAs. In addition, we examined the biological functions of circRNAs in vivo. Finally, we discussed the current clinical applications and challenges faced by circRNA, and proposed future directions for this promising research field.

A DNAzyme-enhanced nonlinear hybridization chain reaction for sensitive detection of microRNA.

As a typical biomarker, the expression of microRNA is closely related to the occurrence of cancer. However, in recent years, the detection methods have had some limitations in the research and application of microRNAs. In this paper, an autocatalytic platform was constructed through the combination of a nonlinear hybridization chain reaction and DNAzyme to achieve efficient detection of microRNA-21. Fluorescently labeled fuel probes can form branched nanostructures and new DNAzyme under the action of the target, and the newly formed DNAzyme can trigger a new round of reactions, resulting in enhanced fluorescence signals. This platform is a simple, efficient, fast, low-cost, and selective method for the detection of microRNA-21, which can detect microRNA-21 at concentrations as low as 0.004 nM and can distinguish sequence differences by single-base differences. In tissue samples from patients with liver cancer, the platform shows the same detection accuracy as real-time PCR but with better reproducibility. In addition, through the flexible design of the trigger chain, our method could be adapted to detect other nucleic acid biomarkers.

Challenges and opportunities for circRNA identification and delivery.

Circular RNAs (circRNAs) are evolutionarily conserved noncoding RNAs with tissue-specific expression patterns, and exert unique cellular functions that have the potential to become biomarkers in therapeutic applications. Therefore, accurate and sensitive detection of circRNA with facile platforms is essential for better understanding of circRNA biological processes and circRNA-related disease diagnosis and prognosis; and precise regulation of circRNA through efficient delivery of circRNA or siRNA is critical for therapeutic purposes. Here, we reviewed the current development of circRNA identification methodologies, including overviewing the purification steps, summarizing the sequencing methods of circRNA, as well as comparing the advantages and disadvantages of traditional and new detection methods. Then, we discussed the delivery and manipulation strategies for circRNAs in both research and clinic treatment. Finally, the challenges and opportunities of analyzing circRNAs were addressed.

Rational design of nonlinear hybridization immunosensor chain reactions for simultaneous ultrasensitive detection of two tumor marker proteins.

Sensitive biomarker detection techniques are beneficial for both disease diagnosis and postoperative examinations. The nonlinear hybridization chain reaction (NHCR) is widely used as an output signal amplification technique for biosensor platforms. A novel hairpin-free NHCR was developed in this study as a flow cytometric immunoassay to detect alpha-fetoprotein (AFP) and prostate specific antigen (PSA). First, the target AFP is captured on magnetic beads (MBs) that are modified with capture antibodies. Then, the prepared biotin-streptavidin-biotin (B-S-B) system, which links biotinylated detection antibodies and biotinylated trigger DNA together through the high affinity between biotin-streptavidin interaction, is added to label the target AFP, forming a sandwich complex with three trigger DNA chains. Each trigger DNA chain grows a dendritic DNA nanostructure following a nonlinear hybridization chain reaction. As the substrate flue chains are labeled with fluorophores, the self-assembly process of dendritic DNA is accompanied by the continuous release of fluorophores. Dendrites with strong fluorescence then form on the surface of MBs. Finally, the target AFP is quantified by analyzing the fluorescent MBs using flow cytometry. The proposed immunoassay has a high selectivity along with isothermal, enzyme-free, and exponential amplification efficiency. It shows a limit of detection (LOD) of 1.74 pg mL-1. The proposed biosensor was also successfully used to quantitatively detect AFP in serum samples. It may be utilized to detect multiple tumor markers simultaneously by changing the size of MBs and antibody-antigen pairs. Most tumor markers are only related to tumor diagnosis but without specificity, so the combined detection of multiple tumor markers can improve the accuracy of early tumor diagnoses.

Trehalose in Biomedical Cryopreservation-Properties, Mechanisms, Delivery Methods, Applications, Benefits, and Problems.

Cells and tissues are the foundation of translational medicine. At present, one of the main technological obstacles is their preservation for long-term usage while maintaining adequate viability and function. Optimized storage techniques must be developed to make them safer to use in the clinic. Cryopreservation is the most common long-term preservation method to maintain the vitality and function of cells and tissues. But, the formation of ice crystals in cells and tissues is considered to be the main mechanism that could harm cells and tissues during freezing and thawing. To reduce the formation of ice crystals, cryoprotective agents (CPAs) must be added to the cells and tissues to achieve the cryoprotective effect. However, conventional cryopreservation of cells and tissues often needs to use toxic organic solvents as CPAs. As a result, cryopreserved cells and tissues may need to go through a time-consuming washing process to remove CPAs for further applications in translational medicine, and multiple valuable cells are potentially lost or killed. Currently, trehalose has been researched as a nontoxic CPA due to its cryoprotective ability and stability during cryopreservation. Nevertheless, trehalose is a nonpermeable CPA, and the lack of an effective intracellular trehalose delivery method has become the main obstacle to its use in cryopreservation. This article illustrated the properties, mechanisms, delivery methods, and applications of trehalose, summarized the benefits and limits of trehalose, and summed up the findings and research direction of trehalose in biomedical cryopreservation.

Biosensors based on functional nucleic acids and isothermal amplification techniques.

In the past few years, with the in-depth research of functional nucleic acids and isothermal amplification techniques, their applications in the field of biosensing have attracted great interest. Since functional nucleic acids have excellent flexibility and convenience in their structural design, they have significant advantages as recognition elements in biosensing. At the same time, isothermal amplification techniques have higher amplification efficiency, so the combination of functional nucleic acids and isothermal amplification techniques can greatly promote the widespread application of biosensors. For the purpose of further improving the performance of biosensors, this review introduces several widely used functional nucleic acids and isothermal amplification techniques, as well as their classification, basic principles, application characteristics, and summarizes their important applications in the field of biosensing. We hope to provide some references for the design and construction of new tactics to enhance the detection sensitivity and detection range of biosensing.

Mulberry leaf polyphenols alleviated high-fat diet-induced obesity in mice.

Mulberry leaf is an important medicinal food plant, which is rich in polyphenol compounds. Mulberry leaf polyphenols (MLP) possess significant lipid-lowering and antioxidant effects, and healthcare functions. In this study, the polyphenol content of mulberry leaf ethanol extract was measured using HPLC. The analysis of mulberry leaf extract resulted in the identification of 14 compounds, of which Chlorogenic acid and Quercitrin were the highest. A high-fat diet (HFD)-induced obese mouse model was developed and treated with MLP for 12 weeks to explore their effect on lipid metabolism in HFD-induced obese mice. The results showed that the MLP could inhibit the weight gain and fat cell volume increase in the HFD-induced obese mice in a dose-dependent manner. Further analysis revealed that the MLP decelerated the fatty acid composition in the adipose tissues of HFD-induced obese mice, and significantly increased the polyunsaturated-to-saturated fatty acid (PUFA/SFA) ratio. The real-time quantitative PCR (RT-qPCR) results indicated that the MLP significantly inhibited the down regulation of uncoupling protein (UCP) 1 (UCP1), UCP3, and PR domain zinc finger protein 16 (PRDM16) caused by the HFD. These beneficial effects of MLP on HFD-induced obese mice might be attributed to their ability to change the fatty acid composition of adipose tissue and increase the expression of thermogenesis genes. Overall, the study results suggested that the MLP could serve as potential lipid-lowering and weight-loss functional food and healthcare products.

Age-related Loss of miR-124 Causes Cognitive Deficits via Derepressing RyR3 Expression.

Epigenetic alterations of brain contribute to age-related cognitive decline. The challenge now is to identify these tractable epigenetic molecules working as the downstream cell-signaling nodes mediating age-related cognitive decline. Here we reported age-related loss of miR-124 in human and rat brains. To further validate these findings, knockout mice in which one of the three miR-124 genes (miR-124-3) was deleted using CRISPR/Cas9-mediated gene engineering were generated. MiR-124-3 knockout mice developed cognitive deficit phenotype. MiR-124 deficiency in the mouse brain resulted in upregulation of the Ryanodine receptor 3 (RyR3) gene, and the cognitive deficits in miR-124-3 knockout mice were ameliorated by knocking down RyR3 expression using RNAi. In addition, miR-124 deficiency facilitated Aß42-induced neuron apoptosis. Our work suggested that age-related cognitive decline, at least in part, was associated with miR-124 deficiency and subsequently upregulated RyR3 expression in inducing neuronal death.

Nonlinear hybridization chain reaction-based flow cytometric immunoassay for the detection of prostate specific antigen.

Sensitive detection of biomarkers is highly desirable for disease diagnosis and postoperative examination. As a signal amplification technique, nonlinear hybridization chain reactions (NHCR) based on DNA self-assembly has been widely adopted to versatile biosensor platforms for signal output and sensitivity enhancement. Herein, we proposed a novel hairpin-free NHCR based flow cytometric immunoassay for prostate specific antigen (PSA) detection. In this study, Ab1-Ag-Ab2-streptavidin-trigger DNA complexes were formed on the magnetic beads (MBs), and each trigger DNA initiated a round of NHCR amplification to form dendritic DNA nanostructures with many fluorescent signal molecules. The robust flow cytometric fluorescent analysis was finally employed for the quantitation of target protein on the MBs. As far as we know, this is the first time to combine the hairpin-free NHCR strategy with fluorescent immunoassay on MBs to detect protein biomarkers. In addition to the high selectivity of immunoassay itself, the characteristics of isothermal, enzyme-free, and exponential amplification efficiency of hairpin-free NHCR endow this developed immunoassay with a detection limit that exceeds 100-folds that of commercially available PSA kits. Moreover, this MBs-based platform of this immunoassay is also amenable to target enrichment and removal of spontaneous NHCR signal through magnetic separation, greatly eliminating the background signal interference. With our efforts, this newly developed biosensor exhibits a detection limit of 1.92 pg/mL and shows high selectivity. It has also been successfully applied to the quantitative detection of PSA in serum samples. With these merits, this convenient biosensor platform has the potential for medical research and disease diagnosis.

Brain-Penetration and Neuron-Targeting DNA Nanoflowers Co-Delivering miR-124 and Rutin for Synergistic Therapy of Alzheimer's Disease.

Alzheimer disease (AD) is the leading cause of dementia that affects millions of old people. Despite significant advances in the understanding of AD pathobiology, no disease modifying treatment is available. MicroRNA-124 (miR-124) is the most abundant miRNA in the normal brain with great potency to ameliorate AD-like pathology, while it is deficient in AD brain. Herein, the authors develop a DNA nanoflowers (DFs)-based delivery system to realize exogenous supplementation of miR-124 for AD therapy. The DFs with well-controlled size and morphology are prepared, and a miR-124 chimera is attached via hybridization. The DFs are further modified with RVG29 peptide to simultaneously realize brain-blood barrier (BBB) penetration and neuron targeting. Meanwhile, Rutin, a small molecular ancillary drug, is co-loaded into the DFs structure via its intercalation into the double stranded DNA region. Interestingly, Rutin could synergize miR-124 to suppress the expression of both BACE1 and APP, thus achieving a robust inhibition of amyloid ß generation. The nanosystem could pro-long miR-124 circulation in vivo, promote its BBB penetration and neuron targeting, resulting in a significant increase of miR-124 in the hippocampus of APP/PS1 mice and robust therapeutic efficacy in vivo. Such a bio-derived therapeutic system shows promise as a biocompatible nanomedicine for AD therapy.

New advances in brain-targeting nano-drug delivery systems for Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease worldwide and its incidence is increasing due to the ageing population. Currently, the main limitations of AD treatment are low blood-brain barrier permeability, severe off-target of drugs, and immune abnormality. In this review, four hypotheses for Alzheimer's pathogenesis and three challenges for Alzheimer's drug delivery are discussed. In addition, this article summarises the different strategies of brain targeting nano-drug delivery systems (NDDSs) developed in the last 10 years. These strategies include receptor-mediated (transferrin receptor, low-density lipoprotein receptor-related protein, lactoferrin receptor, etc.), adsorption-mediated (cationic, alkaline polypeptide, cell-penetrating peptides, etc.), and transporter-mediated (P-gp, GLUT1, etc.). Moreover, it provides insights into novel strategies used in AD, such as exosomes, virus-like particles, and cell membrane coating particles. Hence, this review will help researchers to understand the current progress in the field of NDDSs for the central nervous system and find new directions for AD therapy.HighlightsCharacteristics and challenges based on the pathogenesis of AD were discussed.Recent advances in novel brain-targeting NDDSs for AD over the past 10 years were summarised.

Traditional and new applications of the HCR in biosensing and biomedicine.

The hybridization chain reaction is a very popular isothermal nucleic acid amplification technology. A single-stranded DNA initiator triggers an alternate hybridization event between two hairpins forming a double helix polymer. Due to isothermal, enzyme-free and high amplification efficiency characteristics, the HCR is often used as a signal amplification technology for various biosensing and biomedicine fields. However, as an enzyme-free self-assembly reaction, it has some inevitable shortcomings of relatively slow kinetics, low cell internalization efficiency, weak biostability of DNA probes and uncontrollable reaction in these applications. More and more researchers use this reaction system to synthesize new materials. New materials can avoid these problems skillfully by virtue of their inherent biological characteristics, molecular recognition ability, sequence programmability and biocompatibility. Here, we summarized the traditional application of the HCR in biosensing and biomedicine in recent years, and also introduced its new application in the synthesis of new materials for biosensing and biomedicine. Finally, we summarized the development and challenges of the HCR in biosensing and biomedicine in recent years. We hope to give readers some enlightenment and help.

Mechanisms Regulating Abnormal Circular RNA Biogenesis in Cancer.

Circular RNAs (circRNAs), which are a class of endogenous RNA with covalently closed loops, play important roles in epigenetic regulation of gene expression at both the transcriptional and post-transcriptional level. Accumulating evidence demonstrated that numerous circRNAs were abnormally expressed in tumors and their dysregulation was involved in the tumorigenesis and metastasis of cancer. Although the functional mechanisms of many circRNAs have been revealed, how circRNAs are dysregulated in cancer remains elusive. CircRNAs are generated by a "back-splicing" process, which is regulated by different cis-regulatory elements and trans-acting proteins. Therefore, how these cis and trans elements change during tumorigenesis and how they regulate the biogenesis of circRNAs in cancer are two questions that interest us. In this review, we summarized the pathways for the biogenesis of circRNAs; and then illustrated how circRNAs dysregulated in cancer by discussing the changes of cis-regulatory elements and trans-acting proteins that related to circRNA splicing and maturation in cancer.

Specific and sensitive detection of CircRNA based on netlike hybridization chain reaction.

Circular RNA (circRNA), as a new class of biomarker, plays an important role in the occurrence and development of cancer. However, the limitations of detection methods in recent years have severely restricted the related research of circRNA. Here, we have developed an effective circRNA detection method based on the thermostatic netlike hybridization chain reaction (HCR). It combines reverse transcription-rolling circle amplification (RT-RCA) with well-designed netlike HCR to achieve dual selection and dual signal amplification, which can eliminate the interference of linear isomers. This two-dimensional netlike HCR is composed of an ingeniously designed trigger chain and two hairpin fuel probes, which can generate a stable network structure with RT-RCA products containing multiple sets of repeats at a constant temperature, thereby producing enhanced fluorescent signals. Systematic studies reveal that the optimized netlike HCR system has higher detection efficiency for DNA strands containing multiple sets of repetitive sequences, can detect circRNA as low as 0.1 pM, and has excellent selectivity. By using human tumor cell lines and tissues, it has been verified that the netlike HCR-based method can accurately detect specific circRNA in real biological samples without RNase R enrichment, which provides a simple and useful platform for detecting low-abundance circRNA. Furthermore, the proposed strategy is also a potential method for detecting some genes containing repetitive sequences, such as telomere DNA, centromere DNA and ribosomal DNA (rDNA).