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.

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.

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.

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.

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).

Nonlinear hybridization chain reaction-based functional DNA nanostructure assembly for biosensing, bioimaging applications.

Hybridization chain reaction (HCR) can be divided into two categories: linear HCR and nonlinear HCR. In traditional linear HCR, the relatively slow kinetics and less sufficient sensitivity largely limit its scope of application. In the nonlinear HCR system, under the trigger of the initiator, the judicious designed substrate sequences (hairpin or hairpin-free) will self-assembly to dendritic or branched DNA nanostructures with exponential growth kinetics. Given the advantages of its enzyme-free, high-order growth kinetic, high sensitivity, and simple operation, nonlinear HCR is regarded as a powerful signal amplifier for the detection of biomarkers by integrating with versatile sensing platforms in the past few decades. In this review, we describe the basic features of nonlinear HCR mechanism and classify the nonlinear HCR into several categories based on their self-assembly mechanisms: the branched HCR, dendritic HCR, hydrogel-based clamped HCR, and other types of HCR. Then, we summarize the recent development of nonlinear HCR in biosensing, such as nucleic acid, protein, enzyme activities, and cancer cell detection, etc., and we also review the current applications of nonlinear HCR in bioimaging (mRNA in situ imaging). We choose several representative works to further illustrate the analysis mechanisms via various detection platforms, such as fluorescence, electrochemical, colorimetric, etc. At last, we also review the challenges and further perspectives of nonlinear HCR in the use of bioanalysis.

Two new secondary metabolites from a mangrove-derived fungus Cladosporium sp. JJM22.

Two new compounds (1 and 2), together with six known compounds (3-8), were obtained from the Cladosporium sp. JJM22, an endophytic fungus isolated from the stem bark of the mangrove plant Ceriops tagal collected in South China Sea. Their structures were elucidated on the basis of detailed spectroscopic analysis. The absolute configurations of 1 and 2 were confirmed by the comparison of optical rotation and the CD data with those of known compounds. The inhibitory activities of the isolated compounds against six terrestrial pathogenic bacteria and human cervical carcinoma Hela cell line were evaluated. Compound 3 exhibited a broad spectrum of antibacterial activities.