Engineering DNAzyme cascade for signal transduction and amplification.

Inspired by the natural enzyme cascade reaction, a multiple DNAzyme cascade platform is engineered to imitate the intracellular process of signal transduction and signal amplification. In this design, when particular stimuli appear, an activated upstream DNAzyme will cleave a well-designed intermediary S1, releasing a downstream DNAzyme that can cleave the reporter substrate S2 to output signals. Thus, the signal is passed from the upstream DNAzyme to the downstream DNAzyme through a well-designed intermediary, accomplishing signal transduction and signal amplification. According to the experimental results, the DNAzyme cascades are capable of improving sensitivity for bioassays compared with that for single DNAzyme-based biocatalysis, which holds promise for potential applications, such as biomolecular computing, logic circuits and precision medicine.

Enhanced Imaging of Specific Cell-Surface Glycosylation Based on Multi-FRET.

Cell-surface glycosylation contains abundant biological information that reflects cell physiological state, and it is of great value to image cell-surface glycosylation to elucidate its functions. Here we present a hybridization chain reaction (HCR)-based multifluorescence resonance energy transfer (multi-FRET) method for specific imaging of cell-surface glycosylation. By installing donors through metabolic glycan labeling and acceptors through aptamer-tethered nanoassemblies on the same glycoconjugate, intramolecular multi-FRET occurs due to near donor-acceptor distance. Benefiting from amplified effect and spatial flexibility of the HCR nanoassemblies, enhanced multi-FRET imaging of specific cell-surface glycosylation can be obtained. With this HCR-based multi-FRET method, we achieved obvious contrast in imaging of protein-specific GalNAcylation on 7211 cell surfaces. In addition, we demonstrated the general applicability of this method by visualizing the protein-specific sialylation on CEM cell surfaces. Furthermore, the expression changes of CEM cell-surface protein-specific sialylation under drug treatment was accurately monitored. This developed imaging method may provide a powerful tool in researching glycosylation functions, discovering biomarkers, and screening drugs.

High Signal-to-Background Ratio Detection of Cancer Cells with Activatable Strategy Based on Target-Induced Self-Assembly of Split Aptamers.

Highly sensitive detection of cancer cells with high signal-to-background ratio (SBR) is still urgently needed. Here, a self-assembling activatable probe (SAAP) based on split aptamers was developed to meet this purpose. The SAAP is formed with quenched fluorescence; only when target cells are present would the split aptamers self-assemble together and thus activate fluorescence by intramolecular and intermolecular fluorescence quenching strategies. As proof of concept, a split aptamer pair stemming from an intact aptamer, ZY11, developed by our lab was selected to construct SAAP. Owing to the design of self-assembly and activation strategy, the SBR of our approach could be raised to ∼40 and achieved a very low detection limit of seven target 7721 cells in 100 µL of binding buffer. Meanwhile, one-step detection of target cells was achieved within 15 min without any washing steps and pretreatment, which shows potential for point-of-care detection. Moreover, we succeeded in the specific recognition of target cells in 50% human serum and mixed cell samples, which indicated this strategy had great advantages in detection in complex biological samples. In addition, dual-signal detection was also successfully implemented, which may be helpful for accurate detection of target cells. Therefore, this rapid, facile, specific, and highly sensitive detection method for cancer cells may provide convenience in cancer research and medical diagnosis.

An updated database of virus circular RNAs provides new insights into the biogenesis mechanism of the molecule.

Virus circular RNAs (circRNA) have been reported to be extensively expressed and play important roles in viral infections. Previously we build the first database of virus circRNAs named VirusCircBase which has been widely used in the field. This study significantly improved the database on both the data quantity and database functionality: the number of virus circRNAs, virus species, host organisms was increased from 46440, 23, 9 to 60859, 43, 22, respectively, and 1902 full-length virus circRNAs were newly added; new functions were added such as visualization of the expression level of virus circRNAs and visualization of virus circRNAs in the Genome Browser. Analysis of the expression of virus circRNAs showed that they had low expression levels in most cells or tissues and showed strong expression heterogeneity. Analysis of the splicing of virus circRNAs showed that they used a much higher proportion of non-canonical back-splicing signals compared to those in animals and plants, and mainly used the A5SS (alternative 5' splice site) in alternative-splicing. Most virus circRNAs have no more than two isoforms. Finally, human genes associated with the virus circRNA production were investigated and more than 1000 human genes exhibited moderate correlations with the expression of virus circRNAs. Most of them showed negative correlations including 42 genes encoding RNA-binding proteins. They were significantly enriched in biological processes related to cell cycle and RNA processing. Overall, the study provides a valuable resource for further studies of virus circRNAs and also provides new insights into the biogenesis mechanisms of virus circRNAs.

Multi-omics data integration reveals the complexity and diversity of host factors associated with influenza virus infection.

Influenza viruses pose a significant and ongoing threat to human health. Many host factors have been identified to be associated with influenza virus infection. However, there is currently a lack of an integrated resource for these host factors. This study integrated human genes and proteins associated with influenza virus infections for 14 subtypes of influenza A viruses, as well as influenza B and C viruses, and built a database named H2Flu to store and organize these genes or proteins. The database includes 28,639 differentially expressed genes (DEGs), 1,850 differentially expressed proteins, and 442 proteins with differential posttranslational modifications after influenza virus infection, as well as 3,040 human proteins that interact with influenza virus proteins and 57 human susceptibility genes. Further analysis showed that the dynamic response of human cells to virus infection, cell type and strain specificity contribute significantly to the diversity of DEGs. Additionally, large heterogeneity was also observed in protein-protein interactions between humans and different types or subtypes of influenza viruses. Overall, the study deepens our understanding of the diversity and complexity of interactions between influenza viruses and humans, and provides a valuable resource for further studies on such interactions.

Foxm1 transcription factor is required for maintenance of pluripotency of P19 embryonal carcinoma cells.

Transcription factor Foxm1 plays a critical role during embryonic development and its expression is repressed during retinoic acid (RA)-induced differentiation of pluripotent P19 embryonal carcinoma cells at the early stage, correlated with downregulation of expression of pluripotency markers. To study whether Foxm1 participates in the maintenance of pluripotency of stem cells, we knock down Foxm1 expression in P19 cells and identify that Oct4 are regulated directly by Foxm1. Knockdown of Foxm1 also results in spontaneous differentiation of P19 cells to mesodermal derivatives, such as muscle and adipose tissues. Maintaining Foxm1 expression prevents the downregulation of pluripotency-related transcription factors such as Oct4 and Nanog during P19 cell differentiation. Furthermore, overexpression of FOXM1 alone in RA-differentiated P19 cells (4 days) or human newborn fibroblasts restarts the expression of pluripotent genes Oct4, Nanog and Sox2. Together, our results suggest a critical involvement of Foxm1 in maintenance of stem cell pluripotency.

Polymerization and isomerization cyclic amplification for nucleic acid detection with attomolar sensitivity.

DNA amplification is one of the most valuable tools for the clinical diagnosis of nucleic acid-related diseases, but current techniques for DNA amplification are based on intermolecular polymerization reactions, resulting in the risk of errors in the intermolecular reaction pattern. In this article, we introduce the concept of intramolecular polymerization and isomerization cyclic amplification (PICA), which extends a short DNA strand to a long strand containing periodic repeats of a sequence through cyclic alternating polymerization and isomerization. To the best of our knowledge, this is the first time that a real ssDNA self-extension method without any additional auxiliary oligonucleotides has been reported. By interfacing PICA with external molecular elements, it can be programmed to respond to different targets. Herein, we designed two distinct types of amplified nucleic acid detection platforms that can be implemented with PICA, including cyclic reverse transcription (CRT) and cyclic replication (CR). We experimentally demonstrate the mechanisms of CRT-PICA and CR-PICA using mammalian miRNA and virus DNA. The results showed that this proposed detection platform has excellent sensitivity, selectivity, and reliability. The detection level could reach the aM level, that is, several copies of target molecules can be detected if a small volume is taken into account.

A self-assembled DNA nanostructure as a FRET nanoflare for intracellular ATP imaging.

Due to the incorporation of gold nanoparticles (AuNPs), previously reported AuNP-based FRET nanoflares still have some problems, such as non-negligible cytotoxicity and a time-consuming preparation procedure. In this communication, a novel AuNP-free FRET nanoflare for intracellular ATP imaging is developed based on a DNA nanostructure, which is self-assembled through cyclic U-type hybridization only involving a certain number of DNA strands.

Highly sensitive detection of cancer cells via split aptamer mediated proximity-induced hybridization chain reaction.

Highly sensitive detection of cancer cells is of great importance for evaluating cancer development and improving survival rates. Here, we developed a split aptamer mediated proximity-induced hybridization chain reaction (HCR) strategy to meet this purpose. In this strategy, two split aptamer initiator probes, Sp-a and Sp-b, and two HCR hairpin probes, H1 and H2 were designed. The split aptamer initiator probes contained two components, split aptamer domains being responsible for target recognition, and the split initiator parts serving as the HCR promoter. In the presence of target cells, Sp-a and Sp-b would self-assemble on the cell surfaces, allowing the formation of an intact nicked initiator to activate the HCR reaction. Benefit from low background split aptamers and HCR amplification, this strategy presented high sensitivity in quantitative detection with a detection limit of 18 cells in 150 µL of binding buffer. Moreover, the approach exhibited excellent specificity to target cells in 10% fetal bovine serum and mixed cell samples, which was favorable for clinical diagnosis in complex biological environment. In addition, by changing the split aptamers attached to the split initiator, the proposed strategy can be expanded to detect various kinds of target cells. It may provide a novel and useful applicable platform for the sensitive detection of cancer cells in biomedicine and tumor-related studies.

A DNA molecular diagnostic technology with LAMP-like sensitivity based on one pair of hairpin primers-mediated isothermal polymerization amplification.

With the rapid development of isothermal amplification technology, DNA molecular diagnosis has become an important reference for clinical treatment. In this work, we have designed a DNA molecular diagnostic technology with LAMP-like sensitivity for nucleic acid analysis and detection based on only one pair of hairpin primers. This DNA molecular diagnostic technology consists of Bst DNA polymerase and one pair of hairpin primers, which are designed easily by adding a stem-loop structure to a target binding domain. When the target is present, the polymerization reaction between the hairpin primers and the target generates a specific dumbbell DNA similar to LAMP, which triggers cyclic amplification reactions to extend a series of long dsDNA products with repeated sequences by inserting fluorescent dye Eva Green observed the increase in fluorescence signal. In our method, using the hairpin primers-mediated isothermal polymerization amplification, we can specifically monitor 3-5 copies of the target nucleic acid in the system without labeling and temperature cycling in the reaction. In addition, serum samples from 13 patients with suspected schistosomiasis were targeted; we further demonstrated the ability of the technology to detect complex clinic samples, and its potentially inestimable applicability in clinic early molecular diagnostic research.

Amplified AND logic platform for cell identification.

Herein, we developed an amplified AND logic platform (AALP) on a cell membrane, which integrated two DNA aptamers for cell recognition and localized catalytic hairpin assembly (LCHA) for signal amplification. The AALP could perform "AND" logic computing via a double-checked strategy of two biomarkers on similar cell surfaces and precisely label the target cells with an amplified fluorescence signal.

A DNAzyme cascade for amplified detection of intracellular miRNA.

Inspired by the natural enzyme cascade reaction, an artificial DNAzyme cascade system is developed for the amplified detection of intracellular miR-141. The results showed that the method enormously enhanced the readout of the fluorescence signal and achieved a femtomolar detection limit.

Photocaged FRET nanoflares for intracellular microRNA imaging.

In 2015, we proposed FRET nanoflares for intracellular molecular (RNA, H+, and K+) detection. To improve detection accuracy and achieve on-demand sensing, herein, we developed photocaged FRET nanoflares for spatiotemporal microRNA imaging in living cells. In other words, the probes will not work until they are exposed to UV light.

Intramolecular trigger remodeling-induced HCR for amplified detection of protein-specific glycosylation.

Dynamic changes of protein-glycosylation on cell surface act as an important indicator that reflects cellular physiological states and disease developments. The enhanced visualization of protein-specific glycosylation is of great value to interpret its functions and mechanisms. Hence, we present an intramolecular trigger remodeling-induced hybridization chain reaction (HCR) for imaging protein-specific glycosylation. This strategy relies on designing two DNA probes, protein and glycan probes, labeled respectively on protein by aptamer recognition and glycan through metabolic oligosaccharide engineering (MOE). Upon the same glycoprotein was labeled, the complementary domain of two probes induces hybridization and thus to remodel an intact trigger, followed by initiating HCR assembly. Applying this strategy, we successfully achieved imaging of specific protein-glycosylation on CEM cell surface and monitored dynamic changes of the glycosylation after treating with drugs. It provides a powerful tool with high flexibility, specificity and sensitivity in the research field of protein-specific glycosylation on living cells.

Linear-hairpin variable primer RT-qPCR for MicroRNA.

Here, we present a highly specific, sensitive and cost-effective system to quantify microRNA (miRNA) expression based on two-step RT-qPCR with EvaGreen detection chemistry, called linear-hairpin variable primer RT-qPCR. It takes advantage of the novel designed variable primer, which is initially designed to be linear, extending to form a hairpin structure and replacing the target miRNA for cyclic RT. Then the RT product is quantified by conventional EvaGreen based qPCR. The results show that this method has a dynamic range of 8 logs and the sensitivity is sufficient to directly detect down to 4 target miRNA molecules with a total analysis time of less than 2 hours. It is capable of discriminating between similar miRNAs, leading to an accurate representation of the mature miRNA content in a sample. The RT step can be multiplexed and the 8 miRNA profiles measured in 7 mouse tissues by this method show an excellent correlation with the commercial standard TaqMan RT-qPCR assays (r 2 = 0.9881).

Enhanced visualization of cell surface glycans via a hybridization chain reaction.

Herein, we apply a DNA hybridization chain reaction (HCR) to achieve sensitively amplified imaging of cell surface glycosylation with reduced non-natural monosaccharide units. This method is simple, efficient, sensitive, and possesses great potential to illuminate the pathways via which cell surface glycosylation regulates cell functions.

Real-time investigation of nucleic acids phosphorylation process using molecular beacons.

Phosphorylation of nucleic acids is an indispensable process to repair strand interruption of nucleic acids. We have studied the process of phosphorylation using molecular beacon (MB) DNA probes in real-time and with high selectivity. The MB employed in this method is devised to sense the product of a 'phosphorylation-ligation' coupled enzyme reaction. Compared with the current assays, this novel method is convenient, fast, selective, highly sensitive and capable of real-time monitoring in a homogenous solution. The preference of T4 polynucleotide kinase (T4 PNK) has been investigated using this approach. The results revealed that a single-stranded oligonucleotide containing guanine at the 5' termini is most preferred, while those utilizing cytosine in this location are least preferred. The preference of (T)9 was reduced greatly when phosphoryl was modified at the 5' end, implying that T4 PNK could discern the phosphorylated/unphosphorylated oligonucleotides. The increase of oligonucleotide DNA length leads to an enhancement in preference. A fast and accurate method for assaying the kinase activity of T4 PNK has been developed with a wide linear detection range from 0.002 to 4.0 U/ml in 3 min. The effects of certain factors, such as NTP, ADP, (NH4)2SO4 and Na2HPO4, on phosphorylation have been investigated. This novel approach enables us to investigate the interactions between proteins and nucleic acids in a homogenous solution, such as those found in DNA repair or in drug development.

Metastatic cancer cell and tissue-specific fluorescence imaging using a new DNA aptamer developed by Cell-SELEX.

Metastasis, which derived from a primary tumor, accounts for 90% of mortality caused by cancer. Early diagnosis of cancer metastasis may significantly improve cure rate of patients who are at high risk for developing metastasis. In this study, we successfully achieved metastatic cancer cell and tissue-specific fluorescence imaging by using a new aptamer developed by cell-based systematic evolution of ligands by exponential enrichment (Cell-SELEX). With metastatic colorectal carcinoma LoVo cells as selection target, the aptamer named J3 which bind to metastatic cancer cells with good affinity and specificity was obtained. Then J3 was labeled with Cy5 fluorescent group (J3-Cy5) for imaging metastatic cancer cells, the results demonstrated excellent imaging contrast. Moreover, the results of tissue section imaging revealed that J3-Cy5 probe explicitly recognized lymph node tissue with colorectal carcinoma metastasis with a high detection rate of 73.9%, but showed a low detection rate to colorectal carcinoma tissue with no metastasis or cancer adjacent tissue. Therefore, the targeting reagent J3-based fluorescence imaging possesses great potential for clinical diagnosis of cancer metastasis.

Real-time monitoring of nucleic acid ligation in homogenous solutions using molecular beacons.

Nucleic acids ligation is a vital process in the repair, replication and recombination of nucleic acids. Traditionally, it is assayed by denatured gel electrophoresis and autoradiography, which are not sensitive, and are complex and discontinuous. Here we report a new approach for ligation monitoring using molecular beacon DNA probes. The molecular beacon, designed in such a way that its sequence is complementary with the product of the ligation process, is used to monitor the nucleic acid ligation in a homogeneous solution and in real-time. Our method is fast and simple. We are able to study nucleic acids ligation kinetics conveniently and to determine the activity of DNA ligase accurately. We have studied different factors that influence DNA ligation catalyzed by T4 DNA ligase. The major advantages of our method are its ultrasensitivity, excellent specificity, convenience and real-time monitoring in homogeneous solution. This method will be widely useful for studying nucleic acids ligation process and other nucleic acid interactions.

A signal-on split aptasensor for highly sensitive and specific detection of tumor cells based on FRET.

We present here a signal-on fluorescence biosensor for highly sensitive and specific detection of tumor cells with a split aptamer based on fluorescence resonance energy transfer (FRET). This sensor holds considerable potential for simple, rapid, sensitive and specific tumor cell detection in early clinical diagnosis.