Trans-acting aptazyme for conditional gene knockdown in eukaryotic cells.

Trans-acting hammerhead ribozyme inherits the advantages of being the smallest and best-characterized RNA-cleaving ribozyme, offering high modularity and the ability to cleave any desired sequence without the aid of any protein, as long as the target sequence contains a cleavage site. However, achieving precise control over the trans-acting hammerhead ribozyme would enable safer and more accurate regulation of gene expression. Herein, we described an intracellular selection of hammerhead aptazyme that contains a theophylline aptamer on stem II based on toxin protein IbsC. Based on the intracellular selection, we obtained three new cis-acting hammerhead aptazymes. Moreover, the corresponding trans-acting aptazymes could be efficiently induced by theophylline to knock down different targeted genes in eukaryotic cells. Notably, the best one, T195, exhibited a ligand-dependent and dose-dependent response to theophylline, and the cleavage efficiency could be enhanced by incorporating multiplex aptazymes.

Dysosma versipellis Extract Inhibits Esophageal Cancer Progression through the Wnt Signaling Pathway.

OBJECTIVE: In this study, we aim to investigate the effect of Dysosma versipellis extract on biological behavior of esophageal cancer cells and its underlying mechanisms. METHODS: A total of 30 BALB/C nude mice (class SPF) were equally and randomly divided into the control group, model group, and Dysosma versipellis group. CP-C cell of esophageal cancer was subcutaneously injected into the model group as well as the Dysosma versipellis group, and the same amount of normal saline into the control group, in order to compare the tumorigenesis of nude mice of three groups. Wnt, ß-catenin, and p-GSK3ß/GSK3ß expression in tumor tissues was detected using Western blot. CP-C cells in logarithmic growth were selected and divided into 4 groups, including the control group, podophyllotoxin group, Wnt activator group, and combined group (mixture of podophyllotoxin and Wnt activator). The cell viability, apoptosis, and invasion ability, Wnt, ß-catenin, and p-GSK3ß/GSK3ß expression level of CP-C cells in each group were detected via MTT assay, flow cytometry, transwell, and Western blot, respectively. RESULTS: The tumorigenesis rates of the control group, model group, and Dysosma versipellis group were 0%, 90% (1 tumor-free mouse), and 80% (2 tumor-free mice), respectively. The tumor mass in the Dysosma versipellis group was significant less than that in the model group. Based on the results of Western blot, Wnt, ß-catenin, and p-GSK3ß/GSK3ß expression of the Dysosma versipellis group was lower than that of the control group. The in vitro viability test indicated that there was a significant difference in cell viability exhibited among four groups. Cell viability level in the 3 groups, including the combined group, blank group, and Wnt activator group, was higher than the podophyllotoxin group at each time point. In vitro apoptosis assay revealed that significant differences in cell apoptosis exhibited among four groups. Cell apoptosis rate was higher in the podophyllotoxin group compared to the remaining three groups. The Wnt activator group showed the lowest cell apoptosis rate. The in vitro invasion assay demonstrated that numbers of transmembrane cell in the 3 groups, involving the combined group, blank group, and Wnt activator group, showed a higher level than the podophyllotoxin group. The results of Western blot manifested that the podophyllotoxin group showed lower level of Wnt, ß-catenin, and p-GSK3ß/GSK3ß expression compared to the other 3 groups. CONCLUSION: Podophyllotoxin in Dysosma versipellis has an excellent antiesophageal cancer effect and is able to inhibit cell viability as well as invasion ability and promote apoptosis of esophageal cancer cells by inhibiting the Wnt signaling pathway, which could be potentially used in future clinical treatment of esophageal cancer.

Intracellular Selection of Theophylline-Sensitive Hammerhead Aptazyme.

Hammerhead ribozyme-based aptazyme (HHAz), inheriting the advantages of small size and high efficiency from the RNA-cleaving ribozyme and the specific recognition ability of aptamers to specific targets, exhibits the huge potential to be a transgene expression regulator. Herein, we report a selection strategy for HHAz by using a toxin protein IbsC as the reporter to offer a positive phenotype, thus realizing an easy-operating, time- and labor-saving selection of HHAz variants with desired properties. Based on this strategy, we obtained a new HHAz (TAP-1), which could react sensitively toward the extracellular regulatory molecule, theophylline, both in prokaryotic and eukaryotic systems. With fluorescent protein reporter, the intracellular switching efficiencies of TAP-1 and other reported theophylline-dependent HHAzs has been quantitatively evaluated, showing that TAP-1 not only exhibits the best downregulating ability at high concentration of theophylline but also maintains high activity with 0.1 mM theophylline, which is a safe concentration in the human body.

Frame-shifted proteins of a given gene retain the same function.

Frameshift mutations are generally considered to be lethal because it could result in radical changes of the protein sequence behind. However, the protein of frameshift mutants of a type I toxin (ibsc) was found to be still toxic to bacteria, retaining the similar function as wild-type protein to arrest the cellular growth by impairing the membrane's integrity. Additionally, we have verified that this observation is not an individual event as the same phenomenon had been found in other toxins subsequently. After analyzing the coding sequence of these genes, we proposed a hypothesis to search this kind of hidden gene, through which a dihydrofolate reductase-encoding gene (dfrB3) was found out. Like the wild-type reductase, both +1 and -1 frame-shifted proteins of dfrB3 gene were also proved to catalyze the reduction of dihydrofolate to tetrahydrofolate by using NADPH.

Intracellular selection of trans-cleaving hammerhead ribozymes.

Hammerhead ribozyme is the smallest and best characterized catalytic RNA-cleaving ribozyme. It has been reported as potential therapeutic tools to manipulate the expression of target genes. However, most of naturally occurring hammerhead ribozymes process self-cleavage rather than cleave substrate RNA in trans, and its high intracellular activity relies on the tertiary interaction of Loop II and steam I bulge, resulting in decreased performance as applied in gene silencing. We described a direct intracellular selection method to evolve hammerhead variants based on trans-cleavage mode via using a toxin gene as the reporter. And a dual fluorescence proteins system has also been established to quantitatively evaluate the efficiency of selected ribozymes in the cell. Based on this selection strategy, we obtained three mutants with enhanced intracellular cleaving activity compared to wide type hammerhead ribozyme. The best one, TX-2 was revealed to possess better and consistent gene knockdown ability at different positions on diverse targeted mRNA either in prokaryotic or eukaryotic cells than wild-type hammerhead ribozyme. These observations imply the efficiency of the intracellular selection method of the trans-acting ribozyme and the potentials of improved ribozyme variants for research and therapeutic purposes.

Colorimetric PCR-Based microRNA Detection Method Based on Small Organic Dye and Single Enzyme.

microRNAs (miRNAs) have been a class of promising disease diagnostic biomarkers and therapeutic targets for their important biological functions. However, because of the high homology, interference from precursors (pri-miRNA, pre-miRNA), as well as limitations in the current assay technologies, it poses high demand and challenge for a specific, efficient, and economic miRNA assay method. Here, we propose a new miRNA detection method based on a label-free probe and a small organic dye with sequence dependence, realizing the sequence-specific and colorimetric detection of target miRNA. What is pleasantly surprising, only one enzyme is enough to propel the whole miRNA assay process, greatly simplifying the reaction component and detection process. Together with PCR amplification for the high enough sensitivity and three checks for specificity control, a detection limit of 5 fM was obtained and even one mutation could be discriminated visually. Overall, the new method makes much progress in convenience and economy of PCR-based miRNA assay method so that miRNA assay is going to be more friendly and affordable.

Re-characterization of hammerhead ribozymes as molecular tools for intermolecular RNA cleavage.

A new guideline for the construction of hammerhead ribozymes to achieve trans-cleavage of a single-stranded RNA molecule was developed. The sequence rule of the HHRz cleavage site was highly recommended to be "DWH" with an optimal binding arm length of 8-9nt, which diverged from the former rule of "NUX".