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BHK21 cells were harvested and lysed in 200 µl lysis buffer (0.1 M Tris-HCl, 0.1% Triton X-100, 2 mM EDTA, pH 7.8). Thefluorescence of eGFP was measured using a luminometer. The error bars denote Standard deviation (SD).
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DNAzymes are DNA molecules that can directly cleave cognate mRNA, and have been developed to silence gene expression for research and clinical purposes. The advantage of DNAzymes over ribozymes is that they are inexpensive to produce and exhibit good stability. The "10-23 DNA enzyme" is composed of a catalytic domain of 15 deoxynucleotides, flanked...
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DNAzymes (Dz) 8–17 and 10–23 are two widely studied and well-characterized RNA-cleaving DNA catalysts. In an effort to further improve the understanding of the fragile interactions and dynamics of the enzymatic mechanism, this study examines the catalytic efficiency of minimally modified DNAzymes. Five single mutants of Dz8–17 and Dz10–23 were prep...
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... Based on the N/P ratio-dependent activity of the DZ01/Tat complex, the N/P ratio of DZ01 and lipofectamine 2000 was suggested to be around 1:1. Accordingly if less amount of lipofectamine 2000 was used, the negative effect on DNAzyme activity was decreased. With the suggested amount of lipofectamine 2000, efficient cleavage reactions have been observed in cell-based experiments [36][37][38]. ...
Cationic polymeric materials and cell-penetrating peptides (CPPs) were often used as the delivery vectors in the evaluation of nucleic acid therapeutics. 10-23 DNAzyme is a kind of potential antisense therapeutics by catalytic cleavage of the disease-related RNAs. Here, lipofectamine 2000 and Tat peptide were evaluated for their effect on the catalytic activity of 10-23 DNAzyme, with the observed rate constant, thermal stability, CD spectra, and PAGE analysis, with a duplex DNA mimicking DNAzyme-substrate as a control. It was shown that the cationic carriers had a negative effect on the catalytic performance of the 10-23 DNAzyme. Significantly, the destabilizing effect of the cationic carriers on the duplex formation was noteworthy, as a duplex formation is an essential prerequisite in the silencing mechanisms of antisense and RNAi.
... Therefore, researchers hope to improve the therapeutic effect and prognosis of cancer at the gene level, that is, from the perspective of the fundamental mechanism of cancer pathogenesis. Researchers have previously induced endogenous siRNA process pathways for RNA interference to trigger post-transcriptional gene silencing, or used DNA targeted homologous recombination to achieve gene targeting (including gene knockout or knock-in) (Yi and Liu, 2011). Fortunately, with the emergence of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated nuclease 9 (Cas9) system which acted as an RNAguided genome editing tool, therapeutic gene editing is becoming a viable biomedical tool and was first tested in a person in 2016 (Cyranoski, 2016;Luther et al., 2018). ...
DNA methylation is one of the most essential epigenetic mechanisms to regulate gene expression. DNA methyltransferases (DNMTs) play a vital role in DNA methylation in the genome. In mammals, DNMTs act with some elements to regulate the dynamic DNA methylation patterns of embryonic and adult cells. Conversely, the aberrant function of DNMTs is frequently the hallmark in judging cancer, including total hypomethylation and partial hypermethylation of tumor suppressor genes (TSGs), which improve the malignancy of tumors, aggravate the ailment for patients, and significantly exacerbate the difficulty of cancer therapy. Since DNA methylation is reversible, currently, DNMTs are viewed as an important epigenetic target for drug development. However, the impression of DNMTs on cancers is still controversial, and therapeutic methods targeting DNMTs remain under exploration. This review mainly summarizes the relationship between the main DNMTs and cancers as well as regulatory mechanisms and clinical applications of DNMTs in cancer and highlights several forthcoming strategies for targeting DNMTs.
... DNAzyme has been successfully employed for cellular and in vivo knockdown of target genes. [21][22][23] It has also been well documented that incorporation of LNAs in both arms of DNAzyme significantly enhances its cleavage activity. [24][25][26][27][28] Here, we report for the first time the use of an LNAzyme for in vivo downregulation of miRNA expression in a zebrafish model. ...
MicroRNAs (miRNAs) have crucial functions in many cellular processes, such as differentiation, proliferation and apoptosis; aberrant expression of miRNAs has been linked to human diseases, including cancer. Tools that allow specific and efficient knockdown of miRNAs would be of immense importance for exploring miRNA function. Zebrafish serves as an excellent vertebrate model system to understand the functions of miRNAs involved in a variety of biological processes. We designed and employed a strategy based on locked nucleic acid enzymes (LNAzymes) for in vivo knockdown of miRNA in zebrafish embryos. We demonstrate that LNAzyme can efficiently knockdown miRNAs with minimal toxicity to the zebrafish embryos.
