The Research Of Fluorogenic-Catalytic DNA Screening And Mechanism Of Catalytic Action Using In Vitro Selection Technology

Cancer is one of the most critical threats to the health of human beings.Worldwidely speaking, breast cancer is the most commonly diagnosed cancer in women, accounting for a quarter of all type of cancers. But even worse, the incidence of breast cancer is increasing, especially in developing word because of economy development and adoption of western lifestyles. And, in clinic, different stages represent specific treatment and prognosis.Although there have been several early detection methods, such as mammography,magnetic resonance imaging, but majority of breast cancer cases are diagnosed in very late stage particularly in developing countries, because of limited payment capacity,dependency of medical facilities and long time patient’s endurance of complex procedure. So, developing simple and quick detection method is very necessary to improve breast cancer outcome and survival.Thus, simple quick serum test which take advantage of extracellular secretion mixture of breast cancer cells is the optimal detection method to patient, especially in developing countries.These considerations have promote us to develop a noval detection method based on catalytic DNA molecules(DNAzymes), which are a special class of single-stranded functional nucleic acids that have a catalytic ability. These DNAzyme can be artificially isolated from a random-sequence DNA library by in vivro selection and have been increasingly applied as various molecular tools. Herein, in laboratory, we demonstrate that fluorogenic DNAzymes can be selected from a DNA pool to fluorescently indicate the ESM that is produced by a specific cancer cell, and that such probes can be used for developing simple and quick test to detect this cell line.Thus, we decide to create such fluorogenic DNA probes based on an RNA-cleaving fluorescent DNAzyme(RFD) system because it has been well developed.These DNAzymes are designed to cleave a chimeric DNA/RNA substrate that contains a single ribonucleotide as the cleavage site, which is flanked by a pair nucleotides modified with a fluorophore(F) and a quencher(Q). These features provide a convenient way to link the DNAzyme’s cleaving activity to physical fluorophore-quencher separation, and consequently the increase of fluorescence signal.we have developed a efficient RNA-cleaving DNA molecule--LYF5.This DNAzyme can specifically indicate breast cancer cell line- T47D- with a fluorogenic ability and the minimum limitation of detection is 500 cells per milliliter in vitro. After sequence and 2D structure optimization, the second-generation DNAzyme(2G-LYF5)increase approximately 2 fold cleaved percentage. Finally, we find the intramolecular stem-loop structure play the decisive role in its activity. The development of the DNAzyme with hairpin may offer a rapid simple diagnostic solution toward the early identification of breast cancer.