Detection Of Cancer-related Enzymes And MicroRNAs Based On DNA Probes

Nucleic acid molecular probes are widely used in the detection of biological analytes such as enzymes,proteins,biological small molecules,metal ions,nucleic acids and cells due to their variable structure,simple synthesis,and easy modification.They have become indispensable research tools in the field of biological analysis.The strategy of nucleic acid signal amplification based on nucleic acid molecular probes not only provides an important platform for detecting low-abundance substances,but also greatly improves the sensitivity of nucleic acid molecular probes.This is essential for biomedical research,molecular diagnosis and pharmacogenomics.In recent years,the development of nanotechnology has became more and more rapid and attracted lots of attention.The combination of nanomaterials and nucleic acid molecular probes can improve the stability of nucleic acid molecular probe and promote its application in complex biological environments.This paper has established kinds of methods with simple operation,high sensitivity and specificity for the detection of enzymes and microRNAs by combining nucleic acid molecular probes with nucleic acid signal amplification technology or nanomaterials,the specific work content is as follows:（1）The activity of DNA 3’-phosphatase has been shown to be closely related to a variety of human diseases,and it plays a key role in repairing DNA damage.In this experiment,we developed a simple and sensitive fluorescent method to detect the activity of DNA 3’-phosphatase based on exonuclease III（Exo III）-assisted cascade recycling amplification reaction.Biotin-modified hairpin DNA1 binds with streptavidin-modified magnetic bead（MB）to get MB-DNA1.DNA 3’-phosphatase can hydrolyze phosphate groups on MB-DNA1 to form hydroxyl groups,which leads to the polymerization extension and nicking endonuclease cleavage reaction to obtain the trigger DNA1 fragment（tDNA1）.In the presence of Exo III,the enzyme-assisted cascade recycling amplification reaction of DNA2,FP and tDNA1 occurred,leading to the separation of fluorescent groups labeled on the DNA2 and FP from the quenching groups,and the fluorescence signal of the system is restored,thus achieving the detection of DNA 3’-phosphatase activity.This strategy is applied to determine two typical DNA 3’-phosphatases including T4 polynucleotide kinase（T4PNK）and alkaline phosphatase（ALP）.（2）Telomerase is highly expressed in more than 85%of cancer cells,which is considered a biomarker for early cancer diagnosis and disease prognosis.In this experiments,we developed a simple and sensitive fluorescent method to detect telomerase activity based on the catalytic hairpin assembly cycle amplification reaction.After the MB-DNA complex is successfully prepared,the telomerase-specific probe immobilized on the MB undergoes the extension reaction of the（TTAGGG）_n repeat fragment under the action of telomerase to release the tDNA,and the fluorescence signal of the fluorescent group modified on the probe tDNA is restored.After magnetic separation,the tDNA can catalyze the assembly reaction between the HP1 and HP2,which causes the modified fluorescent groups on the HP1and HP2 to separate from the quenching group,and the fluorescence signal is further superimposed.The released tDNA triggers the cycle reaction again,realizing the amplification of the fluorescence signal.This method has been successfully used to detect telomerase activity in HeLa and HepG2 cell extracts.（3）Deoxyribozyme（DNAzyme）has the characteristics of high catalytic activity,good stability,and easy modification,which is suitable for the detection of low-abundance substances in living cells.Manganese dioxide（MnO₂）nanosheet is a kind of two-dimensional nanomaterial with multiple functions.In this experiment,we designed a ratio type of the Mn²⁺-DNAzyme probe using MnO₂ nanosheets as a carrier to image microRNA-155 in living cells.MnO₂ nanosheets can quench the fluorescence signal of the fluorophore on the probe.When the MnO₂ nanosheets loaded probes are taken up by the cells,the intracellular glutathione（GSH）can reduce the MnO₂ nanosheets to Mn²⁺,and release the probes to restore the fluorescence signal.The generated Mn²⁺as an effective cofactor for the Mn²⁺-DNAzyme probe can activate the catalytic activity of the probe to interact with microRNA-155 in the cells after the cleavage reaction.According to the change of the fluorescence signal,the imaging of microRNA-155 in HeLa and HepG2 cells is achived.（4）Telomerase and microRNA are biomarkers closely related to tumors.Simultaneous detection of both markers can improve the accuracy and reliability of early diagnosis.Based on the mechanism of fluorescence resonance energy transfer（FRET）,we designed two fluorescent DNA probes to detect telomerase and microRNA-21,respectively.The probes are wrapped by gelatin through electrostatic interaction to form nanoparticles.After that,molecularly imprinted coating of transferrin（TrF）on the surface of gelatin nanoparticles and endocytosis into cells by targeted action.Following with the degradation of gelatin in the cells,DNA probes are released to react with telomerase and microRNA-21 and lead to the change of the fluorescence signal.Thereby the simultaneous imaging of telomerase and microRNA-21 are successfully achieved in HeLa cells and HepG2 cells.