Detection And Analysis Of Nuclease Based On Reduced Graphene Oxide Quenched Hairpin Probe

Ribonuclease A?RNase A?is an endonuclease with important research significance in the fields of enzymology,medicine,biochemistry and structure biology.RNase A with significant cytotoxicity can efficiently kill tumor cells.In addition,it can inhibit the replication of Human Immunodeficiency Virus?HIV-1?and has been used as a drug for HIV-1 therapy.Ribonuclease H?RNase H?is an endonuclease that can hydrolysis DNA-RNA heteroduplex endonucleolytically and cleave RNA-DNA junctions.It plays an vital role in regulating the immune defense process and maintaining the stability of the eukaryotic genome.Hence,it has great significance to sensitively detect RNase A and RNase H activity.At present,a variety of methods for RNase A and RNase H have been developed.However,these methods still have some limitations in different extent.Therefore,developing rapid,sensitive and specifical method for ribonuclease is still highly desired.Based on the advantages of small size,good dispersivity and strong quenching ability of reduced graphene oxide?rGO?,we focus on the development of simple,specific and sensitive detection system for ribonuclease assay.The main studies are as follows:1.A new platform for detection of RNase A activity based on rGO quenched fluorescence probe was constructed.Firstly,we develope a method for RNase activity assay.The results showed that the optimal rGO concentration of the system was 8?g/mL,the reaction temperature and time were 37?and 35 min respectively.Under the optimized condditions,a detection limit of RNase A is 0.05 ng/mL.Furthermore,the concentration-dependent inhibitory effect of heavy metal ions on RNase A was observed and the results indicated that Ba²⁺,Co²⁺,Pb²⁺,As³⁺,and Cu²⁺inhibited RNase A activity with IC₅₀0 values of 93.7?M(Ba²⁺),90.9?M(Co²⁺),110.6?M(Pb²⁺),171.5?M(As³⁺),and165.1?M(Cu²⁺),respectively.Next,we further used it to study the interaction between the different natural drugs?which separated from herb?and RNase A in vitro and in vivo.The results showed that all natural compounds could activate the activity of RNase A in a concentration-dependent manner.Intracellular experiments showed that the RNase A activity of MCF-7 cells reduced about 33%compared to that of SMMC-7721.Both of G-10and Chikusetsusaponin V,which could differently regulate RNase A activity in vitro,significantly upregulated RNase A activity of MCF-7 and BEL7404 cells,while no significant effect appeared in SMMC-7721.Finally,it was applied to detect the change of RNase A activity caused by HBV infection on.The results showed that the average level of RNase A in the HBV infection group was significantly inhibited compared with normal group.The above results show that the new method can not only be used for RNase A activity monitoring and high throughput drug screening in vitro,but also can be used for early diagnosis of disease related to the changes of RNase A activity.2.RNase H activity assay and natural effector screening based on rGO-DNAzyme quenching fluorescence probe.Firstly,we developed a RNase H activity detection method based on fluorescent probes,rGO and DNAzyme.The results showed that the optimal graphene oxide concentration of the system was 4?g/mL,and the optimal reaction temperature and time were 37 and 40 min respectively.Under optimal conditions,the we obtained a detection limit of RNase H with 0.01 U/mL.The compounds screening experiments indicated 4 kind of activators and 5 kind of inhibitors for RNase H.Furthermore,5 kind of inhibitors showed an concentration-dependent inhibitory manner.In addition,after using the method for RNase H activity assay of different tumor cells,we found that RNase H levels were varied differently in MCF-7,Hela and HepG-2 cells,and its level in MCF-7 and Hela cells were 1.7 times more than that of HepG-2 cells.By selecting 3 kinds of natural compounds?A,L6,and Y7?to treat tumors of MCF-7,Hela and HEPG-2,we found that compound L6 had the greatest inhibition effect on the RNase H and the inhibition rate in MCF-7,Hela and HEPG-2 cells was 72.05%,68.66%and 56.22%respectively.Compound A inhibited RNase H activity of MCF-7,Hela and HEPG-2 cells with 56.02%,56.96%and 43.05%respectively.The inhibition rates of Y7,with the lowest effect were 24.71%,24.19%and 17.14%for MCF-7,Hela and HEPG-2 cells,respectively.The result of serum assay showed that the activity of RNase H in serum of HCV patients was significantly lower than that of normal group.In conclusion,the high sensitivity and low-cost RNase H detection method of this study holds great potential for RNase H activity detection,drug screening and related function explortion.