Analogous Modified DNA Probe And Immune Competition Method-based Electrochemical Detection Method For RNA N~6-Methyladenosine

Objective:Since the discovery of RNA chemical modifications,researchers have successively discovered more than 150 RNA modifications.N~6-methyladenosine(m~6A)was proved to be one of the most abundant chemical modifications in RNA,which is widely found in eukaryotes.Varies levels of m~6A in organisms play different biological functions,and abnormalities in these biological functions are critical factors leading to tumors and some diseases.Since m~6A does not alter its complementary pairing ability with T and U,it cannot directly detect by standard hybridization or sequencing technology.In addition,there is no chemical method that can effectively distinguish m~6A from A,and RNA unstable,during the chemical processing the degradation of RNA increases.These problems have led to further restrictions on the development of m~6A detection technology,resulting in slow progress in the study of the exact biological regulation mechanisms of m~6A.Therefore,the establishment of a fast and accurate detection method for m~6A is of great significance for the study of m~6A.Methods:A label-free and highly selective electrochemical immune-sensor was developed for the detection of m~6A.This method is established on that the anti-m~6A-antibody can recognize m~6A both in RNA and DNA.Firstly,histidine-tagged recombinant protein G(his-PG)was used to immobilization the anti-m~6A-antibody on the surface of the gold electrode to improve the binding efficiency of antigen and antibody.Secondly,Analogous modified DNA probe(L1)was serves as a signal molecule,by competing with m~6A-RNA for binding to antibodies to broaden the linear range.Then,RNase A was used to hydrolyze the RNA,which bound to the antibodies to eliminate the influences of the target RNA lengths and base sequences on the detection results.Finally,detection of m~6A in RNA is achieved by detecting the EIS signal of the immunosensor.Results:Under the optimal experimental conditions,the proposed electro-chemical immunosensor presented high sensitivity and specificity.Due to the competition mechanism,the immunosensor exhibited a wide linear range from 0.05 to 200 nM with a detection limit as low as 0.016 nM(S/N=3).Furthermore,the developed immunosensor was validated for m~6A determination in HepG2,L02,HBE and A549 cell lines.Conclusions:In this study,his-PG is used to immobilization the anti-m~6A-antibody on the surface of the gold electrode,which not only improves the binding efficiency of antigen and antibody,but also ensures that every antibody can be saturated by antigen,and improves the repeatability of the method.L1 was acts as a signal molecule,by competing with m~6A-RNA for binding to antibodies to broaden the linear range.Then the interferences caused by different RNA lengths and base sequences were eliminated by the hydrolysis of RNase A.Under optimal experimental conditions,m~6A-RNAs from different cell lines were detected.It is proved that the electrochemical immunosensor has the potential to accurately detection the contents of m~6A-RNA in biological samples.