Single-Molecule Detection Of FTO And Ago2

Single-molecule detection technology is a newly emerging technology with the sensitivity of single-molecular level.Single-molecule detection has the advantages of high sensitivity,good specificity,and slow sample consumption,with wide applications in the field of biochemical analysis,drug discovery,and early diseases diagnosis.In this thesis,we constructed two biosensors based on single-molecule detection technology for sensitive detection of demethylase FTO and protein Ago2.1、We developed a rolling circle transcription（RCT）and CRISPR/Cas12a-based method for sensitive detection of FTO in combination with the single molecular detection.N6-methyladenosine（m6A）modification is the most common form of m RNA in mammalian cells,which are dynamically reversible regulated by RNA demethylase,such as fat mass and obesity-associated protein（FTO）and alkylation repair homolog protein 5（ALKBH5）.The aberrant expression of RNA demethylase is closely associated with numerous human diseases,including human obesity and cancers.In this work,we design single-stranded DNA（ss DNA）containing a specific sequence（5’-G-A-T-C-3’）as a substrate,which is modified with biotin at the 3’end.In the presence of target FTO,m6A in substrate ss DNA is recognized and removed.After magnetic separation,the cleaved ds DNA substrate functions as a primer to initiate T7 RNA polymerase-based rolling-circle transcription amplification to generate large amounts of CRISPR RNA（cr RNA）.The resultant cr RNA can trigger the cleavage of the FAM-labeled probe catalyzed by the CRISPR/Cas12a system,releasing a large number of FAM molecules.The released FAM signal can be simply counted by single-molecule detection using total internal reflection fluorescence（TIRF）,and accurate quantification of FTO activity can be achieved by measuring FAM molecules.The cr RNA-initiated cleavage of the FAM-labeled probe can effectively suppresses non-specific background signals,and the introduction of magnetic separation and single-molecule detection further improves the detection sensitivity.This method exhibits good specificity and high sensitivity with a detection limit of 1.20×10^-13 M,and it can even detect FTO at the single-cell level.Moreover,this method can accurately distinguish the expression of FTO in human breast tissue between breast cancer patients and healthy person.It can be applied for the screening of potential inhibitors,holding great potential in clinical diagnosis,biomedical research,and drug discovery.2、We develop a new gold nanoparticle（Au NP）-based single-molecule biosensor for simple and sensitive detection of Ago2 activity.Argonaute 2（Ago2）is an essential component of RNA-induced silencing complex（RISC）and it participates in diverse physiological processes,while the dysregulation of Ago2 activity is closely linked to a variety of human diseases including cancers.The reported Ago2 assays often suffer from laborious procedures,complicated reaction scheme,and unsatisfactory sensitivity.Herein,we constructed an Ago2-responsive Au NP nanoprobe through the self-assembly of multiple Cy5-labeled signal probes onto the Au NP,in which the Cy5fluorescence is efficiently quenched by Au NP.Target Ago2 can bind with guide RNA to form an active RISC,resulting in cyclically cleavage of signal probes and the releasen of Cy5 moieties from the Au NP nanoprobe.The released Cy5 molecules can be quantified by single-molecule counting.The proposed biosensor enables homogenous detection of Ago2 activity with the involvement of only single Au NP nanoprobe,eliminating the use of any antibodies and protein enzymes.This single-molecule biosensor achieves good specificity and high sensitivity with a detection limit of 9.10×10^-12 M,and it can be exploited for the screening of Ago2 inhibitor,analysis of Ago2 kinetic parameters,and measurement of endogenous Ago2 activity in human cells,holding great promise in Ago2-related biomedical researches and clinical diagnosis.