In order to solve the problem of miRNA detection related to metabolic syndrome, we analyzed and compared nucleic acid detection systems such as CONAN and DSN. On this basis, we designed and optimized the DRJ system. In this year's project, our team designed 10 new parts around the DRJ system, 8 of which are basic parts and the rest are composite parts. Browse for details of each part on the AFMU_CHINA 2023 iGEM team part page, or click the part number below.

        Of all the basic parts, we are most satisfied with the probe_β(BBa_K4778008). It is the product of our data analysis and iterative optimization, completes the function of signal conversion, and can realize linear amplification and exponential amplification of the system at the same time. It is a powerful and stable component. Relying on the probe_β, our system accomplishes the detection of trace nucleic acids very well, and brings inspiration for the function of component and structure design of the detection system.

Part number	part
BBa_K4778002	Cas12a_sgRNA_D
BBa_K4778003	sgRNA_D
BBa_K4778004	assistant DNA_D
BBa_K4778005	ssDNA-F_D
BBa_K4778006	DSN
BBa_K4778007	probe-α
BBa_K4778008	probe-β
BBa_K4778009	Circulization Primer

        Our composite parts are designed for signal conversion. scgRNA-F can not only display the fluorescence signal, but also amplify the signal exponentially. Its design is inspired by CONAN system. After cross-linking, colloidal gold particles mainly makes nucleic acid signals visible, which expand the application scope and scene of DRJ system.

part number	part
BBa_K4778013	Colloidal Gold Particles (after crosslinking)
BBa_K4778015	scgRNA-F

        Our detection system is composed of Cas12a_sgRNA_D, assistant DNA_D, ssDNA-F_D, probe-β_CP, DSN .And our system skillfully integrates the above components.If the microRNA target is present, it will hybridize to the probe. And duplex-specific nuclease (DSN) will cleave DNA in DNA-RNA hybrid duplex, re-releasing the microRNA and a complete sgRNA sequence will be released. The released microRNA can bind to a new probe for a new round of reaction. The released sgRNA sequence then self-folds to form a functional sgRNA.
        Then, the released sgRNA binds to the Cas12a protein, and then recruits the assistant DNA (later called aDNA), and Cas protein cleaves the aDNA with its trans-cleavage activity. After cleaving, Cas12a-sgRNA-aDNA forms a stable ternary complex, giving Cas12a trans-cleaving activity (non-specific ssDNA cleavage). After the trans-activity of Cas12a is activated, the DNA sequence of the circular DNA-RNA chimeric probe will be cleaved to release a new sgRNA sequence. The sgRNA sequence will self-fold to form a mature sgRNA and the newly generated sgRNA can activate a new Cas12a. Through this process, cas12a amplifies exponentially. Activated cas12a will also cleave the ssDNA-F and let out fluorescence signals.
        In brief, our system utilizes the double-strand (ds) DNA cleavage activity of DSN enzymes to achieve linear signal amplification and DNA trans-cleavage activity of Cas12a to achieve exponential signal amplification.In a nutshell,we collect all the five parts and realize the trace detection of microRNA. -->