Regulation And Imaging Detection Of Circular RNAs Using A Dual-functional Nanocomplex

Circular RNA(circular RNA)is a kind of non-coding RNA without 5' cap and 3'poly(A)end.Compared with linear m RNA,circular RNA has a stable structure and is not easily degraded by RNA hydrolase.The in vivo expression of circular RNA is tissue-specific and plays an important regulatory role in the process of life activities.Studies have shown that the dysregulated expression of some specific circular RNAs in cancer cells can accelerate the proliferation and metastasis of cancer cells and increase the difficulty of tumor treatment.Therefore,regulating the expression of circular RNAs has become one of the current research hotspots in the study of assisting tumor therapy.At present,technologies for regulation(such as si RNA interference)and characterization(such as quantitative PCR)of circular RNAs generally suffer from disadvantages such as high cost,poor stability,cumbersome and time-consuming,and have not been widely used.Aiming at the above bottlenecks in circular RNA research,this paper established a low-cost,stable,simple and efficient method by constructing a class of bifunctional nanocomplexes,which circular RNAs can be regulated and visualized real-time monitoring of circular RNA(CDR1as)function by live cell imaging.The main research contents and achievements are summarized as follows:1.Designing DNAzymes for regulation of intracellular CDR1asCDR1 antisense(CDR1as)is a Circular RNA that is abundantly expressed in the mammalian brain.It has more than 70 potential binding sites for mi R-7 in its sequence,and is associated with many diseases such as neurological diseases and some cancers such as gastric cancer,breast Cancer,etc.This part of the work designed 10-23 DNAzyme and 8-17 DNAzyme for specific cleavage of CDR1 as.RNA-DNAzyme reactions were analyzed by polyacrylamide gel electrophoresis(PAGE)and agarose gel electrophoresis(Agarose).The results show that the designed DNAzyme can effectively cleave the target substrate in vitro.In order to improve the cutting efficiency,the effect of combined cutting of multiple DNAzyme was explored.The results show that the combined action of multiple DNAzyme can effectively improve the cleavage efficiency,but it is related to the position of the target cleavage site.At the same time,the work further quantitatively detected the expression level of CDR1 as in cells after DNAzyme acted on cells by q RT-PCR.The results showed that the cleavage efficiency was the highest when DNAzyme 1 at the splice junction site of CDR1 as was combined with DNAzyme 2 near the splice junction site.The results of exploring the biological function of CDR1 as showed that CDR1 as could affect the expression of downstream m RNA of mi R-7.2.Construction of nanomaterial-loaded DNAzyme as an intracellular CDR1 as regulatory platformZIF-8 is a porous crystalline nanomaterial that self-assembled with zinc ions coordinated with 2-methylimidazole.It has the advantages of large specific surface area,high porosity,convenient synthesis,controllable size,good biocompatibility,and has outstanding advantages in the encapsulation and transportation of functional substances.In this part of the work,DNAzyme was loaded on the surface of ZIF-8 by electrostatic adsorption to synthesize nanocomposite DNAzyme@ZIF-8.SEM,DLS,and Zeta characterizations proved that the synthesized nanocomposites had uniform particle size and good dispersibility.MTT cytotoxicity experiments proved that the composite material has good biocompatibility.Laser confocal microscope observation showed that the DNAzyme@ZIF-8 complex had good cellular uptake,and the DNAzyme could be released by acidolysis after entering the lysosome for a period of time.q RT-PCR showed that DNAzyme@ZIF-8 could effectively inhibit the expression level of CDR1 as and the expression of downstream genes of mi R-7 after acting on U87 MG cells overexpressing CDR1 as.3.Construction of bifunctional nanocomplexes to realize the regulation and monitoring of circular RNAs in living cellsThere are more than 70 potential binding sites for mi R-7 on CDR1 as,and the expression changes of CDR1 as can be indirectly monitored by fluorescence detection of changes in mi R-7 levels before and after CDR1 as regulation.This part of the work designed a molecular beacon(MB)to detect the changes in the content of mi R-7.Both extracellular and intracellular experiments demonstrated that the designed MB could recognize mi R-7 and its fluorescence was enhanced with the increase of mi R-7concentration.Next,a bifunctional nanocomplex(DNAzyme/MB@ZIF-8)was constructed and prepared,and its intracellular function was investigated.The results showed that the bifunctional nanocomposite could inhibit the expression of CDR1 as in living cells,and at the same time,the content of free mi R-7 in the cells was detected to increase,indicating that the inhibition of CDR1 as resulted in the release of mi R-7,constructing a system to monitor the expression changes of CDR1 as through changes of mi R-7,explaining the mechanism by which CDR1 as affects the expression of downstream m RNAs.and enriched the Circular RNA-mi R-7-m RNA regulatory network system.We have also demonstrated the method and cell universality of the bifunctional nanocomplexes in multiple cell systems.