Molecular Mechanism Of Small Fragment Circular DNA Enhancing CGAS-STING Signaling Pathway Activation

Innate immunity is the natural defense of the body.Microbial infection is recognized by the innate immune system through pattern recognition receptors(PRRs)encoded by germ cells.Because most microbial pathogens contain DNA and/or RNA during their life cycle,nucleic acid induction has become an important strategy in host innate immune defense.cGAS-STING pathway is known to be a major signaling pathway that senses cytoplasmic DNA and activates natural immune signals.cGAS can recognize a variety of linear DNA in the cytoplasm from viruses,apoptosis,mitochondrial DNA,and exosomes.After recognizing linear DNA,cGAS forms phase separation and catalyzes AMP and GMP reactions to generate the second messenger cGAMP,which then induces the formation of STING-TBK1-IRF3 complex located in endoplasmic ream,resulting in phosphorylation of IRF3 into the nucleus.At this time,STING transfers to the Golgi body and palmitoylation occurs.TBK1 phosphorylates STING,which in turn recruits IRF3-mediated TBK1 phosphorylation,at which point IRF3 enters the nucleus and activates transcriptional expression of interferon and other immune stimulators.Therefore,the key step of this pathway lies in the recognition of ds DNA by cGAS.At the same time,there is also a negative feedback regulatory mechanism in cells.TREX1 is an exonuclease,which can degrade DNA in the cytoplasm and thus negatively regulate the cGAS-STING pathway.Recently,it has been reported that cGAS is separated from DNA formation,thus mediating the catalytic function of cGAS.In the cell,there is not only linear DNA,but also extra-chromosome circular DNA,one is a large fragment of circular DNA with transcription elements,and the other is a small fragment of circular DNA,and it has been shown that ecc DNA mainly distributed in human tissue and pregnant women’s plasma is mainly a small fragment of circular DNA.In previous studies,most thought that the primary stimulator of the innate immune system to activate cells was double strand DNAs(ds DNA),but now what is interesting is that the latest research shows that small fragments of circular DNA are produced in the process of apoptosis.Such DNA induces a stronger innate immune response than linear DNA,but the mechanism of action is unclear.Therefore,the purpose of this study is to explore the molecular mechanism of small fragment circular DNA enhancing the activation of cGAS-STING signaling pathway,so as to provide a deeper insight for a better understanding of the functional mechanism of ecc DNA.Through the study,we confirmed the enhanced effect of ecc DNA on the activation of cGAS-STING pathway.The effect of different size ecc DNA was compared and analyzed.It was found that 200 bp circular DNA could cause more obvious innate immune response.The transfection efficiency and degradation efficiency of linear DNA and circular DNA were compared and analyzed.It was found that there was no significant difference in the transfection efficiency and short time degradation efficiency of linear DNA and circular DNA.At the same time,the affinity of linear DNA and circular DNA with cGAS was compared,and there was no significant difference.The phase separation ability of linear and annular induced cGAS was compared,and it was found that this aspect may be the main influence of circular DNA.At the same time,we are further verifying the function of circular DNA specific interacting proteins by MS analysis.