Screening Of G-quadruplex In Vitro By Rolling Circle Amplification

Objective:In addition to the classical B-type configuration,the secondary structure of DNA has many non-classical configurations,such as G-quadruplex(G4)structure,i-motif,etc.G4 structure is often considered to be related to telomere regulation,DNA replication and gene expression regulation.Identification and detection of the secondary structure of DNA is the premise of analyzing its structure and function,especially the identification and detection of the advanced structure of DNA in living cells is of great significance in the innovative diagnosis and treatment of major diseases and the development of new drugs.At present,G4 structure identification technology mainly includes biophysics and biology.The principles of these two methods are similar.The former identifies G4 sequence by recognizing the spatial structure of G4,and the latter identifies G4 sequence by using the biological characteristics of G4 structure.Traditional detection methods rely on large instruments,which are time-consuming and cumbersome.It can be seen that the common detection methods of G4 structure still have technical bottlenecks.Based on the existing detection methods of G4,we innovatively propose a new method for screening G4 in vitro,which aims to provide theoretical basis and experimental support for the "screening and identification of intracellular G4 structure" that needs to be solved at present,and provide a new research idea for the screening and identification of other types of nonclassical DNA secondary structure in cells.Methods:Using the classical G4 structure TBA as the model,two circle templates were prepared by using the locking probe in the rolling circle amplification technology.One circle template contains TBA sequence,and the other circle template contains TBA complementary sequence.The long single strand was cyclized by T4 DNA ligase,and then purified by exonuclease III.The effect of different concentrations of TMPy P4 on G4 structure was characterized by polyacrylamide gel electrophoresis.The effects of different concentrations of TMPy P4 on the RCA reaction system were discussed,and the appropriate concentration of TMPy P4 was selected for the screening of G4 structure.Due to the different rigid structures of linear template and circle template,the differences of G4 structure formed under three different conditions in the screening process were compared using the appropriate TMPy P4 concentration selected above.The first is that the linear template first forms the structure with TMPy P4,then cyclization and purification;The second is the cyclization of the linear template and TMPy P4 edge formation structure,and then purification;The third is that the linear template is cyclized and purified to form a circle template,and then forms a structure with TMPy P4.Use the circle template obtained above,add primer for rolling circle amplification,detect the fluorescence signal through the enzyme marker,and distinguish the formation of G4 structure according to the presence or absence of the signal,so as to achieve the purpose of screening G4.Results :1.The circle template constructed by the locking probe can be used as the template for the experiment after cyclization and purification.2.Polypropylene gel electrophoresis showed that when the concentration ratio of G4 and TMPy P4 was 1:5,1:10,1:20,1:50,there was obvious G4 structure formation.3.When the concentration ratio of G4 to TMPy P4 is 1:5,1:10 and 1:20,there is no effect on the RCA reaction system.When the concentration ratio of G4 to TMPy P4 is 1:50,there is an effect on the RCA reaction system.4.After incubating with linear template,TMPy P4 will reduce the stability of the template and the connection efficiency of the template;5.TMPy P4 will reduce the efficiency of T4 DNA ligase;6.The linear template is cyclized and purified to form a circle template,and then forms a structure with TMPy P4.This method is more conducive to the screening of G4 structure;When the concentration ratio of G4 to TMPy P4 is 1:20,the formed G4 structure can completely block the movement of DNA polymerase with chain displacement,thus realizing the screening of G4 in vitro.7.When the G4 structure is formed after the circle connection for screening,the optimal time for incubation of the circle template with TMPy P4 is 1 h.8.When the concentration ratio of G-containing sequence to TMPy P4 is 1:20,the G4 formed can completely block the movement of phi 29 DNA polymerase(P<0.05)and realize the screening of G4 in vitro.Conclusions:This study established a new method for in vitro screening of G4 based on rolling circle amplification technology.In this study,the formation of G4 structure after circle connection can achieve in vitro screening of G4;When screening,the concentration ratio of the circle template to TMPy P4 was 1:20 and incubated at 25 ℃ for1 hour;The G4 formed can completely block the movement of phi 29 DNA polymerase and realize the screening of G4 in vitro.