Construction And Targeted Detection Of Rolling Circle Amplification-based Poly-molecular Beacon

Molecular Beacon（MB）is a kind of hairpin fluorescent probe,which has been widely used in the recognition of proteins and antisense oligonucleotides.However,most MB/protein interactions are nonspecific and interfere with selective target binding.And nucleic acid aptamer can be based on the unique three-dimensional conformation to identify specific targets.By introducing the aptamer into the molecular beacon,the shortcoming of MB’s inability to specifically identify the target is solved.As a result,MB has been widely used in the detection of proteins and peptides,m RNA monitoring,real-time imaging of living cells,etc.,which improves its advantage in the identification and detection of biomolecules in the fields of chemistry,biology and medicine,and has a potential application prospect in the field of disease diagnosis and treatment.However,the detection sensitivity of simple MB is not high,and the signal amplification method is usually needed to improve the sensitivity of the detection target.As an isothermal enzymatic reaction,rolling circle amplification technology（RCA）can reach hundreds or thousands of bases in molecular weight,and they are all repeated base sequences.When this technology is introduced into the structure construction of MB,hundreds or thousands of repeated detection units can be obtained,so as to achieve signal amplification and improve detection sensitivity.According to the characteristics of the MB and aptamer,this paper first constructs three different lock-key structure of single molecular beacon.Then rolling circle circle amplification technology is introduced into the lock-key molecular beacon,which can construct a poly-molecular beacon with multiple repeated detection units.Due to the poly-molecular beacons are composed of multiple repeating single structure of MB,the fluorescence signal of poly-molecular beacons are significantly enhanced compared with single MB,which can be used to detect thrombin and cancer cells.The specific content is as follows:1.Construction of aptamer-based molecular beacons with varied lock-key structures and targeted detection of thrombinThe binding-induced fluorescence turn-on assays using aptamer-functionalized molecular beacons（MBs）were designed.Three molecular beacons named MB（8+8）,MB（15+8）and MB（15+6）were consisted of two single-stranded oligonucleotides.One long single-stranded oligonucleotide（abbreviated as SS）contained thrombin aptamer sequence and modified with fluorescence group and quenching group on each end side.Another short single-stranded oligonucleotide（written as c DNA）was partially complementary to the long SS.Gel electrophoresis and fluorescence spectra showed that MB（8+8）was complementary by one SS and one c DNA（8+8）,which called one-to-one combination.While MB（15+8）was two-to-two combination and MB（15+6）was one-to-two combination.MB（8+8）and MB（15+8）could form the quenching molecular beacon,while MB（15+6）could not.Once they encountered thrombins,the G-quadruplex structures would form and lead to the collapse of the complementary of SS and c DNA,then the MBs were turned on and fluorescence signal was released.Under optimized experimental conditions,the detection limit of MB（8+8）for thrombin was 0.19 n M,and the detection limit of MB（15+8）was 1.2 n M.Due to the introduction of the aptamer,the molecular beacon was selective for thrombin,and other tested proteins did not interfere with thrombin detection.2.Construction of poly-molecular beacons MB_RCA/Thr（8+8）and targeted detection of thrombinIn this part,a locked-poly molecular beacon MB_RCA/Thr（8+8）based on rolling loop amplification was constructed,which consisted of three oligonucleotide chains.One was the product chain of RCA,with multiple repeats and up to thousands of bases in length.One was an oligonucleotide chain(abbreviated as SS_Thr（8+8）)containing thrombin aptamer,both ends of which were modified with FAM and DABCYL groups respectively,and each end has 8bases complementary paired with part of the sequence base of the repeating unit in the long chain of RCA.There was also a fixed short chain,referred to as FC-43,which complemented the 43 base sequences in a repeating unit of the RCA long chain and played a role in strengthening the rigidity of RCA.Fluorescence quenched poly-molecular beacon MB_RCA/Thr（8+8）can be formed by hybridization and pairing of the three chains.When MB_RCA/Thr（8+8）encountered thrombins,the aptamer in SS_Thr（8+8）would specifically bind to thrombins,resulting in the destruction of the poly-molecular beacon structure.Then the FAM and DABCYL re-separated,and the release of fluorescence signals to realize the detection of thrombin.In this part,the influence of the construction method and the ratio of RCA/SS_Thr（8+8）/FC-43 on the construction of MB_RCA/Thr（8+8）was explored,and the differences of the construction and detection of thrombin between the single-molecular beacon MB_{c DNA-63/Thr}（8+8）and the poly-molecular beacon MB_RCA/Thr（8+8）were compared.The experimental results showed that the two-step annealing procedure was beneficial to the construction of MB_RCA/Thr（8+8）.The optimum ratio of RCA to FC-43 was 1:120.And the optimum ratio of RCA to SS_Thr（8+8）was 1:3.The optimal ratio of short c DNA-63（the same as one repeat of RCA）to SS_Thr（8+8）was 30:1 for the construction of a single-molecular beacon MB_{c DNA-63/Thr}（8+8）.The binding efficiency of RCA to SS_Thr（8+8）was 11.75 times higher than that of c DNA-63 to SS_Thr（8+8）,and the effect of RCA was significantly better than that of c DNA-63.In addition,the detection limit of MB_RCA/Thr（8+8）for thrombin detection was 0.1 n M and the fluorescence signal was high,while MB_{c DNA-63/Thr}（8+8）for thrombin detection was not linear and the fluorescence signal was low,which reflected the superiority of RCA technology.3.Construction of poly-molecular beacons MB_RCA/Thr（12+8）and targeted detection of thrombinIn the previous system,the binding efficiency of SS_Thr（8+8）and RCA was low.It was speculated that the base number of complementary pairs between SS_Thr（8+8）and RCA was too few.Therefore,a main chain was redesigned in this part,which was called SS_Thr（12+8）,that was,four bases paired with RCA were added at the DABCYL group end.The corresponding fixed short chains were designed as two（FC1-12,FC2-31）,and the poly-molecular beacon MB_RCA/Thr（12+8）was constructed by hybridization and pair of four chains.In this part,the influence of the ratio of RCA/SS_Thr（12+8）/（FC1-12,FC2-31）on the construction of MB_RCA/Thr（12+8）was explored,and the differences of the construction and detection of thrombin between the single-molecular beacon MB_{c DNA-63/Thr}（8+8）and the poly-molecular beacon MB_RCA/Thr（8+8）were compared.The experimental results showed that the optimum ratio of RCA to two fixed short chains was 1:100:100,and the optimum ratio of RCA to SS_Thr（12+8）was 1:20.Compared with the optimum ratio of 1:3 of the previous system,the binding efficiency was significantly improved.The optimal ratio of short c DNA-63 to SS_Thr（12+8）was 1:1 for the construction of single molecular beacon MB_{c DNA-63/Thr}（12+8）.The binding efficiency of RCA was significantly better than that of c DNA-63.In addition,the detection limit of MB_RCA/Thr（12+8）for thrombin detection was0.13 n M,while MB_{c DNA-63/Thr}（12+8）for thrombin detection was not linear and had a low fluorescence signal,thus reflecting the advantages of poly-molecular beacon MB_RCA/Thr（12+8）.4.Construction of poly-molecular beacons MB_RCA/MUC1（12+8）and targeted detection of cancer cellsOn the basis of the third part,a poly-molecular beacon MB_RCA/MUC1（12+8）that can specifically detect cancer cells was constructed,which provided a new idea and method for the early diagnosis and treatment of cancer.MB_RCA/MUC1（12+8）consisted of three oligonucleotide chains,one of which was the RCA product chain.An oligonucleotide chain(abbreviated as SS_MUC1（12+8）)containing the S2.2 aptamer sequence that specifically targeted MUC1 protein,both ends of which were modified with FAM and DABCYL groups respectively,with 12 and 8 bases complementary to RCA,respectively.The other fixed short chain FC-41,was complementarily paired with 41 bases of one repeat of RCA.Since MUC1protein was overexpressed on the surface of the cell membranes of breast cancer cells（MCF-7）and cervical cancer cells（Hela）,when MB_RCA/MUC1（12+8）meet MCF-7 and He La cells,the S2.2 aptamer sequence in MB_RCA/MUC1（12+8）will specifically bind to the MUC1protein on the surface of cancer cells,destroyed the structure of MB_RCA/MUC1（12+8）,and enhance the fluorescence signal.In this part,exploring the influence of the ratio between RCA/SS_MUC1（12+8）/FC-41 on the construction of MB_RCA/MUC1（12+8）,discussing the specificity of MB_RCA/MUC1（12+8）for detection of cancer cells,and comparing the ability of single-molecular beacon MB_{c DNA-63/MUC1}（12+8）and poly-molecular beacon MB_RCA/MUC1（12+8）for detection of cancer cells.Fluorescence spectra showed that the best ratio of RCA to FC-41 was 1:100,and the best ratio of RCA to SS_MUC1（12+8）was 1:20.MB_RCA/MUC1（12+8）can specifically identify MCF-7 and He La cells,but had a lower effect on fibroblasts（L929）.Compared with the single molecule beacon MB_{c DNA-63/MUC1}（12+8）,the fluorescent signal of MB_RCA/MUC1（12+8）was stronger,indicating the superiority of signal amplification of MB_RCA/MUC1（12+8）.