Studies On Construction And Optimization Of Bioluminescence Resonance Energy Transfer System Based On Protein-DNA Interactions

With the development and progress of bioanalytical technology,biomedical analysis puts forward higher requirements for the detection and analysis of various targets.However,traditional analysis methods,such as the fluorescence analysis represented by fluorescence resonance energy transfer(FRET),suffer from the drawbacks of biological auto-fluorescence,photobleaching,phototoxicity,and co-excitation of donors,which largely influences the sensitivity and specificity of corresponding analytical methods.Therefore,there is an urgent requirement for the establishment of analytical methods with high sensitivity,specificity,convenience,low-costs,and generality.In this thesis,we designed a series of bioluminescence resonance energy transfer(BRET)systems by using protein structural elements which can specifically recognize the DNA molecules.Based on this design,the BRET systems proposed here have the potential to be compatible with the nucleic acid-based detection methods.The following works have been done in this thesis:1.Construction of DNA-templated BRET moduleBy respectively fusing zinc finger domain Zif-268 and AZP-4 to the Nanoluciferase(Nluc)and yellow-green fluorescent protein(mNeonGreen),we constructed a DNA-templated BRET signal module.In this module,the zinc finger domain-fused energy donor and acceptor could simultaneously binds to the same double-stranded DNA molecule with sequence specificity.This binding process brought the energy donor and acceptor into close proximity,which satisfied the critical distance required for the energy transfer,and therefore produced detectable BRET signals.The DNA-templated BRET module proposed here has the potential to be compatible with the nucleic acid-based detection methods,and provide foundation for design of novel BRET-based detection methods.2.The optimization of DNA-templated BRET signal moduleBased on the work mentioned above,we further investigated the influence of space orientation and distance between donor and acceptor on the efficiency of energy transfer,and finally optimized the key parameters of DNA-templated BRET module.By fusing Zif-268 and AZP-4 to the N-terminus and C-terminus of Nluc and mNeonGreen respectively,we obtained different donors and acceptors.By further pairing the donors and acceptors into different BRET modules,we found that the binding orientation and affinity of zinc finger domain to the DNA template plays an important role in the efficiency of energy transfer.Furthermore,the distance and molar ratio between energy donor and acceptor also have significant influences on the final efficiency of energy transfer.By analyzing the rules proposed above,the optimized configuration of BRET module with the highest energy transfer efficiency was concluded as:1)Energy donor: Zif-268 fused at N-terminus of Nluc;2)Energy acceptor: AZP-4 fused at C-terminus of mNeonGreen;3)The distance between donor and acceptor: 20 bp;4)The molar ratio of donor and acceptor: 1:10.3.Construction of DNA-templated BRET/FRET signal moduleBecause of emission spectral overlap between Nluc and mNeonGreen,the corresponding BRET systems have the problem of low signal to noise ratio.To solve this problem,we designed a DNA-templated BRET/FRET signal module.In this module,the Zif-268 domain,Nluc,and mNeonGreen were fused into a trimer protein.The binding of this trimer protein to the DNA molecules intercalated with red fluorescent dye BOBO-3 not only realized the BRET between Nluc and mNeonGreen but also realized the FRET between mNeonGreen and BOBO-3.Because of nearly neglectable spectrum overlap between Nluc,mNeonGreen,and BOBO-3,the signal to noise ratio can be significantly improved in the resulting DNA-templated BRET/FRET module.Unlike traditional FRET-based technology,the DNA-templated BRET/FRET module avoids the problems associated with the use of outer excitation light,such as auto-fluorescence,photobleaching and phototoxicity.4.Construction of transcription activator like effector(TALE)protein-based bioluminescence systemBecause of the drawbacks of zinc finger domain,such as short recognition sequences,low affinity,and poor maneuverability,we further constructed an novel protein/DNA bioluminescent system by using TALE protein as DNA recognition element.For this,we fused the N-and C-terminus of TALE protein to the Nluc,and finally constructed fusion protein with both of DNA recognition and bioluminescent properties by using Golden Gate cloning techniques.This work improves the compatibility of bioluminescence and DNA-based detection systems,and provides the foundation for future development of BRET-based detection methods.