Novel Methods For Protease Activity Detection And Nucleic Acid Transport Based On GFP

Proteases are ubiquitous in organisms and play an important role in various life activities.The detection of proteases is of great significance both for disease diagnosis and therapy.Gene therapy achieves precise medical treatment by introducing foreign genes into cells.Gene therapy is mainly aimed at a number of serious diseases that threaten human health,including genetic diseases,cancer,infectious diseases?such as AIDS?.Currently,one of the major challenges in gene therapy is the design of safe and efficient vectors for gene delivery,which enables targeted delivery of nucleic acids.Based on the fact that the imidazole group in the side chain of histidine can bind to metal ions and is charge tunable by pH,new analytical sensing methods for protease activity detection were developed and intelligent multi-responsive delivery of nucleic acids was realized,utilizing H₃₉GFP with polyhistidine on the surface and GFP containing an N-terminus His-tag.The main research contents are as follows:?1?A new analytical method for the detection of alkaline phosphatase activity was constructed based on H₃₉GFP.Histidine has the ability to coordinate with metal ions,and the polyhistidine on the surface of H₃₉GFP makes it high affinity with Cu²⁺.At the same time,Cu²⁺is a common quencher that can effectively quench the fluorescence of H₃₉GFP.Pyrophosphate?PPi?can combine with Cu²⁺to form complexes,which allows fluorescence recovery of H₃₉GFP.Thereby the detection of PPi can be realized.As a substrate of alkaline phosphatase?ALP?,PPi can be hydrolyzed by ALP to generate phosphate and lose its ability to bind to Cu²⁺.Therefore,this method can be used for detection of ALP activity and analysis of its inhibitors.The detection limit of this method is 0.08 U/L with a linear range of 0.1–250 U/L.The autofluorescence of H₃₉GFP makes this method with the advantages of labele-free and simple operation.?2?RNAi was realized by using H₃₉GFP to intelligently deliver siRNA into cells.Our previous work showed that with either H⁺or Ni²⁺,H₃₉GFP can be super-positivly charged and carry nucleic acids into cells as a vector through electrostatic interaction.To further improve that the delivered nucleic acid is functional,H₃₉GFP delivery with siRNA as the cargo was carried out.It was found that with the help of H₃₉GFP,siRNA can be uptaken by Hela cells,and effective RNA interference with the target protein was achieved on both mRNA and protein level.Given the weakly acidic nature of the cancer tissue microenvironment,H₃₉GFP is expected to serve as an effective vector for the intelligent delivery of functional biological macromolecules and further for cancer therapy.?3?We explored the interaction between hemin and GFP with a His-tag at the N-terminus and developed a new detection method for thrombin activity based on GFP/hemin complex.Because the His-tag can coordinate with transition metal atoms and hemin has a Fe atom in the porphyrin ring structure,hemin can bind to His-tag.Meanwhile,hemin acts as a good electron acceptor so it can quench the fluorescence of GFP in a photo-induced electron transfer manner.We successfully apply the GFP/hemin complex to the detection of thrombin activity based on the thrombin cleavage site between GFP and hemin.