Development And Application Of A Novel BRET-based Live Imaging Technique For Apoptosis Detection

Apoptosis,also called programmed cell death,is an important bioprocess involved in normal tissue development and diseases.The R&D of new apoptosis detection techniques are always one of active fields in scientific research.Annexin V belongs to a family of phospholipid binding proteins which is able to bind to negatively charged phospholipids such as phosphatidylserine(PS)in the presence of a high affinity Ca²⁺ion.In normal cells,PS is only distributed inside the lipid bilayer of the cell membrane.When apoptosis occurs,even in the early stage,PS will be exposed to the outside of the cell surface from the cytoplasm side of membrane leaflets.Therefore Annexin V has been suggested as abio-marker for apoptosis detection for early apoptotic events.Although many fluorescent labeled Annexin V probes have been used in vitro to detect the occurrence of early cell apoptosis,the fluorescent probes themselves require excitation light,and are also usually phototoxic and easily interfered by the auto-fluorescence from the biologic samples,which may generate additional background.These fluorescent probes are not suitable for the study of deep tissue in vivo.We propose a new BRET(Bioluminescence Resonance Energy Transfer,BRET)-based Annexin V probe,which can be used for both fluorescent and bioluminescence apoptosis detection.The dual-signal function of the new sensor allows it to be used for conventional fluorescence-based in vitro cell apoptosis detection and bioluminescence-based live animal imaging to track apoptotic cells in vivo.Because bioluminescence signal has high sensitivity and almost zero background,the biosensor can evaluate the apoptosis and recovery process caused by diseases at in vivo level directly.Firstly,based on the principle of BRET,we constructed a fused apoptotic probe(Annexin V Probe,An VPb,containing Annexin V,m Neon Green fluorescent protein,and te Luciferase)and a control probe(mutant Annexin V Probe,m An VPb,containing E72Q/D144N/E228Q/D303N four sites mutanted Annexin V,m Neon Green fluorescent protein,and te Luciferase),respectively expressed in E.coli,and used Ni-NTA was subjected to affinity purification to obtain two fusion probe proteins.Subsequently,we measured the excitation and emission spectra of the constructed An VPb and its control probe m An VPb in a specific range.At the same time,we selected the best substrate diphenylterazine(DTZ)reported in the literature for te Luc,and measured the bioluminescence intensity of An VPb/m An VPb-DTZ pairing.As we expected,the emission spectrum of the probe was red-shifted,and the bioluminescence spectrum reached a peak at 517 nm.We characterized the spectrum properties of An VPb and m An VPb in the range of 350-650nm,and verified the good linear dose-dependent bioluminescence intensity of these probes.So far,we have obtained the ideal Annexin V probe and its control probe,which can be used for subsequent in vitro and in vivo apoptosis detection.Next,we treated with 200?M H₂O₂ for 12 hours to establish an oxidative stress injury model of glial cells(A172 cells),and induced apoptosis in vitro.Then,the constructed An VPb,m An VPb,commercial mitochondrial superoxide red fluorescent probe,or Annexin V-Alexa Fluor 647-propidium iodide apoptosis detection kit were used to co-stain the cells after H₂O₂ treatment.Fluorescence microscopy results show that:under the same oxidative stress stimulation level,An VPb can bind to PS to detect early apoptotic cells,while m An VPb cannot.After demonstrating the detection ability of Annexin V probe at the cellular level,we further established the in vivo model-the mouse acute kidney injury(AKI)model,which used the probe's targeted binding ability and small amount of apoptotic cells.Animal in vivo imaging technology assesses the apoptosis of cells in the AKI model at the in vivo level and then tracks the development of the lesion.The results of in vivo imaging in mice proved that the luminous intensity of the probe was sufficient to penetrate mammalian tissues to realize the visualization of lesions at the in vivo level.Next,we also established a mouse stroke model to evaluate cell apoptosis in the lesion area under the skull and track the evolution of lesions in different time courses at the same in vivo level.Using Image J to calculate the imaging gray levels of the focal region at different time courses,we found that in the acute phase of stroke,the apoptosis signal gradually increased,and then gradually reduced.By the 5th day,the apoptosis of the focal area had basically ended.This result once again proves that An VPb can effectively evaluate disease models at the in vivo level.We further used this technique to evaluate the neuroprotective effects of the mouse protein Serpina3n and its human homolog,SERPINA3,which have been screened in previous another project,and compared with the evaluation method of MRI.The results showed that the focal area(MRI)and apoptosis signal(small animal live imaging based on An VPb)in mice brain after Serpina3n or SERPINA3 treatment was significantly smaller and weaker than those in the control group.In summary,in order to develop a bioluminescent probe for detecting cell apoptosis and use it to assess the level of apoptosis in disease models at the in vivo level,we combined Annexin V with a BRET-based m Neon Green-te Luc fusion protein to construct An VPb,a highly sensitive and low background apoptosis biosensor.An VPb not only provides a simple and rapid method for detecting the occurrence of early apoptosis at the cellular level,but also provides a convenient and reliable method for tracking apoptosis and lesion evolution at the in vivo level.The successful construction of this method can provide help for the dynamic monitoring of the development of the disease and the in vivo screening and evaluation of new drugs.