Activatable T7 RNA Polymerase-based Sensor For Protease Activity Detection

Protease is widely found in bacteria,plants,animals and other organisms,and is involved in different physiological and pathological processes.Development of protease activity assays with high specificity and sensitivity,and fast response is important for the study of their biological functions,and c an provide powerful methodological support for diagnosis and treatment of the related diseases.Nucleic acids have the advantages of easy being edited,modificated,and amplificated,which make them widely used in the field of biosensing.In particular,fl exible nucleic acid signal amplification methods,such as strand displacement,rolling circle amplification,hybridization chain reaction,etc.,provide great solution for highly sensitive detection.Here,we developed a novel sensing method for protease activity detection based on a activatable T7 RNA polymerase,where the signal of protease detection is converted to nucleic acid signal by T7 RNA polymerase,and an appropriate signal output mode is designed as needed.T7 lysozyme,a natural inhibitor of T7 RNA polymerase,was fused with T7 RNA polymerase using a flexible linker containing a protease substrate sequence.Normally,T7 lysozyme is close to T7 RNA polymerase,and the transcriptional activity of T7 RNA polymerase is inhibited.In the presence of the target protease,the substrate sequence is specifically recognized and cleaved.Subsequently,T7 lysozyme is separated from T7 RNA polymerase,and restores the transcriptional activity.Then,the mRNA can be transcribed and detected.The main contents of the study are as follows:?1?Cloning,expression,and characterization of LP_Thrombin:through molecular cloning,prokaryotic expression and purification,a protease-responsive recombinant protein LP_Thrombin?T7 Lysozyme-linker-T7 RNA Polymerase,where the linker contains the cleavage sequence of thrombin?was obtained.After being hydrolyzed by the target protease,the transcriptional activity of T7 RNA polymerase is recovered.?2?Detection of thrombin activity based on LP_Thrombin:we design a chain substitution reaction as a signal output of the sensor for thrombin activity sensing.It allows the RNA transcribed from T7 RNA polymerase to generate a fluorescent signal.When thrombin is presented,T7 RNA polymerase activity is restored,and transcription of the target RNA is initiated,followed by strand displacement reaction.The RNA separates the DNA strand labeled with a fluorescent group from the DNA strand labeled with a quenching group,and the fluorophore is recovered.After optimization of a series of experimental conditions,we successfully simplified the three-steps process of digestion,transcription,and strand substitution into"one pot method"reaction,and the detection was completed within 1 hour,with a good linearity?0.5-100 nM?,a low detection limit of 0.5 nM,and good specificity.?3?Development of the recombinant protein LP_MMP-2 for MMP-2 activity detection:to verify the universality of protease-sensing methods for activated T7RNA polymerase,the linker portion of the recombinant protein was redesigned with a substrate sequence of MMP-2 to obtain LP_MMP-2.We verified the good response of LP_MMP-2 to MMP-2 through fluorescence experiment.Thus,it indicates that the method has good potential and versatility for measurement of differe nt kinds of protease activities.