Construction Of PUF Protein Reporter Probe And Application In Imaging MiRNA/siRNA In Vivo

Non-coding RNAs are a class of RNA molecules that are widely found in eukaryotes and prokaryotes which are not translated into proteins.They can regulate expression at the gene and chromosomal levels and play an important role in epigenetic modification.Mi RNA and siRNA,as typical and widely studied short-chain small molecules of non-coding RNAs with regulatory roles,either as emerging biomarkers or through RNA interference technology,influence and control a variety of cellular processes and metabolic pathways in all aspects.The functional diversity of non-coding RNAs suggests that changes in their expression levels are also of great significance in physiological and pathological processes.At present,the research on non-coding RNA quantification mainly focuses on conventional detection methods,such as RT-PCR,RNA sequencing,Northern blotting,fluorescence in situ hybridization and other technologies.These methods may cause damage and injury to tissues,animals and plants Moreover,scientific research and clinical practice have put forward new requirements for the detection of non-coding RNA.Dynamic real-time reflection of non-coding RNA content is necessary to make timely and effective evaluation of the stages in the development of physiology and pathology.Reporter gene systems are highly programmable systems that encode an enzyme or protein which can be easily detected.The signal can be directly detected or act on appropriate ligands after fusion with a target gene.As an alternative platform for gene expression sensing drive devices,there are new breakthroughs and applications in biotechnology,basic research and clinical medicine.In this manuscript,we coupled the increased abundance of miRNA or siRNA with targeted gene expression events to dynamically and non-invasively monitor the expression changes of noncoding RNAs in vitro and in vivo,by designing and developing reporter gene system to provide a non-invasive visualization detection method for the quantitative monitoring of non-coding RNAs.The main research work of this paper is as follows:1.PUF/N-miR reporter probes monitor the expression of specific miRNA during neural differentiation.Based on the property of PUF protein bind specifically to its target NRE sequences to repress downstream gene expression,we designed and developed PUF/N-miR reporter probes to dynamically and noninvasively monitor the expression of specific miRNA-124 a and miRNA-9 during the process of neural differentiation.The probes were named PUF/miR-124 a and PUF/miR-9 reporter probes,respectively.A series of biological elements are connected to form a new vector by molecular cloning.When the miRNA of interest is not expressed or the content is low in the cell,the PUF protein binds to the 6×NRE sequence and inhibits the expression of the downstream output module Fluc protein.In contrast,when the miRNA of interest is expressed in cells,the binding of the miRNA to the 4× miR target sequence inhibited the expression of the PUF protein.Binding of PUF protein to NRE does not occur,thereby releasing inhibition of the output module luciferase.Changes in the expression and content of miRNA molecules were visualized by induced activation of the luciferase gene.The specificity of PUF protein binding to NRE and the specificity and sensitivity of miRNA were evaluated by bioluminescence in vitro,both probes showed dose-dependent responses to target miRNA only,and the detection limit was as low as 1.25 n M.Real-time dynamic monitoring of neural differentiation specific miRNA expression was realized at the cellular level and in vivo with living animals.2.Application of PUF/M-miR reporter gene probes in monitoring muscle differentiation specific miRNA and muscle injury model.After the dynamic monitoring of neural differentiation-related miRNA expression using the designed reporter system in the previous part of the study,the probe was further used to explore its performance in detecting muscle-specific miRNA.The original PUF/N-miR system was modified by molecular cloning,and its miR target was replaced by the target of muscle-associated miRNA.The PUF/M-miR reporter systems were constructed,which named PUF/miR-133 a and PUF/miR-1,respectively.The feasibility and sensitivity of PUF/M-miR reporter probes in sensing miR-133 a and miR-1 were confirmed by bioluminescence imaging in vitro,and the probes could detect miRNA expression at concentrations as low as 1.25 n M.The miRNA expression was monitored dynamically in real time in a successfully induced cellular muscle differentiation model,and the process was visualized using bioluminescence imaging in an animal subcutaneous implantation model.Using this reporter probe,we visually demonstrated the development of mice disease in an animal model of muscle injury in vivo,and the results were consistent with the gold-standard q RT-PCR technique for miRNA detection.The reliability and feasibility of the reporter probe applied to the diagnosis of related diseases was confirmed.3.Application of PUF/siRNA reporter probes image of siRNA delivery and silencing efficiency in vivo.After the successfully molecular imaging of miRNA expression by using the reporter probes in the previous two chapters,it was further explored whether another class of non-coding RNA small molecule siRNAs could be detected using similar principles.Using molecular cloning,the PUF protein in the original vector is fused with the target gene of interest,the original miR targets biological element were removed,and a new reporter gene system PUF/siRNA was constructed to detect the delivery and silencing efficiency of the target gene of interest siRNA.PUF/EGFP and PUF/p53 reporter probes were constructed to detect silencing targeting EGFP and p53 proteins,respectively.The toxicity of the reporter probes was examined using CCK-8 in vitro,and the cell viability was maintained above 80%,which confirmed that it did not have any effect on cell growth.The specificity and detection limits of the constructed PUF/EGFP and PUF/p53 reporter probe were evaluated by bioluminescence,both reporter probes could detect siRNA delivery as low as 2.5 n M.Optical imaging of siRNA delivery has been achieved in both subcutaneous cell models and liver depth imaging in live animals.Finally,the silencing efficiency of siRNA delivered by the currently mature commercial transfection reagents was compared using the developed PUF/EGFP reporter probe,the silencing efficiency of these transfection reagents ranged from 1.7 to 11.7 fold,and all of them could deliver siRNA to silencing target genes to different degrees.Different targets of siRNA against p53 protein coding region were also screened using the PUF/p53 reporter probe in vitro and in vivo.In conclusion,based on PUF protein,this work developed the reporter gene systems PUF/miR and PUF/siRNA for miRNA expression and siRNA delivery detection,respectively.The performance of these two types of reporter probes in imaging neural differentiation,muscle differentiation specific miRNA expression and siRNA silencing efficiency in vitro and in vivo was investigated,and the application in muscle-related diseases and practical research was preliminary explored.This study provides a theoretical and experimental basis for the development of a pervasive non-invasive dynamic monitoring platform for non-coding RNA expression delivery efficiency.