| As the expression product of genetic information,protein is an important biological macromolecule,which undertakes multiple activities from cell structures to functions.By precisely regulating the expression level of specific proteins,the cellular protein functions or signaling pathways can be reprogrammed.At present,CRISPR-dCas9 and siRNAs have shown their capacity in accurate regulation of target genes through the design of relevant RNA sequences.Therefore,they play a significant role in the mechanism research and gene therapy of diseases caused by the disorder of gene expression.The common protein secondary structure a helix showed clear sequent-structure relationship.It has been widely utilized in biomaterials,drug delivery,protein self-assembly,and especially,modular regulation of protein interactions.In this paper,the construction of programmable CRISPR-dCas9 transcriptional activators mediated by peptides strand-displacement based on α-helical coiled coil was mainly investigated,as well as its applications in disease diagnosis and treatment with non-coding RNAs.With the continuous development of CRISPR-dCas9 transcriptional activation system in recent years,it has realized the efficient activation of target genes.However,higher activation efficiency was required in some special applications like cell reprogramming.Nuclear localization signals(NLS),which are short peptides guiding nuclear localization processes of proteins,have been widely used in the construction of CRISPR-dCas9 transcriptional activators.Nevertheless,its necessity and effects on transcriptional activation have not been studied yet.Therefore,in Chapter 2,by changing the position and number of SV40 NLS in dCas9-VPR activator,it was confirmed that both the position and number of NLS have a certain influence on the transcriptional activation level.Contrary to expectations,direct deletion of additional NLS in the system increased transcriptional activation efficiency by 2-4 times.Subsequently,this conclution was further verified by changing experimental conditions including targeted genes,cell lines,transfection dosage,cell division speed,NLS sequence,methods of component delivery and activator types,respectively.It indicated that NLS has a negative effect on CRISPR-dCas9-related transcription activators,and direct deletion of NLS can significantly improve the transcriptional activation efficiency.This study should be beneficial to the design of NLS based CRISPR-dCas9 transcription activators.DNA nanomolecular devices constructed based on toehold-mediated DNA strand-displacement reaction have been widely applied in the field of nanomolecular assembly and detection of biomarkers in vitro.However,the properties and functions of DNA limited the relevant applications in vivo.As a common protein secondary structure motif,α helix peptide can change into heterodimer by sequence design,which called coiled coil(CC)with similar structures and properties with double strand DNA.In Chapter 3,the peptide mediated dCas9-VPR transcriptional activator constructed in Chapter 2 was further designed and modified,for establishing the toehold-mediated peptide strand-displacement reaction,which was subsequently confirmed by imitating the construction strategy of DNA strand-displacement reaction.Then the impact of toehold properties on the strand-displacement efficiency was studied.Subsequently,the reliability of the above implementation conclusions was further verified by FRET system.This study established a new regulatory response element for protein interaction regulation and laid a solid foundation for building cellular complex signal networks.The characteristics of logical sensing element enable it to sense external stimulus signals and only respond to specific combinations of input signals.Additionally,a cellular physiological process is usually simultaneously regulated by multiple molecules.For example,transcription initiation is regulated by different transcription factors.Constructing of multi-input logic system in cells is meaningful to achieve precise regulation of cellular signaling pathway or physiological process.In Chapter 4,based on the peptide strand displacement reaction mediated programmable transcription system constructed in Chapter 3,multiple two-input logic gates such as AND,NAND,OR,NOR,XOR,and INHIBIT,and three-input cascaded logic gates(A OR B)AND C were successfully designed and constructed,respectively.Subsequently,a programmable activator that can simultaneously respond to multiple microRNAs was constructed by controlling the expression of INHIBIT gate with microRNA-mediated expression regulation.Finally,specific cell recognition sensors targeting HEK293T cells and HCT116 cells were designed according to the expression level of endogenous microRNAs in different cells,for the aim of achieving accurate detection of target cells.This study showed important implications for fields in protein logic computation,cell reprogramming and biomedicine.SiRNAs are a class of non-coding RNAs,which can be used to achieve targeted cleavage of the complementary mRNA by utilizing endogenous proteins of cells.They have shown important value in gene therapy in recent years.SARS-CoV-2 is a classical RNA virus,and its rapid worldwide spread seriously endangered human health.Therefore,in the study of Chapter 5,an effective prevention and treatment platform for SARS-CoV-2 was established by combining neutralization strategy and RNAi technology.In order to protect normal cells from infection,HEK293T cells were customized to stably express the antigen receptor of SARS-CoV-2 ACE2.The modified cells were used as bait to specifically trap the SARS-CoV-2 virus.The released viral genomic RNA is then targeted and broken down by cytoplasmic siRNA complementary to the viral gene fragment.The feasibility and effectiveness of this strategy were verified by using pseudovirus reconstructed from HIV lentivirus as virus model in vitro.This study provides an effective foundation for building general solutions for intractable RNA viruses in the future. |
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