Genetic Engineering Of RNA Activated Fluorescent Proteins And Multicolor Imaging Of RNA In Living Cells

The transformation from DNA to protein is a complex,multi-stage process centered on RNA.RNA transcription,splicing,localization,translation and degradation are highly coordinated and strictly regulated in space and time.Visualization technology of RNA in cells can help us to further study the metabolic process.Gene encoded RNA imaging systems mainly include two strategies,fluorescence protein labeling based RNA imaging method or fluorescence RNA aptamer based RNA imaging method.The RNA labeling method based on the fusion of RNA binding protein and fluorescent protein has achieved RNA imaging with single molecular resolution for the first time,but it also has the problem of high background caused by overexpression of fluorescent fusion protein.Emerging genetically encoded molecular tools have created useful platforms enabling high-contrast or low-background RNA imaging.Light-up RNA aptamers can specifically bind and turn on nonfluorescent small-molecule dyes,which solves the problem of high background of traditional methods.Target RNA can be genetically engineered with light-up RNA tags and tracked through activated fluorescence signals from the dye-aptamer complexes.Direct imaging of endogenous RNAs has been achieved using genetically engineered RNA sensors based on split or structure-switched light-up RNAs.An intrinsic limitation of light-up RNA approaches is that the dyes require exogenous additions for RNA imaging.Recently,a new concept of RNA aptamer stabilized fluorogenic protein has been developed using fluorescent proteins fused with a Tat peptide-based degron(t Deg).The degron-fused fluorescent proteins are rapidly degraded,but become stabilized and fluorescent when the degron binds to a Tat peptidebinding RNA aptamer.This fluorescent protein strategy is completely genetically encoded,without exogenous addition or high background,but requires genetic engineering of target RNAs via inserting degron-binding RNA motifs.In Chapter 2,we developed a new genetically encoded sensor using conformationswitching induced fluorogenic protein(csiFP),which enables direct imaging of endogenous RNAs.Fluorescent proteins that incorporate t Deg were rapidly degraded and controlled by the degron-binding RNA aptamer(deg Apt),we modulated the aptamer structure using target endogenous RNAs.We speculated that misfolding the stemloop conformation of deg Apt via sequence extension could abrogate its degron-binding ability.In the situation,target RNA hybridization can restore its active conformation to bind the degron,protecting the degron-fused fluorescent proteins from degradation.A high expression RNA sensing module was constructed using the Tornado system,and t Deg-fused EGFP was used as a protein reporting module.By the target RNAs induced stabilization of fluorogenic RNA-protein complexes,survivin m RNA was successfully quantified.It provides a reliable platform for quantitative detection and continuous realtime imaging of mRNA in mammalian cells.In Chapter 3,To expand the color palette of csiFP sensor for multicolor imaging applications,we explored the possibility of using fluorescent proteins with distinct colors as the reporter.The tandem of fluorescent proteins was challenging in plasmid construction.In order to achieve signal amplification of low abundance RNA,we used split GFP to construct a protein reporting module.We further engineered a series RNA sensing module,and the 24× signal amplifier showed the potential of single molecule imaging.The imaging detection and relative abundance analysis of c-myc m RNA and mi R-21 in living cells were realized by using csiFP sensor,while the imaging and kinetic tracking of lnc RNA MALAT-1 were performed.csiFP sensor could provide a new paradigm for highly sensitive,multicolor imaging of endogenous RNA in live cells and animals.In Chapter 4,we developed a multicolor orthogonal RNA sensor based on RNA stabilized fluorescent protein.The existing methods for RNA imaging based on RNA stabilized fluorescent protein can only detect and track single RNA.To enable simultaneous imaging of multiple RNAs,we fused fluorescent proteins and RNAbinding proteins(RBPs)with degron.A variety of fluorescent proteins of different wavelengths and naturally occurring multiple pairs of RNA and RNA binding proteins provide the basic guarantee for the selective multi-color orthogonal imaging.The distance between the RNA hairpin and the degron was shortened by the methods of degradation subsequence optimization and circular permutation RBP,and the RBP-Deg label with the best degradation subsequence shielding effect and the best activation signal was obtained.The multiple selectivity of fluorescent proteins and RNA-RBP enables the orthogonal recognition and detection dynamic tracer imaging of RNA,which can be used as a universal label for RNA stabilized fluorescent protein.