Novel Biosening Method Based On Geneticaly Endcoded RNA Aptamer

In recent years,an important breakthrough in the field of analytical chemistry and life sciences is the development of light-up RNA aptamers binding fluorescence dyes as new biosensing strategies,in which the fluorophore dye and RNA aptamer pairs are called as "light-up" probes.Light-up RNA aptamer probes are not just used for in-situ fluorescence imaging in living cells or detecting the target in the environment,such as heavy metals,small metabolism molecules and proteins,they are also used as the "tag" elements of the interest proteins,with a function similar to green fluorescence protein(GFP),for example the well-known "Spinach" system.Besides Spinach,RNA aptamer for fluorophore sulforhodamine B(SR)has also provided a simple and useful platform for the development of light-up RNA aptamer biosensors in bacterial cells,of which the three-way junction structure binding the sulforhodamine-quencher conjugate leads to the recovery of fluorescence of fluorophore.Light-up RNA aptamer can not only be obtained from in vitro transcription,but also could be genetically encoded in living cells.Using the genetic mechanism of host cells,artificial gene sequence module can be expressed as proteins or RNA aptamer sequences.Combing with the mechanism of synthetic biology and fluorescence imaging techniques,it would afford novel approaches for the development of light-up RNA biosensing strategies.In this thesis,some new biosensing methods bases on light-up RNA aptamers have developed for the detection of microRNA(miRNA)and PSA in vitro,for the ratiometric fluorescence imaging of microRNA and mRNA in the living tumor cells,as well as for the living-cell visualization of CRISPR/Cas9 system-activated transcription.The main contents are described as follow:In Chapter 2,we have developed a novel Spinach-based fluorescent light-up biosensor to generate unmodified Spinach sequences for miRNA detection.In this strategy,it uses two adjacent miRNA-specific DNA oligonucleotides to capture target miRNAs followed by T7-mediated in-vitro transcription,consequently generating numerous Spinach RNA aptamer and malachite green RNA aptamer(MGA)transcripts to light up the dyes of DFHBI-1T and MG dye,respectively.Using such strategy realized simple,label-free,and highly sensitive detection of miRNA 21 and miRNA 141 with a detection limit as low as 3 pM.In Chapter 3,target-induced proximity ligation transcription assay based on RNA aptamer has developed for the sensitive detection of prostate-specofic antigen(PSA).The binding events between the DNA-connected ligands and the target molecule mediates the formation of DNA-DNA splint complex and the short bridging DNA connector chain,which further induces T7-mediated transcription of the RNA aptamer and significantly enhanced fluorescence signals.In PSA assay,a detection limit of 0.4 ng/mL was achieved for the developed light-up RNA biosensing strategy.In Chapter 4,we have reported a novel genetically-encoded RNA sensor for fluorescent imaging of miRs in living tumor cells using a light-up RNA aptamer which binds to sulforhodamine and separates it from a conjugated contact quencher.The RNA sensor can be stably expressed within a designed tRNA scaffold in tumor cells and deliver light-up response to miR target.We also realize the RNA sensor for dual-emission,ratiometric imaging by co-expression of RNA sensor with GFP,enabling quantitative studies of target miR in living tumor cells.In Chapter 5,we have further reported a novel strategy of genetically coexpressing RNA sensor with its fluorescent reference GFP under a single transcription event in living tumor cells.We reasoned that simultaneous transcribing RNA sensor with GFP in a single transcription event could contribute to a consistent expression level.Therefore,in this method,a single promoter-driven plasmid has constructed to coexpress GFP and light-up RNA biosensor for the ratiometric imaging of survivin mRNA in living cells.Results verified that such a biosensor enabled robust ratiometric mRNA imaging in tumor cells and it was also capable of quantative analysis of the copies of mRNA.In Chapter 6,we have developed a biosensing strategy for the visualization of CRISPR/Cas9 system-activated transcription in living tumor cells.Two different plasmids are constructed to express a closed-gRNA and dcas9-VP64 protein,separately.In the present of target mRNA,the closed-gRNA opens and binds with dCas9-VP64 protein to further induce transcription activation of the CRISPR system and produce abundant transcripts of light-up RNA aptamer,therefore,achieving visualization of such a transcription event in living cells.