Study On Polychromatic RNA Aptamers For Nucleic Acid Molecular Detection And Cellular Imaging

Nowadays,more and more attention paid to the new biosensors for diagnosis and basic research.With the rapid development of newly aptamer technology,aptamer-based biosensor is expected to be one of the most promising devices.At present,aptamer is a potential recognition element in the construction of biosensors.The biggest difference between aptamer and traditional affinity reagent-monoclonal antibody is the range of detection.It can bind to almost any target selectively,ranging from small molecules to whole cells and microorganisms.Therefore,it has significantly broadened the application range of corresponding biosensors,including medical diagnosis,food analysis,environmental detection and many other fields.Among them,the aptamer based on RNA fluorescence module,as a new kind of genetically encoded biosensor,which can activate fluorescence by binding to its homologous fluorophore and widely used in the field of RNA vitro and vivo imaging.It has become the focus of biology and biochemistry research.In recent years,this kind of sensors developed from malachite green to spinach aptamers,formed RNA fluorescence module covering the whole visible spectrum gradually.Some fluorescent modules are even brighter than the green fluorescent protein(GFP),which becomes an excellent imaging tool.RNA is one of the main participants among the cell function,not only in protein synthesis,especially for non-coding RNAs,such as Small nuclear RNAs(snRNAs),Long non-coding RNAs(IncRNAs),MicroRNA(miRNAs)and so on.As more and more RNA are found,the number of transcripts far exceeds that of proteome.However,the direct measurement of intracellular RNA function and its robust research are still in development.Among them,miRNAs,as small endogenous RNAs,can degrade or inhibit translation by targeting specific mRNA,which plays a extremely important role in animal and plant cells,including cell proliferation,cell death,fat metabolism,hematopoiesis and many other functions.In this paper,some newly sensing methods with simple operation,high sensitivity and strong selectivity are established by biosensor construction based nucleic acid aptamer,which can detect small molecules of nucleic acid metabolism.The specific contents of the study are as follows:In chapter 2,we developed an aptamer biosensor based on RNA fluorescence module for label-free and selectivity detection of miR21.This approach utilized the T7 in vitro transcription process to generate a large amount of unmodified spinach aptamers triggered by the target.Actually,the kind of homologous fluorophore DFHBI-1T has no fluorescence signal itself,which lead to low fluorescence background.The fluorophore can bind to the aptamer and produce a recognizable fluorescence signal immediately.By measuring,the high sensitivity detection of miRNA can be realized.Compared with the traditional spinach biosensor,three or four orders of magnitude improvement in detection limit was achieved by the newly biosensor combined with amplification strategy,up to 3 pM.It can be expected to provide a new idea for the study of low abundance targets.In chapter 3,we proposed a novel genetically encoded RNA sensor strategy.Theoretically,the aptamer based on RNA fluorescence module can provide a valuable platform for the RNA sensor-based living cells imaging.Therefore,in this chapter,we continue to focus on the construction of RNA sensors for dual-emission by aptamer SRB2 and GFP.In order to minimize the differences in different promoters and the interference of intramolecular folding,a splittable fusion expression scheme of SRB2 RNA aptamer and GFP mRNA was designed,which based on the single promoter plasmid system.It can realize the synchronous expression of RNA aptamer and GFP mRNA and provide a new method for the development of live cell ratiometric imaging.