Study On L-Functional Nucleic Acids Fluorescent Sensors For Detection In Complex Biological Matrix

Several types of metal ions and organic molecules hold significant roles in environment safety,as well as in health of the human beings.Therefore,to obtain biostable sensors for sensing these targets in complex biological matrix is of great importance.Functional Nucleic Acids(FNA),aptamers with ability to specifically recognize targets and DNAzymes with capacity to catalyze certain reactions mainly included,usually obtained through in vitro Systematic Evolution of Ligands by Exponential Enrichment(SELEX).However,the development and application of the natural FNA(D-FNA)are impeded since the vulnerability to nuclease digestion,the easy interferences with proteins and non-targets nucleic acids binding,which leads to off-targets effect.Hence,finding simple yet effective methods to construct biostable FNA is highly desired.Based on the reciprocal chiral specificity,left-handed FNA(L-FNA),as the mirror image of the D-FNA,would have the same ability to recognize achiral targets.Based on this theory,we construct a series of biostable fluorescent sensors for detecting and imaging achiral targets in complex biological matrix.In the second chapter,we chose Cu2+that is important to environment and humans.Then,we simply change the chirality of the Cu2+-dependent DNAzyme to obtain L-DNAzyme.Based on L-DNAzyme,a fluorescent sensor for Cu2+ detection and imaging is developed.The resultant L-DNAzyme shows almost the same ability to catalyze the reaction as D-DNAzyme.However,L-DNAzyme is demonstrated to have excellent biostability,which is resistant to interferences in complex biological media and resistant to nuclease digestion.The fluorescent sensor firstly used for sensing Cu2+in buffer,it shows good linearity with limit of detection 10 nM and excellent selectivity.Afterwards,it is used to detect Cu2+ in biological matrix(FBS)and image Cu2+ in Hela cells.The L-DNAzyme fluorescent sensor shows excellent stability in complex biological matrix,indicating that it holds great potential for sensing metal ions in biomedical science.In the third chapter,firstly small organic molecule that related to several types of diseases,EA is chosen as achiral targets.By changing the chirality of the DNA aptamer of EA,L-EA Aptamer is obtained.Based on L-EA Aptamer,we build an“OFF-ON"fluorescent sensor.L-EA Aptamer and D-EA Aptamer shows similar capacity to bind EA.Meanwhile L-EA Aptamer shows great robustness in nuclease digesting experiment and excellent ability to resist the interferences from proteins and natural nucleic acids,demonstrating that L-DNA Aptamers are suitable developing biostable sensors.Also,L-DNA Aptamers show great potential in the fields of medicine.In the second part of chapter 3,we chose MG as the target,which is universally applied in fishing industry but with high toxicity and carcinogenicity to human beings.By altering the chirality of its RNA aptamer,biostable L-MG aptamer(L-MGA)is obtained.Due to the ubiquitous presence of RNase,RNA can be easily digested,resulting in few reports with RNA FNA.The experiments demonstrate that L-MGA and D-MGA have similar capacity to recognize and bind to MG.However,L-MGA is stable both in fetal bovine serum(FBS)and Cryonase cold-active nuclease.Also,L-MGA can maintain intact over a long period of time,which helps solving the storage difficulty facing D-RNA.Then,L-MGA is applied to detect MG in buffer,showing good linearity with limit of detection 65 nM.Afterwards,L-MGA is applied to detect MG both in water samples of Taozi Lake and fish tissue extraction samples.Similar results with detection in buffer are obtained.This strategy provides a more simpler way to solve the problem of inherent instability of RNA and provides a method to construct biostable RNA.