Screening And Applications Of Nucleic Acid Aptamers Of Berberine

Berberine(BBR)is a natural small molecule compound which can be extracted from medicine plants.BBR has found broad applications due to its ubiquity,low-cost,and safety to human.Nucleic acid aptamer generally refers to a fragment of RNA,DNA or nucleic acid analog that binds to a target with high specificity and affinity.RNA aptamers can manifest diverse secondary structures,and RNA fragments can be obtained through transcription in microorganisms,and thus can be used to construct genetic regulatory elements.Compared with RNA aptamers,DNA aptamers are more stable,as they cannot be degraded by the ubiquitous RNases.Therefore,DNA aptamers are more appropriate for interaction with small molecule ligands in vitro and can be applied to broad fields such as molecular diagnostics,nucleic acid probes,biochips and biosensors.In this study,the Systematic Evolution of Ligands by Exponential Enrichment(SELEX)method was implemented to screen RNA and DNA fragments that can bind BBR with high specificity and affinity.Notably,a BBR-inducible genetic switch was engineered based on the RNA aptamer,and gene expression regulation was achieved through the interplay between BBR and the aptamer.As a novel inducer,BBR was expected to replace the current expensive inducers,and thus reduce industrial production cost.Namely,this study extends the applications of BBR in microbial fermentation.Apart from aforementioned studies,based on AuNPs colorimetric method,I developed a simple,rapid and inexpensive biosensor to monitor BBR,and this work provides a paradigm for development of biosensors in other fields.The main research contents are as follows:1.Using BBR as a target molecule,an RNA aptamer fragment R1:5'-CAUCAUUGCCUAGUCUGAUCUCAUCUCCGUCAGUGUGU GCUCUUCUCUCUCAGAUUGAUG-3',which bound specifically to BBR with high affinity,was screened from a random library by 16 rounds of SELEX cycles.The secondary structure of the aptamer R1 was simulated by mfold software,which showed that it was easy to form a stem-loop structure.The dissociation constant Kd of the aptamer R1 and BBR was measured by equilibrium permeation method,and it was 764± 15 nM.R1 has high affinity with BBR and can be harnessed to construct genetic elements.2.A BBR-inducible riboswitch element based on the ribonucleic acid aptamer R1 was constructed to regulate gene expression.The resulting recombinant strain E.coli(pTAC15A-Aptamer-N10RBS-egfp)was fermented,and BBR-induced EGFP expression was measured by addition or absence of BBR.We investigated the fluorescence intensities of 28 recombinant strains during fermentation,with wild-type E.coli BL21 and E.coli(Ptac-egfp)as reference strains.Results showed that the engineered riboswitches functioned better in recombinant C25.The shutdown and opening efficiency was 97%and 77%,respectively,and the ON/OFF ratio was 8.94.It is concluded that this riboswitch can be easily induced by BBR and thus can be applied to regulate downstream gene expression.3.DNA aptamers were achieved through a total of 8 rounds of SELEX screening.The resultant DNA aptamers can combine BBR with high specificity and affinity.The primary structure analysis and secondary structure simulation of 8 aptamers with higher repetition rate revealed that the aptamer sequence was rich in GC and easily formed into stem ring and G-quadruplex structure.The affinity of 8 aptamers to BBR was determined by equilibrium permeation method.Results showed that the aptamer S23 had the smallest Kd value(414 nM)and the highest affinity with BBR,which provides a basis for the development of BBR-mediated biosensors.4.An Aptamer-AuNPs-mediated colorimetric sensor was developed for the simple,fast and low-cost detection of BBR.The effects of NaCl concentration,aptamer concentration,binding time and binding temperature on the aggregation phenomenon of AuNPs in the reaction system were investigated.The optimal NaCl and aptamer concentrations were 0.4 mol/L and 500 nM,respectively.The optimum binding time was 1 h,and the optimum reaction temperature was 35?.Different concentrations of BBR were added to the system to investigate the sensitivity of the aptamer-AuNPs system to BBR.The results showed that the absorbance of the system decreased significantly after the addition of BBR,and the Aptamer-AuNPs colorimetric sensor could detect the BBR in the sample.However,no significant correlation between the absorbance and BBR concentration was observed.The fitting curve is y=-1.06×10-4x+0.1263,and the R square is only 0.776.Hence,the detection sensitivity needs improvement.