New Techniques For Detection Of D-Gal And Other Small Molecules Based On Strand Displacement Amplification Regulated By Transcription Factor

Objective:Metabolites are found in body fluids and can serve as important biomarkers of medical significance.Metabolite detection is one of the research hotspots in the field of biology and medicine.At present,many metabolite detection methods have shortcomings such as low sensitivity,easy interference,cumbersome operation,dependence on expensive instruments and professional operation,so it is very important to develop simple,fast and easy to operate metabolite biosensors.Transcription factor-based biosensors（TFBs）play an important role in metabolic engineering and synthetic biology.TFBs can sense the metabolite concentration signal and convert it into a specific output signal.They are widely used in the detection of target metabolites because of their high sensitivity,strong specificity and fast analysis speed.Strand displacement amplification（SDA）is one of the most important isothermal nucleic acid amplification techniques,which is widely used in recent years because of its simple principle and operation procedures.Most of the application fields of SDA technology are the detection of proteins and nucleic acids,and its detection of small molecule metabolites remains to be explored.In this study,we selected a prokaryotic transcription factor,Galactose repressor（GalR）,and used its regulatory chain replacement amplification technology to establish a new detection technology for galactose and other metabolites.Methods:1.The SDA template and primer with GalR recognition sequence are designed to inhibit SDA amplification by GalR recognition and binding to DNA,and D-galactose（D-Gal）specifically binds to GalR to relieve the inhibition and resume SDA amplification,forming a"switch"mechanism of biosensing.So GalR can regulate SDA reaction specifically to detect galactose.We named this biosensor repressor-SDA.（1）GalR protein was purified by prokaryotic expression.（2）SDA template and primer with GalR recognition sequence were designed to establish SDA reaction system.（3）The repressor-SDA technology is established by regulating the SDA reaction with GalR,and the conditions of this technology are optimized.（4）Methodological characterization of D-Galwas detected by repressor-SDA technique.2.SDA technology not only has high amplification efficiency,short reaction time and strong specificity,but also can be easily coupled with other technologies to improve detection efficiency.Catalyzed hairpin assembly（CHA）isothermal amplification is an in vitro enzyme-free amplification technique,which is widely used in modern analytical fields because of its simple operation and sensitive response.Based on repressor-SDA,this study further coupled CHA technology to further improve the detection of D-Gal.（1）H1/H2 hairpins and fluorescent probes in CHA reaction were designed to establish CHA reaction system.（2）The buffer and H1/H2 concentration in CHA reaction were optimized.（3）The repressor-SDA coupling CHA technology was established to improve the detection efficiency of D-Gal.（4）Methodological characterization of D-Galwas performed by repressor-SDA technique coupled with CHA technique.3.The proof-of-concept of repressor-SDA technology relies on the transcription factor GalR,and the detection object is limited to galactose.In order to enable the biosensor to detect more targets and expand its application range,we adopted the strategy of hydrolytic enzyme-coupled repressor-SDA to detect other metabolites.Lactose and meliobiose were used as detection models,and the repressor-SDA reaction was triggered by lactase and meliobiase to decompose lactose and meliobiose into galactose,which expanded the detection target range.（1）Lactase is used to decompose lactose according to the principle of enzymatic reaction.（2）Using repressor-SDA technology to detect galactose produced after lactose decomposition,lactose can be quantitatively detected.（3）Meliobiose was decomposed by meliobiase and qualitatively detected by repressor-SDA.4.Inhibitor screening helps to reveal the molecular mechanism of action of inhibitors,as well as their application in drug development and disease treatment.Inhibitor screening of transcription factors is not only meaningful to reveal their regulatory mechanisms,but also plays an important role in medical practice such as drug development.The feasibility of screening GalR transcription factor inhibitors was verified by repressor-SDA technology,and the inhibitor of GalR was screened,and the half inhibitory concentration of the inhibitor,IC50,was further detected by repressor-SDA technology.（1）To verify whether the presence of IPTG can inhibit GalR reaction to D-Gal.（2）Screen whether different kinds of substances have inhibitory effect.（3）The half inhibitory concentration of IPTG,IC₅₀,was verified.Results:The active GalR protein was successfully obtained,a new biosensor was established,the fluorescence signal was detected by Molecular beacon（MB）,and the sequence,the concentration of two enzymes and the concentration of GalR in the repressor-SDA system were optimized.The specific detection of small molecule metabolite D-Galwas realized,and the detection limit of D-Galwas 2.70μM.The detection limit of D-Galwas further improved through the coupled CHA amplification technology.The buffer used in the system and the concentration of two hairpins were optimized during the combined CHA reaction.Under the optimal conditions,the detection limit of D-Galdetected by the combined amplification of the two technologies reached 1.05μM.Moreover,the specificity of other types of monosaccharides was significantly improved.The detection of lactose and meliobiose was extended to lactose and meliose by an additional enzymatic reaction.The detection limit of lactose was 5.34μM.Moreover,three inhibitors of GalR transcription factor were further screened by this technique,namely IPTG,D-Glu and L-Fuc,and the half inhibitory concentration of IPTG was detected to be 4 m M.Conclusion:In this study,a variety of novel biosensors were successfully constructed using the repressed transcription factor GalR,and a variety of different metabolic small molecules were successfully detected and GalR inhibitors were screened through these detection techniques.