Research On The Analysis Method Of Conformational Changes Originated From Nucleic Acid Aptamer Binding To Theophylline Molecule

Theophylline promotes the release of adrenaline and norepinephrine,reduce the tension of smooth muscle,and expand the respiratory tract.It is a common drug used to treat asthma,bronchitis,and chronic obstructive pulmonary disease.However,due to the narrow therapeutic window(20?100 ?M),the dosage of theophylline must be monitored carefully when it is applied in practice.Traditional detection methods will be interfered by structural analogs,which will hinder theophylline detection.In recent years,aptamers characterized by low cost and easy synthesis,are often used as specific capture agents for target molecules to improve their detection sensitivity and selectivity.So,efficient and specific aptamer becomes the key to accurately detect the concentration of theophylline.Understanding the conformational changes of the aptamer in the process of binding to theophylline is of great significance for screening,designing and modifying the aptamers with high affinity and selectivity to.Therefore,it is urgent to establish a new method to detect the conformational change of aptamer efficiently and accurately.This study intends to establish an analytical method based on biolayer interferometry(BLI),surface-enhanced Raman spectroscopy(SERS)and molecular dynamics(MD)simulation to confirm,detect,and simply explain the conformational changes of aptamer during the binding process,so as to better understand the mechanism of the interaction between aptamer and theophylline.First,the affinity between the theophylline and aptamer selected in this study was determined by BLI experiments,which laid a foundation for detecting the conformational changes of the aptamer.And then,according to the fingerprint characteristics of SERS,the SERS spectrum changes before and after the combination between aptamer and theophylline were detected with high sensitivity,and the conformational changes of the aptamer were interpreted accordingly.Finally,the dynamic process of aptamer binding to theophylline was simulated by MD simulations,which was used to supply for the experiments,and many parameters obtained in the binding process were calculated to visually and concretely show the changes of the conformation.Such an analytic mode combined with experiments and calculations can provide a well-rounded and accurate descriptions of the conformational changes of the aptamer in the process of binding to theophylline,and the causes of the conformational changes can be simply attributed.Compared with the classical techniques such as nuclear magnetic resonance(NMR)or X-ray crystallography,which inevitably suffer from being time-consuming,requiring large samples,and being complicated to operate,this method is simple,rapid,and accurate,acquiring a comprehensive analysis and causes of the conformational changes of the aptamer.This study is the first time to establish an analytical mean of combining BLI-SERS-MD simulation,which detects and illustrates the interactions between aptamer and ligand,as well as the conformational changes of aptamer during the binding process comprehensively.This method overcomes the shortcomings of the traditional application that only one or two techniques are used to study the conformation is not sufficient and rounded.This method is not only applied to the study of aptamer-theophylline,also can be used to study other aptamer-ligand interactions.Meanwhile,our study forms a new mode of screening,designing and modifying aptamers with high affinity and specificity,so that it can be better applied to analysis,detection,sensing,clinical diagnosis and treatment and other fields.