Screening Of Nucleic Acid Aptamers Of Geniposide Based On SELEX Method And Its Preliminary Application

ObjectiveGeniposide,a small molecule,is one of the key active ingredients from Gardenia jasminoides fructus widely used in traditional Chinese herbal medicine.It has been shown in many studies exhibiting a variety of biological activities.However,the precise mechanisms of its various effects remain poorly understood.Our research group has carried out experiments on studying its targets previously.It was predicted that glucocorticoid receptor may be the target of geniposide by virtual screening method,pull-down by affinity chromatography and drugs competitive experiments in vitro cells.All these results suggested that geniposide has interaction with glucocorticoid receptor.However,it is now still in doubt that whether they still have interaction when geniposide playing a neuroprotective role in vivo.The aim of this thesis was to generate aptamers that binds geniposide with high affinity and specificity via the Systematic Evolution of Ligands by Exponential enrichment(SELEX),and preliminary experiments were carried out to study the relationship between glucocorticoid receptor(GR)and geniposide in the mouse brain using the aptamer.This study provides experimental data for the further study of molecular mechanism for geniposide and provides the new ideas and new methods for studying the targets and molecular mechanism of small molecules in Chinese Medicine.MethodsThis thesis mainly included four parts:the first part was to generate aptamers that binds geniposide via SLEX in vitro.Geniposide was the positive selection target,and genipin and glucose were the counter selection targets.The stragety used in SELEX process was called structure-switching method.By calculating the coupling rate,optimizing the PCR cycle condition,the percentage of the eluted nucleic acid sequences,the comparison of the eluent and the supernatant,and the quantitation of eluents by real time PCR,we examined the enrichment effect,screened the process,and optimized the conditions if needed;In the second part,when the nucleic acid library was enriched to a certain extent,the high-throughput sequencing was performed to the selected library.The sequencing results offered the copies numbers and the base information of each sequence.Selected the domain oligonucleotides and analysed their secondary structures,affinities and specificities.Then,we got the aptamers with high affinity and specificity;In the third part,the geniposide in the brain homogenates should be identified by using ultra high performance liquid chromatography-mass spectrometry and then aptamerbased co-precipitation were carried out to co-precipitated geniposide and glucocorticoid receptor complex in the mouse brain by using biotin-labeled aptamers.SDS-PAGE and Western Blot were followed to detect the existence of glucocorticoid receptor;In the fourth part,the colocalization was carried out by using the fluorophore aptamer and inmmune antibodies of glucocorticoid receptor.Under the fluorescence microscope,the position relationships between glucocorticoid receptor and geniposide were observed in frozen brain slices.Results1.The immobilization rate of single strand oligonucleotide library was 89.9%,and it showed the library was fixed well;The PCR conditions were optimized,the best amplification of PCR were got when annealing temperature was 55℃ and extension time was 10 min,showing the reaction conditions of PCR were suitable for each stage,and the specific amplification products could be obtained effectively;The percentage of elution against the geniposide in the 10th round was significantly higher than that of the initial library,the percentage of elution against buffer was approximately the same during the SELEX process and the percentage of elution against the geniposide in the 10th round was the same of those against the genipin and glucose.All the results showed that the selection process was effective but poor specific,and the counter selection should be introduced;In the 16th round,the percentage of elution against geniposide was gradually significantly increasing,up to 25%,while the percentage of the eluent against counter targets was stable,at 3%,showing the selection process should be finished.2.GP1(geniposide aptamer 1)was 934 copies and GP2(geniposide aptamer 2)was 713 copies in the oligonucleotides pool.The rest oligonucleotides were much fewer,less than 300 copies;The Mfold Web Server predicted that the secondary structure of GP1 contains a hairpin and an internal bulge,and GP2 has two hairpins and a three-way connecting element.The internal bulge element of GP1 and the 3-way junction motif of GP2 were suspected to be the binding site for geniposide.The Kd,eff1 of GP1 and GP2 were 17.7 nM and 16.8 nM,respectively.The Kd.eff2 of GP1 and GP2 were 0.0029 and 0.0086,respectively.The Kd values were calculated from the equations Kd=Kd,eff1/Kd,eff2.The of GP1 and GP2 were～6.1 μM and～2.0 μM,respectively.Genipin,glucose and mouse brain homogenates did not induce the increase of fluorescence intensity,which was significant different with geniposide.The affinity of GP1 and GP2 were around micromoler level,and both of them would not be interact with genipin,glucose and mouse brain homogenates.3.The results of mass spectrometry showed that the C-G groups contained 3,7,5,8 and 7 quasi-molecular ions which were the same as those of the geniposide standard product in the same retention time,and the characteristic quasi-molecular ions of each geniposide group were consistent with the references.It showed there were detectable geniposide in the mouse brain samples under our administration;The results of co-percipitation showed that there were bands for input and the supernatants of the brain homogenate in all groups,but only the eluent of geniposide group had a light band while the other two eluents had no bands.The aptamer-based co-precipitation results suggested that the GP2 and the agarose resin did not cause false positive,and the GP2 specifity with the mouse brain homogenates results were in line with expectations.There were interactions between geniposide and glucocorticoid receptor in the mouse brain.4.There were no specific fluorescence signals for GP2 staining the frozen mouse brain sections of control groups,but specific staining with the mouse brain slices of geniposide group,which showing GP2 could identify the geniposide in mouse brain slices,suggesting GP2 could be used to locate geniposide in the frozen mouse brain tissue sections;There were also no specific fluorescence signals for GP2 after GR immune antibodies stained frozen mouse brain sections of control groups,which showing glucocorticoid receptor staining did not cause GP2 nonspecific staining,suggesting that the fluorescent aptamers and glucocorticoid receptor antibodies can be used for double labeling and the aptamer-based co-localization method was feasible.The results of co-localization experiment results showed the green signals and the red signals merged into yellow signals,suggesting that the geniposide and glucocorticoid receptor have potential interactions in the mouse brain.ConclusionsIn conclusion,the structure-switching method was well designed and suitable for geniposide.We isolated the high affinity and specificity DNA aptamers of geniposide for the first time,and then we tried to apply the aptamer GP2 to study the relationships of geniposide and glucocorticoid receptor in the mouse brain.The results showed there were interactions between glucocorticoid receptor and geniposide in the mouse brain.The SELEX technique could be used as a platform to generate aptamers for the other small molecules like geniposide.It is suggested that the aptamer could be applied to the field of studying the small molecules and proteins relationships research,which provides new tools and new methods for studying the molecular mechanisms of active ingredients from Chinese medicine.