Orientation Selection,Truncation And Application Of Broad-spectrum Aptamer Against Lipopolysaccharides

Lipopolysaccharide(LPS),also refered as endotoxin,is an inherent component of outer membranes for most Gram-negative bacteria.It is a non-secretory substance with biological toxicity to the host.LPS molecules are released to the environment when gram-negative bactiria were killed and died,which can cause toxin effects,such as fever,inflammatory responses.LPS is considered as the main pathogenic factors of septic shock and multiple organ dysfunction syndrome(MODS).LPS contamination is hard to avoid and habitually found in food and drinking water,medicine and medical products of bacterial origin.These have potential threats and harms to human health.Therefore,screening specific LPS binding molecules and antagonistic substances deserves strong attention by researchers.Aptamers have attracted much attention in recent years because of their high affinity,strong specificity,easy tmodification,large amount of chemical synthesis,and no need for animal epxeriments,and have been widely used in safety detection,biological analysis,disease treatment and other fields.In this study,a broad-spectrum aptamer against LPSs was screened by the improved capture-SELEX,then was truncated and the sequence structure-function,binding mechanism were studied based on fluorescence polarization,circular dichroism,isothermal titration calorimetry and molecular simulation technology.Rapid and homogeneous assay and the anti-inflammatory activity of cells were also tested.These researches laid a solid foundation for the rapid detection of LPS and the development of antagonistic molecules in the future.The contents and results are as follows:Firstly,an orientation screening strategy was estabilised based on capture-SELEX technology to obtain the broad-spectrum aptamers against lipopolysaccharides.According to the principle of complementary base pairing,a complementary sequence was designed as a bridge for ssDNA library immobilization on magnetic beads nanoparticles(MNPs).Following the incubation between target LPS(from salmonella typhimurium)and immobilized ssDNA library,the candidate aptamers were released from MNPs and binding to the target forming free LPS/ssDNA complex.Magnetic centrifugation was carried out to obtained PCR template for amplification,then generated ssDNA through lambda exonuclease digestion method for the next selection round.After the first six rounds of selection,the library got enriched,then performed orientation selection with two bacteria.With the continue nine rounds of positive selection,counter selection and orientation selection,a broad-spectrum aptamer EA7 with K_d value of 102±17 nM was obtained,which can binding to four tpyes of LPS from different bacterial sources.Secondly,to further verify the performance of EA7,a structure-switching aptasensor assay for LPS detection was established.A short complementary sequence qCS labeled with a3'-end DABCYL quenching group was designed in the upstream of EA7 sequence labeled with a 5'-end FAM fluorescence group.In the absence of the target LPS,EA7 was hybridizated with qCS,so FAM and DABCYL are close to each other and the fluorescence resonance energy transfer was generated,the fluorescence was quenched.After the target appeared,the aptamer is released from the qCS and becomes bound to the target.Thus,DABCYL moves away from the FAM and the fluorescence was recovered.As a result,the target can be detected according to the fluorescence changes.Under the optimal condition,the linear ranges of LPSs from Salmonella enterica serotype typhimurium(StLPS),Salmonella enterica serotype enteritidis(SeLPS),Pseudomonas aeruginosa 10(PaLPS),Escherichia coli 055:B5(EcoliLPS)were 5-250 ng/mL,0.025-10?g/mL,0.05-5?g/mL,0.1-10?g/mL,respectively.The LODs of them were 3.0 ng/mL,21.30 ng/mL,47.70 ng/mL,99.31 ng/mL,respectively.The standard recovery rate of drinking water was 95.1-105.1%.This assay is simple operation,no separation,sensitive and reliable.Thirdly,EA7 was truncated based on the primary and secondary structure,and the structure-function was analyzed.The obtained 7 truncated aptamers were identified with fluoresecence polarization and isothermal titration calorimetry assay.It was found that by eliminating the nonessential regions,LA27 was the best aptamer which still retained broad-sepctrum capability and has doubled the binding affinity to LPS(K_d=46.2±9.5 nM).Moreover,the open loop formed by CC at the 5'end and CGA at the 3'end of LA27 may be the support region for LPS binding,while the stem ring structure formed by the remaining bases may be the main binding region for LPS.CD results further indicate that LA27 may bind to LPS in an embedded manner.Fourthly,graphene oxide(GO)-enhanced fluorescence polarization assay was established for rapid detection LPS to further analyze the performance of LA27.The aptamer probe was adsorbed on the prepared-GO surface formed GO/aptamer complex and got a higher FP signal.When there is a target,the aptamer was captured by LPS and formed aptamer/target complex,resulting in a decrease in FP value.Therefore,the target can be detected in homogeneous way and without separation based on the changes of FP signals.The results showed the detection limits of LA27 probe for StLPS,PaLPS and EcoliLPS were 38.7 ng/mL,88.7 ng/mL,154ng/mL,respectively,which were 4.8,29 and 18 times higher than the LOD of EA7(LA80).The FP real-time kinetic test results showed that one sample can be detected within 30 min.The standard recovery of sodium chloride injection samples was 92-112.5%.These results indicate that the truncated and optimized aptamer LA27 has better performance due to its short sequence and better affinity.Finally,the anti-inflammatory activity of truncated aptamer LA27 and interaction mechanism were analyzed.Four types of LPSs were used to stimulate HepG2 cells to produce inflammatory cytokines,and the expression levels of three factors(TNF-?,IL-1?and IL-6)were detected by ELISA.The results showed that LA27 the expression levels of inflammatory cytokines(TNF-?,IL-1?and IL-6)in StLPS,EcoliLPS and PaLPS groups were significantly reduced(p<0.01),while there is not affected in SeLPS groups(p<0.01).These indicate that LA27 exhibits a considerable anti-inflammatory activity.Furthermore,these also further verifies its ability to recognize LPS,EcoliLPS and PaLPS.MOE Molecular simulation results showed that under the combination of the hydrophobic interaction,hydrogen bonding and electrostatic interaction,the fatty acid chain of LPS may interaction with the big hydrophobic region which mainly formed by seven T bases including T3,T4,T6,T16,T17,T19 and T20.Because of these,LA27 binding to LPSs specificity and forms a stable compounds like“T”type.