Based On GO-SELEX Screening And Identification Of Aptamers For Exosomes From Lung Cells

Research background and purpose:Exosomes（Exo）are cup-shaped lipid bilayer membrane vesicles（30-150 nm in diameter）released outward from cell membranes and are widely present in various body fluids and cell cultures.Therefore,the detection of Exosomes from different cancer cells in blood plays an important role in tumor screening,diagnosis and treatment.Currently,the detection of exosomes is mainly based on exosome-specific proteins and immunoaffinity principles with high specificity for antigen-antibody interactions;however,antibodies are costly to manufacture and require high storage requirements,and there is still a need to find alternatives to antibodies that are accurate,reliable,and can be used for clinical applications.Aptamers are single strands of oligonucleotides with a small size of 30-100 bp（6-26 k Da）obtained by in vitro screening technique-Systematic Evolution of Ligands by EXponential Enrichment（SELEX）.In recent years,aptamers have been widely used in biosensors to detect small molecules,cells and exosomes due to their easy synthesis,easy storage,low cost and high stability properties and the ability to be labeled by a variety of molecules（e.g.fluorescent dyes,enzymes）.With the development of aptamer research,aptamer screening methods have gradually matured,especially with the development of new nanomaterials,which not only greatly shorten the screening period but also improve the specificity of aptamers.Graphene oxide（GO）has a strong distance-dependent fluorescence quenching ability and can be attached to single-stranded DNA and RNA throughπ-πelectron stacking interactions.GO has a high screening efficiency and can distinguish unbound and target-bound ss DNA easily and clearly.In order to visualize the aptamer screening procedure of GO-based exosomes,this study was conducted to obtain aptamers that specifically recognize exosomes,analyze their specificity and affinity,and elucidate the spatial structure of novel DNA aptamers by GO-SELEX screening method,using non-small cell lung cancer（NSCLC）A549 cell exosomes as targets.The spatial structure of the new DNA aptamer was elucidated.This work will provide a theoretical basis for the construction of novel biosensors and provide technical support for liquid biopsy and early diagnosis of lung cancer.Materials and Methods:（1）Isolation,extraction and identification of exosomes from NSCLC A549 cellsIn this experiment,A549,a non-small cell lung cancer cell from our laboratory,was used as the source for the preparation of exosomes.Exosomes from human breast cancer cells MCF-7 were used as the target for negative screening to prepare exosomes from both cell lines.The cell culture method was strictly in accordance with the ATCC operating guidelines.When the cells grew to approximately 50%coverage,the supernatant was aspirated,rinsed twice with 1×PBS,and incubated with serum-free medium for 48 h.The cell supernatants were then collected and incubated using the ultracentrifugation method and a combination of ultracentrifugation（Ult）with a kit(Exo Quick ^TM)method（U-EQ）to isolate exosomes from the cell culture supernatant.The concentration of A549 exosomes was determined by BCA,and the morphology and particle size of A549 exosomes were observed by transmission electron microscope（TEM）,Nanoparticle tracking analysis（NTA）and Di I staining.The expression of four transmembrane proteins CD63 and Heat Shock Protein 70（HSP70）on the surface of A549 exosomes was analyzed by Western blotting（WB）.（2）GO-SELEX-based screening and identification of aptamers for exosomes in NSCLC A549 cellsAptamer screening against target A549 cell exosomes by establishing graphene oxide-enriched ligand phylogenetic technique（GO-SELEX）.Firstly,positive screening was performed;the prepared screening library ss DNA was first immobilized on the GO surface and incubated on a shaker,and the precipitated GO/ss DNA complex was obtained by centrifugation after a certain time of incubation,and then the GO/ss DNA complex was co-incubated with the isolated and purified A549 cell exosomes at 4°C for a certain time of incubation,and the ss DNA unbound to the target exosomes was adsorbed by theπ-πconjugation bond of GO The ss DNA that is not bound to the target exosomes is adsorbed by theπ-πconjugation bond of GO,and the ss DNA that has bound to the target（Exo/ss DNA）is left in the supernatant,and the A549 cell exosome-ss DNA complex in the supernatant is obtained by centrifugation.The same incubation time was used for the first three rounds,and the incubation time was gradually reduced starting from the fourth round.Negative screening:The 5th and 9th rounds are counter-screening,and the exosomes of the negative screening,i.e.,exosomes of breast cancer cells MCF-7,are used as the target of negative screening.The prepared ss DNA library was first added to GO solution and incubated on a shaker,and the precipitated GO/ss DNA complex was obtained by centrifugation after a certain time of incubation,and then the GO/ss DNA complex was co-incubated with the isolated and purified MCF-7 cell exosomes at 4℃（the incubation time increased with the number of screening rounds）,and the MCF-7cell exosomes in the supernatant were discarded by centrifugation after a certain time of incubation.The exosome/ss DNA complex was added to the purified A549 cell exosomes and incubated at 4°C.After incubation for a certain time,the A549 cell exosome-ss DNA complex in the supernatant was obtained by centrifugation.The ss DNA recovered by centrifugation from the Exo-ss DNA complex of each screening round was used as a template for asymmetric PCR amplification,and the double-stranded DNA（ds DNA）of the PCR product was completely cleaved to single-stranded ss DNA by thermal denaturation（95°C,5 min）,which was used as a secondary ss DNA library for the next screening round.The recovery rate was calculated once for each round of screening,and in rounds 5 and 9,the counter-screener breast cancer MCF-7 cell exosomes were introduced to remove ss DNA that bound poorly to A549 cell exosomes specifically.ss DNA was removed when the recovery rate reached saturation and screening was stopped.A total of 12 rounds of nucleic acid aptamer screening were performed in this study.After the libraries bound by multiple rounds of screening reached saturation,PCR amplification was performed on the screened libraries with primers without markers,and the amplified products were used for TA cloning and sequencing,with the TA cloning process done by Ta Ka Ra kit and the sequencing process done by Shanghai Bioengineering Co.The sequences measured were analyzed statistically and evolutionary trees were drawn with the software Bio Edit,and secondary structures were analyzed with Mfold（http://mfold.rna.albany.edu/?q=mfold/dna-folding-form）.Finally,the specificity and affinity of the candidate aptamers obtained from the screening were evaluated.Result:1.In this study,we applied the U-EQ method combining ultracentrifugation and kits and ultracentrifugation to isolate and extract exosomes,and identified the exosome concentration,quantity,morphology and protein expression of A549 cells by BCA protein assay kit,NTA,TEM,orange-red fluorescent probe staining of cell membrane（Di I）and WB;the data showed that the yield of exosomes isolated and extracted by U-EQ method was higher than that of The data showed that the yield of exosomes isolated by U-EQ method was higher than that of ultracentrifugation method,and the enzyme standard results showed that the concentration of（A280）reached 1.355 mg/m L,the number of particles/m L reached 4.65×10⁹ particles in NTA results,the peak size distribution ranged from 81.5 to 165.5 nm,Di I and TEM showed that the morphological size was one nanometer vesicles,and WB was applied to verify the presence of CD63and HSP70 expressed in the isolated extracted exosomes protein.2.The PCR results showed that the desired target ss DNA fragments were obtained after 12 rounds of screening.The products of the 11th round of screening were cloned and sequenced,and a total of 43 nucleic acid aptamer sequences were obtained.After secondary structure,free energy（△G）prediction and affinity analysis of the 43nucleotide sequences,a total of 6 candidate nucleic acid aptamers,Exo-1,Exo-2,Exo-11,Exo-17,Exo-20 and Exo-34,were finally obtained.The same number of exosomes from lung cancer cells A549,breast cancer cells MCF-7,pancreatic cancer cells PANC-1,breast cancer cells 4T1,human colon cancer cells HCT116,and human colon cancer cells HT29 were used for the specificity analysis of the 6 candidate aptamers,and the results showed that all 3 candidate aptamers,Exo-2,Exo-11,and Exo-20,could be used with A549 The specificity of the three candidate aptamers was determined by measuring the dissociation constants（Kd values）of the three more specific candidate aptamers and their affinity to A549 exosomes.The results showed that the Kd values of the three candidate aptamers Exo-2,Exo-11 and Exo-20 were at56.15 n M,55.92 n M and 73.25 n M,respectively;and the aptamer Exo11 had a stronger affinity effect with A549 cell exosomes and could be used as a specific nucleic acid aptamer for A549 cell exosomes.Conclusion:1.In this study,the U-EQ method combining ultracentrifugation with a kit to isolate and extract exosomes was used to isolate and purify exosomes from cell culture supernatants.Exosomes were successfully isolated from the cell culture supernatant of A549 and were analyzed and characterized.The shape,particle size and protein expression of exosomes were identified by TEM,NTA,fluorescent staining and Western Blot.The data showed that the exosomes isolated and extracted by U-EQ method were highly concentrated and homogeneous in particle size,and high-quality exosome extraction is expected to be further studied.2.Screening of A549 exosome-specific aptamers by using the GO-SELEX method.A total of 12 rounds were screened,and six aptamers with high repeat rate were screened in this study.For lung cancer A549 cell exosomes,the obtained aptamer Exo-11 had a high specificity and a low Kd value of 55.92 n M,indicating that aptamer Exo-11 is a specific aptamer for target A549 cell exosomes.