Selection And Analysis Of Aptamers Against Serum Of Primary Hepatic Carcinoma

Background and objectives:The mean lifetime of patients with advanced primary hepatic carcinoma (PHC) is only several months. The key to improve the prognosis is earlier discovery and treatment. Currently, tumor markers is one of the principal diagnostic methods for early hepatocellular carcinoma. Because the heterogeneity of tumor cells, AFP and the other classic HCC markers are not satisfied in the early diagnosis of PHC. Recently, proteomics and other biological high-techs greatly promoted the hepatocellular carcinoma biomarker researches. For example, GPC3and GP73were found by proteomic technique and with good application prospect. Therefore, using biological high-tech to explore new methods to diagnose PHC has become the research hotspot. SELEX is a kind of combinatorial chemistry technology, by which nuclear acid aptamers with high specificity and affinity to targets could be screened out from a random oligonucleotide library. The function of aptamers is similar to antibodies but better than antibodies in some characteristics, so that aptamers are a potential tool in disease diagnosis. Screening out specific aptamers to unknown target molecules is the outstanding characteristic of SELEX. Because there are lots of tumor biomarkers (known and unknown) in the serum of PHC, it is possible to select a group of aptamers against PHC serum markers known and unknown by using PHC sera as a compound target to perform SELEX and then found new methods for the early diagnosis of PHC. Therefore, in the present study we used PHC sera as SELEX target to select aptamers, aiming to lay the foundation for novel PHC diagnostic methods based on aptamers.Methods:(1) Target serum collection:20cases of preoperative sera from patients with PHC confirmed by surgery and pathology and20cases of normal sera from health subjects were collected. The mixed sera of PHC and normal subjects were prepared in equal proportion, respectively (to eliminate individual differences), and used as targets of SELEX.(2) Nucleic acid library and primers design and synthesis:the single-stranded random oligonucleotide library and its corresponding primers designed and applied in our previous studies were synthesized by a biotechnology company.(3) Subtractive SELEX:three round subtractive SELEX were carried out by mixed normal serum to remove ssDNAs in the library binding to normal serum components. The library was incubated with mixed normal serum, and then the unbound ssDNAs were separated and recovered by neutral polyacrylamide gel electrophoresis (PAGE), amplified by asymmetric PCR. The ssDNAs in the PCR products were separated and recovered by denature PAGE, quantified by real-time PCR and used as next round of SELEX.(4) Selection of aptamers to PHC serum: about10rounds of SELEX with the mixed PHC serum were performed to select ssDNAs specifically binding PHC serum. The sub-library after the subtractive SELEX was incubated with mixed PHC serum, and then the bound ssDNAs were separated and recovered by neutral PAGE, amplified by asymmetric PCR. The ssDNAs in the PCR products were separated and recovered by denature PAGE, quantified by real-time PCR and used as next round of SELEX. The selection was repeated until the binding band was no longer densified.(5) Isolation of individual aptamers:the sub-library after the last round SELEX was amplified by PCR. The PCR products were sent to biological company cloning and sequencing. The secondary structures of the isolated sequences were imitated by RNA Structure4.5software. The aptamer sequences were clustered and aligned by Clustal software.(6) Specificity analysis of aptamers:aptamer solutions in gradient amounts (2-6pmol) were incubated with mixed PHC and normal sera, respectively, and the binding status was displayed by PAGE. The gray values of unbound bands were measured by Quantity One image analysis software, and its ratios between normal and PHC sera were calculated to evaluate the specificity of aptamers in binding to mixed HCC serum.(7) Affinity measurement of aptamers:aptamer solutions in gradient amounts (1-8pmol) were incubated with mixed PHC serum, and then the gray values of bound bands were measured after PAGE. The dissociation constants were calculated by Graphpad prism5.01software based on the gray values.(8) The specificity analysis of aptamers binding single PHC serum:the representative aptamers were incubated with single PHC sera and normal sera, and the binding of aptamer to serum was observed after PAGE. Free band gray values were measured and its ratio to aptamer controls were calculated. The diagnostic values of aptamers were assessed by ROC curve.Results:(1) Screening and separation of aptamers to mixed PHC serum:After3rounds of subtractive SELEX, the nucleic acids bound to normal serum reduced. After9rounds of SELEX with mixed PHC sera,209sequences were isolated by cloning. The sequences at two sites were identical to the library and in the middle were different each other. It indicated a group of HCC serum aptamers were selected successfully.(2) Aptamer structure analysis:imitating analyses of secondary structure of aptamers showed lots of types, including pocket, hairpin, bar, bat, etc. Aptamers could divided into three families by clustering analysis. Multiple motifs were identified in aptamers by alignment.(3) The specificity of aptamers:most aptamers showed stronger bound bands to PHC serum than normal serum, and the free bands were opposite. The largest ratios of free bands were from1.07to6.61(average1.90±0.77). Those results indicated that aptamers binding to HCC serum in different ranges.(4) The affinity of aptamers:10aptamers with obvious bound band were randomly selected to analyze their affinity in binding to mixed HCC serum. Their Kd value were between46nM-640nM.(5) The specificity analysis of aptamers binding single HCC serum:two representative aptamers were observed. ROC curve analysis showed that the AUC, sensitivity and specificity of AP-HCS-9-25were0.969,94.1%and94.1%, and the AP-HCS-9-41were0.945,94.1%and88.2%.Conclusions:(1) We use primary hepatic carcinoma serum as target to perform complex-target subtractive SELEX and separate more than200nucleic acid sequences which are consistent with the library. It demonstrates that we successfully established the method of aptamer selection using serum target and select out a group of aptamers against the serum of primary hepatic carcinoma.(2) The sequences in middle region are different among the aptamers. Imitating analysis shows that the secondary structures of these aptamers were abundant. These aptamers could be divided into3families by cluster analysis, and each family has different motifs. These suggest the aptamers against different targets and epitopes in PHC serum.(3) Most aptamers have stronger bound bands and weaker free bands in primary hepatic carcinoma serum than in normal serum, which is consistent with the band gray value. These results indicate that these aptamers could specifically bind primary hepatic carcinoma serum to different extent;(4) The affinity analyses in some aptamers with clear bound bands to primary hepatic carcinoma serum show their Kd value at nM level, indicating that aptamers could combine the serum with high affinity.(5) Two representative aptamers applied to react with a small sample of single clinical serums show they could specifically bind primary hepatic carcinoma serum, which preliminarily prove their diagnostic value in primary hepatic carcinoma.