Establishment Of Subtractive Systematic Evolution Of Ligands By Exponential Enrichment Method

Malignancy is one of the main reasons of people death at present, the main reasons of which are the lack of specificity in inchoate diagnose and treatment availably. Compared with the normal cells, there is no qualitative but only quantitative difference for tumor marker found and used in inchoate diagnose. Until now, the combination of operation resection radiative treatment and chemical drug treatment is still the first consideration in tumor treatment. There are lots of side effects for these treatments. Operation resection will cause great wound. Radiation and chemical drugs will kill tumor cells and normal cells without distinguishing. So it is important to find a way to search new tumor markers.Systematic evolution of ligands by exponential enrichment (SELEX) is a combinatorial chemistry methodology for in vitro selection of specific aptamers — oligonucleotide ligands. Considerable progress has been achieved in this field since the establishment of SELEX in 1990. Aptamers specific for a wide variety of targets have been selected, demonstrating that aptamers for almost every target can now be obtained. Nucleic acid aptamers selected by SELEX show high specificity for their targets and could distinguish between homologous proteins or nearly identical low molecular weight compounds. SELEX has been widely used in searching for new oligonucleotides and has many potential applications in basic research, diagnostic and therapeutic purposes.In this work, subtractive SELEX, a new SELEX procedure incorporating the principle of subtraction, was established and successfully used in selecting of ssDNA aptamers, which could recognize differentiated PC 12 cells from undifferentiated PC 12 cells specifically.For establishing of subtractive SELEX method targeted the whole cell, it's necessary to investigate firstly the feasibility that random ssDNA pool was used to perform SELEX selection targeted pure protein. We found that the eletrophoresis action was different in the 10% of denatured polyacrylamide (0.5% of bisacrylamide) gel containing 7M of urea between the same random ssDNA pool and random dsDNApool, the eletrophoresis rate of ssDNA pool was slower than that of dsDNA pool because ssDNA could form complex steric structure. Based on this, firstly we amplified ssDNA and dsDNA using asymmetric PCR method, then ssDNA product was purified with 10% of denatured polyacrylamide (0.5% of bisacrylamide) gel containing 7M of urea for the next round of SELEX selection. The pool capability of the random ssDNA pool used in this work was determined by cloning and the nucleotide sequering. The four bases in the central random region had about equal distribution, and A+T and C+G were about equal in each of the central random position except exceptional position. Then we carried out SELEX process to select streptavidin ssDNA aptamers with this random ssDNA pool. After 12 round selections, we found that the aptamers of final round pool had been enriched by Flow cytometry. The structure analyzed by software RNAstructure 3.5 showed a single stem-loop secondary structure. We approve streptavidin — specific ssDNA aptamers using biotin. It is feasible to determine the enrichment and specificity of the beads or cells using FITC (fluorescein isothiocyanat, FITC) -labeled aptamers by Flow cytometry.Incorporating the principle of subtraction, we established subtractive SELEX method targeted the whole cell. Subtractive selection was performed by firstly incubating ssDNA pools with undifferentiated PC 12 cells in selection buffer prior to each round of selection. Partitioning of bound and unbound ssDNA was done by centrifugation. The subtracted SELEX pools were incubated with differentiated PC 12 cells in selection buffer, cell-bound aptamers were eluted by elution, and were amplified by PCR. PCR products were used as templates to prepare ssDNA by asymmetric PCR for the next round of SELEX. After 6 round of subtractive SELEX selection, Radiolabeled aptamer binding assays results showed that the amount of radioactivity bound to differentiated PC 12 cells was higher than that to undifferentiated PC 12 cells in the 3rd and the 6th round. Flow cytometry with FITC-labeled aptamers showed a slow but steady increase of binding to differentiated PC 12 cells from round 2 to round 6. There was no significant change for the fluorescence intensity bound to undifferentiated PC 12 cells. Compared to the starting pools, G-base content in round 6 became higher, which had about equal distribution of all the four bases. Individual cloned aptamers were evaluated by flow cytometry. All of the 23 sequences showed specific binding to differentiated PC12 cells. No binding to undifferentiated PC 12 cells was observed. Aptamers from the starting pool did not bind to either cell. Among 23 ligands tested, aptamer 17 displayed the most intensive fluorescence binding to differentiated PC 12 cells. Two separate peaks of nearly equal counts resulted from aptamer17 binding, presumably one of the differentiated and theother parental PC 12 cells. These results indicate that aptamer 17 specifically recognizes the differentiated PC 12 cells from a mixture of various cellsThe completion of the aptamer selection process typically yields a high affinity and specific antagonist of the targeted protein. Several postselection optimization steps general must be performed to translate a molecule from an in vitro antagonist to a molecule that can be tested for pharmacologic effect in animals or used in vivo in target-validation studies. Chemical synthesis is required to produce the quantities of aptamer needed for most if not all in vivo experiments. Currently, for efficient and cost-effective chemical synthesis, the size of an aptamer must be reduced to fewer than 45 nucleotides, from a starting molecule of approximately 80 to 100 nucleotides. We truncate aptamer 17 from subtractive SELEX to 25 nucleotides (AP17-25) 38 nucleotides (AP17-38)and 48 nucleotides (API7-48) respectively by cutting defined primer-binding sequences partially or overall. AP17-25 AP17-38 and AP17-48 showed specific binding to differentiated PC 12 cells. No binding to undifferentiated PC 12 cells was observed using both Flow cytometry and fluorescent microscope. Two separate peaks of nearly equal counts resulted from API7-38 binding using Flow cytometry. AP17-38 showed specific binding to cytoplasm of differentiated PC12 cells by laser cofocalize microscope.Above all, we have established subtractive SELEX, and selected successfully ssDNA aptamers with the specificity to distinguish closely related cells. If the specific oligonucleotide ligands could be found to distinguish a certain type of cell from others, e.g. tumor cells from normal cells or cells of different developmental stages, these aptamers might be of important usefulness in basic research and clinical tumor diagnosis. This method may be useful in tumor diagnosis and therapy, and studies of cell differentiation. It also can be applied in finding novel proteins if combined with functional cloning.