Selection And Characterization Of SsDNA Aptamers To Ras Protein In Vitro

Part One Design and synthesis of a random ssDNA library and Optimization of PCR conditionsObjective To synthesis a random ssDNA library and optimize the PCR conditionsMethods A random ssDNA library with 78 bp in length containing a random sequence of 35 bp which was flanked by a 23 bp constant region and a 20 bp constant region was synthesized by a biological engineering company in vitro. The PCR conditions including the appropriate heat cycles and annealing temperatuer in symmetric PCR and the best proportion between forward primer and backward primer in asymmetric were optimized. The agarose electrophiresis was applied to observe the products of PCR products.Results After the agarose electrophiresis, the 78 bp ssDNA was observed near th 40-50 bp dsDNA ladder and 78 bp dsDNA, the 80 bp dsDNA. In symmetric PCR, the appropriate heat cycles were 24 and annealing temperatuer is 60℃. In indirect asymmetric PCR, the proportion of two primers was 100:1 and the heat cycles were 40. the direct asymmetric PCR amplification did not get ssDNA but by-products.Conclusion It was obtained that a a random ssDNA library, primers and optimized symmetric and indirect asymmetric PCR conditions. In the optimized PCR conditions, the both can get more objective products and less by-products. Direct asymmetric PCR conditions failed to be optimized, but they did not affect the following experiments. Part Two Selection of ssDNA aptamers to Ras protein in vitroObjective To abtain the high affinity ssDNA aptamers to Ras protein by selection using systematic evolution of ligands by exponential enrichment(SELEX) in vitro.Methods The synthesized original library was incubated with Ras protein coated on Nunc96 in SELEX binding buffer. The bound ssDNAs were collected and amplified by asymmetric PCR or streptavidin magnesphere paramagnetic particles to prepare the sub-library for the next round of selection. The selection and amplification were reiterated until the binding rate no longer increased significantly. The binding rate of every round aptamers bound to Ras protein was measured. The binding affinities between the aptamers and Ras protein were measured by ELISA.Results The sub-library could be satisfactorily obtained by asymmetric PCR or streptavidin magnesphere paramagnetic particles. The binding rate of aptamers bound to Ras protein increased gradually from 2.3% to 32.4% after 11 rounds selection and is the highest between the 11th aptamers and Ras protein. OD increased gradually from 0.215 to 1.033.Conclusion The high affinity ssDNA aptamers to Ras protein have been successfully selected.Part Three Clone, identification and sequencing of ssDNA aptamers to Ras proteinObjective To clone, identificate and sequence the 11th ssDNA aptamers to Ras protein and summarize the relationship between their structures and binding affinities.Methods The 11th aptamers were cloned and 50 of them were randomly selected to sequence. The primary structures of the sequences were analyzed and the secondary structures were imitated by DNAMAN 5.29 software. The biotin-labeled sequences were synthesized and the binding affinities to the Ras protein were measured by ELISA. The relationship between structures and binding affinities was summarized.Results The constant regions of 50 sequences were all identical to identical to the designed. The number of the base pair in random region was 35 in 45,34 in 3,33 in one,36 in one, and two of 45 sequences were same. The primary structure analysis showed that there were 10 families in 45 sequences. There were different conserved sequences from 1th to 9th family and no ones in 10th. The secondary structure analysis showed that the conformations were different and there was only one type:stem-loops. The binding affinities showed that OD was from 0.742 to 1.213. Aptamer Ral and Ra2 had more small stem-loops and their OD was 1.213 with the highest affinity. Aptamer Ra27 had less small stem-loops and its OD was 0.742 with the lowest affinity. The most conserved sequences located at stem-loops with angles.Conclusion The high affinity ssDNA aptamers to Ras protein have been successfully selected. The more small stem-loops maybe is the binding site of aptamers to Ras protein and conserved sequences maybe play an important role in the binding. Our research work that have been done makes a good foundation for the future studies in this regard.