Study On The Aptamer Protein Ligands Recognizing Amino Acid Sequences

We have designed and developed a technology that could capture proteins based on the amino acid sequences (CPAS). Mouse cathepsin D was used for the proof of principle experiment. Four tripeptides, LAS, DGI, GEL, and KAI, were selected based on the published sequence of mouse cathepsin D. DNA aptamers against the tripeptides were isolated using Systematic Evolution of Ligands of Exponential enrichment method. We have demonstrated that the aptamers targeted to sythesized tripepetide was able to recognize tripeptides in protein by structure-switch signaling method. Furthermore, cathepsin D could be captured by the magnetic beads which were coated with biotin-labeled aptamers. The aptamers have also been successfully used to detect cathepsin D protein at fM level using rolling circle amplification method. This technology is potential to be applied for protein array development.Isolation of DNA aptamers against tripeptides.The tripeptides were synthesized and coupled to HiTrap NHS-activated column. The starting DNA pool was a mixture of 96-mer single-stranded DNA (ssDNA) molecules that had randomized 60-nucleotide inserts. The ssDNA molecules that bound to the tripeptides were eluted and amplified by PCR. The PCR products were further selected using the tripeptide affinity columns for sub-library generation. The selection procedure was repeated twelve times and the final products were cloned and sequenced. Four aptamers, GEL-aptmer, KAI-aptmer, DGI-aptamer and LAS-aptamer, were used for proof of principle analysis of CPAS technology.Detection of aptamer-protein interaction.We postulated that the DNA aptamer targeting to tripeptide should be able to interact with proteins containing the tripeptide sequences. The structure-switching method was used to test the interaction. In this assay, the fluorescence signals can be detected when the aptamers interact with proteins. Our results demonstrated that addition of the protein resulted in the elevation of fluorescence signals in a dose response manner while the added BSA did not induce any fluorescence signal even at relatively high concentration. These results suggested that DNA aptamers targeting to tripcpetidcs could interact with the proteins containing the tripcptide sequences and the interactions between the aptamers and proteins were specific.Capture of the mouse Cathepsin D protein by combination of several aptamers.We postulated that the combination of four aptamers could interact with all four tripeptide sequences of Cathepsin D, and therefore had the highest affinity for the protein. Similarly, three aptamers should have higher affinity than two aptamers and so on. The partially purified recombinant mouse Cathepsin D protein in a solution containing excess amount of BSA and lysozyme was applied to the magnetic beads which was coated with streptividin and was able to bind biotin-labeled aptamers. The beads were washed with increasing concentrations of salt and finally the protein was eluted with NaOH solution. Our results showed that four aptamers could capture more Cathepsin D protein than three or less aptamers. Next, we directly applied recombinant E. coli iysis containing the mouse Cathepsin D protein to the beads. Three aptamers, LAS, KAI and GEL, could capture the protein as efficiently as four aptamers with the same stringency wash condition.Detection of Cathepsin D protein by RCA based proximity-dependent multiple aptamers ligation assay.Based on proximity-dependent DNA ligation principle, all four aptamers binding to Cathepsin D protein would be ligated to a circle and then can be amplified by rolling circle amplification. We postulated that structure-switch arms, two arms annealing to the aptamer itself, will decrease the background. Four aptamers with structure-switch arms were synthesized. The Cathepsin D protein could enhance the ligation at fM level and can be detected by rolling circle amplification. Recombinant Cathepsin D protein with 3 tripeptieds could not generate any signal even at nM level. The background ligation was very low and the assay was much more sensitive and specific for the Cathepsin D protein.