Development of ss DNA aptamers for c-Myc:Max by SELEX (Systematic Evolution of Ligands by Exponential Enrichment)

摘要

Cancer, a complicated disease, results from various causes including abnormal overexpression of oncogene products. The c-myc gene was found as the cellular homolog of v-myc oncogene. Its deregulated expression can result in a wide range of tumors in human and mouse. c-Myc, as a transcription factor, requires the Max protein to form a heterodimer that binds to its target DNA sequence, CACGTG, termed an E box, to drive some cancer-related downstream gene expression. Here a strategy of developing DNA aptamer(s) using SELEX (Systemic Evolution of ligands by Exponential Enrichment) was performed to isolate single-stranded DNA molecules that could recognize the c-Myc:Max protein and inhibit c-Myc\u27s activity in vitro and in vivo , and therefore to develop a new type of reagent for cancer therapy.;Aptamers are small single-stranded DNA or RNA molecules that can bind to a wide range of targets from small molecules, such as ATP, to proteins and even to whole cells, with high specificity and affinity. In the procedure of SELEX, we attempted to combine the \u22decoy\u22 approach to develop an anti-c-Myc aptamer. In the design of the original pool, we put a complement of the E box into the 5\u27 primer followed by a 42 base random sequence. We expected that the selected anti-c-Myc aptamer includes a primary c-Myc binding site that may include a loop-like structure. We also expected that the other complement of E box would be selected from the random region and form a stem structure with the one included in the 5\u27 primer. This E box could then bind to the E box binding domain of the c-Myc:Max heterodimer. So the expected anti-c-Myc aptamer would bind to the target protein by two mechanisms - primary aptamer binding and decoy. The combination of primary binding site of the aptamer to c-Myc:Max and the interaction between the decoy component with c-Myc:Max will make the affinity of the anti-c-Myc aptamer higher than only with one primary binding site or only with the E box.;The data we obtained suggest that the single-stranded DNA aptamers prepared by this \u22decoy\u22 method, including full length and truncated sequences, can bind to the c-Myc:Max heterodimer with very high affinity (Kd\u27s from 100-500 nM) and specificity for the target protein, but can not compete with the E box sequence to bind to c-Myc:Max. It was interesting to find that none of the single-stranded DNA molecules we selected included E box sequence, which means that the selected DNA molecules bind to c-Myc:Max at some other sites instead of an E box binding domain.;There are several directions we could go to optimize the current selected DNA molecules so as to develop anti-c-Myc aptamers. First, we could gradually truncate the current aptamers to determine the minimum sequences for each aptamer. Second, an extra E box sequences could be linked to the DNA sequences by rational design to increase the binding affinity. Third, some random mutations could be introduced into the previously selected DNA sequences by doping. Doping means that when the candidate aptamers were synthesized, the percentage of the four bases, A, T, C and G, is not evenly distributed among each position, so some random variations will be added to the original aptamer sequences. By introducing the new mutations, certain members of the degenerate aptamer pool could have higher binding affinity to target protein than the starting aptamer.