Optimization and Modifications of Aptamers Selected from Live Cancer Cell Lines

Aptamers are single-stranded oligonucleotides derived from an in vitro evolution process called SELEX (systematic evolution of ligands by exponential enrichment). 2] The selected aptamers can recognize their target molecules with high affinity and specificity by folding into well-defined three-dimensional shapes. Recently, using a human T-cell acute lymphoblastic leukemia cell line as target, we generated a group of aptamers for the specific recognition of leukemia cells. The aptamers have an equilibrium dissociation constant (Kd) in the nm to pm range. They can specifically recognize target leukemia cells that have been mixed with normal human bone marrow aspirates, can identify cancer cells closely related to the target cell line in clinical specimens, and can also be used to enrich target cells spiked in blood samples. 6] These aptamers are very promising for molecular recognition in a variety of applications. Unlike antibodies, aptamers have low molecular weight, fast tissue penetration, and low toxicity, and can be reproducibly produced with a DNA synthesizer. They can be easily labeled with radioscopic, fluorescent, or other reporters. Moreover, aptamers remain stable during long-term storage and sustain reversible denaturation. These advantages make aptamers uniquely suitable as molecular probes for diagnosis or as drugs for targeted cancer therapy. Even though regular DNA molecules are sturdier than antibodies in vitro, in vivo stability is still a serious problem. To serve as effective therapeutic and diagnostic tools, aptamers must resist rapid degradation by exoand endonucleases. In this paper, we present the results of experiments for the design of aptamers with excellent biostability, affinity, and specificity for the study of diseases. Full-length aptamers generated by the SELEX process contain 70–100 nucleotides, which include two fixed primer sequences at each terminus for PCR amplification. Generally, not all nucleotides are necessary for direct interaction with the target or for folding into the structure that facilitates target binding. There is no doubt that longer sequences result in lower yield and higher cost in synthesis. The unnecessary nucleotides could also possess a higher probability of forming various secondary structures that destabilize the target-binding conformation of the aptamer. Additionally, reduced size has been reported to increase tissue penetration rates. Thus, in practical usage, it is always beneficial to obtain minimized sequences of aptamers that possess the same or better binding affinity to the target compared to the original full-length aptamer. For RNA aptamers, RNase footprinting or partial hydrolysis has been performed to determine the boundary and binding site of aptamers. For DNA aptamers, the minimal sequence has been determined by partially fragmenting a full-length aptamer, and then selecting the fragments that retained high affinity for the target. In these methods, radioactive labeling was needed to detect the aptamer fragments, and then the predicted potential minimal sequences had to be synthesized to confirm binding capacity. In this study, we utilized a relatively easy method to determine the secondary structures of aptamers as well as the critical sequences for target binding. At the end of the selection process, the enriched pool with high affinity to the target was sequenced. After alignment, the sequences were found to distribute into different families based on their sequence similarities, and many repeats were observed in each family. In the same family they sometimes showed very different affinities for the target even though sequences had the same motif and the difference was only a few nucleotides. The relationship ACHTUNGTRENNUNGbetween the sequences of the same family and their affinities can help us deduce the secondary structure of the aptamer that is needed for target binding. The potential secondary structures of DNA sequences can be predicted by a few algorithms. The free energy change (DG) of each structure was also calculated as an indication of structural stability. However, the lowest DG did not necessarily correspond with the actual target-binding structure, since target-induced refolding of the aptamer is common in aptamer–target interactions. On the other hand, sequences of the same family should usually bind to the same target with similar secondary structures but different Kd values. Thus, when searching for the target-binding structure, we gave higher priority to the calculated structures that were present for every sequence in the same family. Further analysis determined the target-binding structure which correlated well with the differences in the Kd values of the ACHTUNGTRENNUNGsequences. As an example, the two sequences Sga16 (Kd=5 nm) and Sgc8 (Kd=0.8 nm) in one of the sequence families, only differed by two nucleotides (G38, A69 in Sga16, and A38, T69 in Sgc8; Figure 1A). There are 11 potential structures of aptamer Sgc8 and thirteen of Sga16, as predicted by a program available on the internet. Among these structures, only two pairs of structures (Figure S1 in the Supporting Information) were commonly present. By further comparing the two structure pairs, we found that one structure pair could explain the different Kd values of Sga16 and Sgc8 (Figure 1A). In this structure pair, the differences in base sequence are in the stem section between loops 1 and 2 (indicated by arrows in Figure 1A). Namely, aptamer Sgc8 has a perfect-match stem while aptamer Sga16 has a one-base-mismatch stem. The mismatched base [a] Dr. D. Shangguan, Dr. Z. Tang, P. Mallikaratchy, Z. Xiao, Prof. Dr. W. Tan Department of Chemistry and Shands Cancer Center UF Genetics Institute and McKnight Brain Institute Center for Research at Bio/nano Interface, University of Florida Gainesville, FL 32611 (USA) Fax: (+1)352-846-2410 E-mail : tan@chem.ufl.edu Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.