분자유전학실험실 (단국대학교 분자생물학과)



 이성욱 ( 2014-04-23 13:58:30 , Hit : 2500
 Tales of Aptamer Selection: Astute Observation and Thoughtful Experimentation or Serendipity?

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Research Update


Selection methods for aptamers use large libraries of random oligonucleotides (~1 X 1015 oligomers).  The theory behind the use of these huge, non-discriminate libraries lies in probability – the probability that within each library is at least one candidate that targets your protein of interest. Thus, the number of molecules that are highly selective for other proteins is vast, many of which must be prime candidates for research, therapeutic or diagnostic tools. While researchers enter an experiment looking for an aptamer targeted to a specific protein, in a recent report, From Ugly Duckling to Swan: Unexpected Identification from Cell-SELEX of an Anti-Annexin A2 Aptamer Targeting Tumors, Frédéric Ducongé and colleagues show that through careful observation and thoughtful experimentation even a failed round of selection can lead to the discovery of a disease-relevant aptamer.  

Nucleic acid aptamers are short sequences of single-stranded RNA or DNA that target a desired molecule, similar to an antibody. However, aptamers contain superior qualities to antibodies because they are more stable, exhibit favorable toxicity profiles, have a simpler and more robust production process, are highly discriminate and can be engineered to contain various properties, such the use of 2′ Fluoro NTPs for nuclease resistance (Keefe and Cload, 2008). First described in 1990, the traditional selection process involved an iterative selection-amplification method aptly named ‘systematic evolution of ligands by exponential enrichment’ (SELEX) (Ellington and Szostak, 1990; Tuerk and Gold, 1990). Since then, alternative methodologies have been developed. One such method, Cell-SELEX, utilizes the same selection-enrichment process, but the target is within its cellular environment, thus containing a heterogeneous mixture of proteins and other cellular components (Morris et al., 1998). Recently, Cibiel et al. used the Cell-SELEX method to screen for an aptamer against human Endothelin Type-B Receptor (ETBR)(Cibiel et al., 2014), a G-coupled protein receptor that mediates vasodilation through the binding of Endothelin-1. Notably, Cell-SELEX allows for membrane proteins to retain their native conformation, essential when targeting receptors, and is useful to target cell-specific receptors and proteins. Important to Cell-SELEX, there must be a negative selection step against a cell type that does not express the desired target (Ohuchi, 2012).

Initially, the authors incubated a random RNA oligo library with wild-type (wt) CHO-K1 cells (for negative selection) and CHO-K1 cells transformed to stably overexpress ETBR (CHO-ETBR) to positively select for ETBR specific aptamers. After 15 rounds of selection and amplification, they found 26 sequences represented 1-7% of the total population and seven of those were found to have significantly higher binding than a scramble sequence when assayed against CHO-ETBR cells and MCR-7 cells, a cell line that naturally expresses the type A and type B endothelin receptors. All seven aptamers also bound to wt CHO cells, suggesting that none of these aptamers are specific for ETBR. Interestingly, three candidates failed to bind after pre-incubation of Endothelin-1. This suggests that while these may not specifically target the ETBR protein, they must interact closely with it. Competitive binding assays demonstrated that two of the aptamers, ACE4 and ACE26, target the same motif of an unknown membrane protein, with ACE4 having the highest binding affinity.

Despite the fact that none of the candidates passed the initial criteria (they bound to both CHO-ETBR and wt CHO-K1 cells), the authors concluded that ACE4 may be an attractive candidate to target cancer specific cells due to that fact that it exhibited 10 times higher binding affinity on MCF-7 cells (a well-studied breast cancer model) as compared to wt CHO-K1 cells. The authors next set out to determine the target protein of ACE4. Initial results showed that ACE4 binds preferentially to Annexin A2, and further studies determined it specifically targets the hetero-tetrameric form, AnxA2t. Interestingly, Annexin A2 had previously been reported to be increased in various cancer models. The authors went on to show that ACE4 efficiently and preferentially binds to several cancer cell lines in vitro. Furthermore, fluorescently-labeled ACE4 is able to be internalized within 30 minutes of incubation with MCF-7 cells. Finally, it was demonstrated that ACE4 binds to MCF-7 tumor xenografts in vivo using fluorescence diffuse optical tomography (fDOT).

In this report, the authors demonstrate that while they did not obtain an aptamer to the intended target, through thoughtful experimentation they found they had a specific and high affinity aptamer to a membrane protein upregulated in many cancers. While its usefulness as a diagnostic or therapeutic tool remains to be seen, the results are promising and they convincingly demonstrate that active investigation into undesirable candidates may result in exciting and relevant aptamers.  Whether considered the fruit of astute observation or simple serendipity, in the wise words of Louis Pasteur, “Fortune favors the prepared mind.”







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  Tales of Aptamer Selection: Astute Observation and Thoughtful Experimentation or Serendipity?  이성욱 2014/04/23 2500
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