Summarizing recent chemical literature
Authors: Hui Shi, Xiaoxiao He, Kemin Wang, Xu Wu, Xiaosheng Ye, Qiuping Guo, Weihong Tan, Zhihe Qing, Xiaohai Yang, Bing Zhou
Affiliation: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Institute of Biology, Hunan University, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, Changsha, China
Journal: Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 3900-05.
Systematic Evolution of Ligands by EXponential enrichment (SELEX) is a technique commonly used to develop and select single-stranded oligonucleotides that specifically bind target molecules with high affinity. Aptamers are the oligonucleotides selected by SELEX that have unique intramolecular interactions that bind to their target molecule with great specificity. Recently, aptamers have been developed for therapeutic applications such as imaging cancer cells in vivo.
Aptamers used for imaging have been described as “always-on” probes because they bind to their target molecule and accumulate around the cells. This increases the signal, making it difficult to distinguish beyond the high background. To overcome this problem, the researchers have designed an activatable aptamer probe (AAP) for cell membrane proteins of living cancer cells for enhanced imaging of cancer in mice. The AAP consists of three portions: a cancer-targeted sequence (A-strand), a poly-T linker (T-strand), and a short DNA sequence complementary to the cancer-targeted sequence with a fluorophore and a quencher at each terminus (C-strand). The researchers hypothesize that once the AAP, which is quenched in absence of a target, is recognized by the target receptor, it undergoes a conformation change that results in an activated fluorescence signal, as indicated in the figure below.
To test their hypothesis, the researchers used an established aptamer, sgc8, which interacts with the cell membrane protein tyrosine kinase-7 (PTK7), as a model system. Sgc8 was selected by SELEX against a human actual lymphoblastic leukemia, CCRF-CEM cell line. The researchers designed an AAP that had good signal-to-background, a near-infrared dye (Cy5), and an efficient quencher (BHQ2). Before testing the AAP in vivo, the activation and specificity of the probe was tested on different types of cell lines. Using flow cytometry to detect fluorescence in four different cells lines, the researchers found that the AAP was indeed specific to it target CCRF-CEM cell line.
The researchers tested the AAP in vivo using fluorescence imaging of CCRF-CEM tumors in mice. Time-dependent images, in comparison to controls indicated that the AAP circulated rapidly through the animal. The probe had prominent fluorescence in the tumor site starting at around 5 min. At 60 min, while the AAP had mostly been cleared from the non-tumor tissues in the mouse, the tumor was still fluorescent. The researchers attributed this to the high affinity of the probe to the target cells, which protect it from degradation. Overall, the AAP strategy has shown to have diagnostic potential with relatively high sensitivity.