Abstract
Cancer biomarker discovery constitutes a frontier in cancer research. In recent years, cell-binding aptamers have become useful molecular probes for biomarker discovery. However, there are few successful examples, and the critical barrier resides in the identification of the cell-surface protein targets for the aptamers, where only a limited number of aptamer targets have been identified so far. Herein, we developed a universal SILAC-based quantitative proteomic method for target discovery of cell-binding aptamers. The method allowed for distinguishing specific aptamer-binding proteins from nonspecific proteins based on abundance ratios of proteins bound to aptamer-carrying bait and control bait. In addition, we employed fluorescently labeled aptamers for monitoring and optimizing the binding conditions. We were able to identify and validate selectin L and integrin α4 as the protein targets for two previously reported aptamers, Sgc-3b and Sgc-4e, respectively. This strategy should be generally applicable for the discovery of protein targets for other cell-binding aptamers, which will promote the applications of these aptamers.
Highlights
Cancer is the leading cause of morbidity and mortality worldwide, with ϳ14 million new cases and 8.2 million cancer-related deaths in 2012, and the number of new cases is expected to rise by ϳ 70% over the two decades [1]
Aptamer Sgc-3b displays selective binding toward T-lineage leukemia cells (e.g. Molt-4, Sup-T1, or Jurkat E6 –1) [6] and nearly no binding toward normal hematopoietic cells or lymphoma and myeloma cells, whereas aptamer Sgc-4e can bind to several types of cancer cells [15] (Table S1)
Because there are multiple steps involved in aptamer target identification, including protein pull-down, separation, and identification, and the protein targets for aptamer are not known until the last step, we employed 3Ј-fluorescently labeled aptamers for monitoring the aptamer-cell binding so as to optimize the experimental conditions
Summary
Cancer is the leading cause of morbidity and mortality worldwide, with ϳ14 million new cases and 8.2 million cancer-related deaths in 2012, and the number of new cases is expected to rise by ϳ 70% over the two decades [1]. The use of reliable cancer biomarkers for early detection, staging, and individualized therapy may improve patient care. A large number of aptamers exhibiting specific binding toward a variety of cells has been identified by employing cell-based SELEX [6]. These aptamers can recognize the molecular signatures of certain types of cancer cells; cell-surface protein targets of aptamers may serve as candidate biomarkers for these cells. Recent studies have led to the selection of more than 100 cell aptamers, protein targets for only a very limited number of these aptamers have been identified [7], which greatly hampered their applications. By employing crosslinking with the use of an aptamer harboring a photochemically activatable nucleoside, Mallikaratchy et al [11] identified
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.