Abstract
Nucleic acid aptamers have attracted considerable attention as next-generation pharmaceutical agents and delivery vehicles for small molecule drugs and therapeutic oligonucleotides. Chemical modification is an effective approach for improving the functionality of aptamers. However, the process of selecting appropriately modified aptamers is laborious because of many possible modification patterns. Here, we describe a hybrid-type systematic evolution of ligands by exponential enrichment (SELEX) approach for the generation of the artificial nucleic acid aptamers effective against human TROP2, a cell surface protein identified by drug discovery as a promising target for cancer therapy. Capillary electrophoresis SELEX was used for the pre-screening of multiple modified nucleic acid libraries and enrichment of TROP2 binding aptamers in the first step, followed by functional screening using cell-SELEX in the second step for the generation of cell-internalizing aptamers. One representative aptamer, Tac-B1, had a nanomolar-level affinity to human TROP2 and exhibited elevated capacity for internalization by cells. Because of the growing interest in the application of aptamers for drug delivery, our hybrid selection approach has great potential for the generation of functional artificial nucleic acid aptamers with ideal modification patterns in vitro.
Highlights
Nucleic acid aptamers are single-stranded oligonucleotides that are functionally comparable to antibodies
We aimed to develop a new approach, a hybrid-type systematic evolution of ligands by exponential enrichment (SELEX) protocol, for the discovery of artificial nucleic acid aptamers with modification patterns that maximize the affinity to the target protein and allow efficient cell internalization
We have proposed a new approach for discovering artificial nucleic acid aptamers by utilizing the advantages of capillary electrophoresis (CE) and conventional hybrid-SELEX technology
Summary
Nucleic acid aptamers are single-stranded oligonucleotides that are functionally comparable to antibodies. Aptamers can be obtained through the in vitro screening of random nucleic acid libraries by the process known as the systematic evolution of ligands by exponential enrichment (SELEX) [1,2]. (1) separation of sequences bound to the target from a mixture of random oligonucleotide libraries and the target molecule, (2) PCR amplification of target-bound sequences, and (3) purification of single-stranded oligonucleotides for the round of selection. A variety of separation methods have been developed, including conventional protein bead-based SELEX [3], capillary electrophoresis (CE) SELEX [4,5,6,7], microfluidic SELEX [8,9], and flow cytometry SELEX [10].
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