Selection-by-function: efficient enrichment of cathepsin E inhibitors from a DNA library |
| |
Authors: | Naimuddin Mohammed Kitamura Koichirou Kinoshita Yasunori Honda-Takahashi Yoko Murakami Marina Ito Masato Yamamoto Kenji Hanada Kazunori Husimi Yuzuru Nishigaki Koichi |
| |
Affiliation: | Rational Evolutionary Design of Advanced Biomolecules (REDS), Saitama Small Enterprise Promotion Corporation, SKIP city, 3-12-18 Kamiaoki, Kawaguchi, Saitama 333-0844, Japan. naimuddin@janusys.co.jp |
| |
Abstract: | A method for efficient enrichment of protease inhibitors out of a DNA library was developed by introducing SF-link technology. A two-step selection strategy was designed consisting of the initial enrichment of aptamers based on binding function while the second enrichment step was based on the inhibitory activity to a protease, cathepsin E (CE). The latter was constructed by covalently linking of a biotinylated peptide substrate to each of the ssDNA molecule contained in the preliminarily selected DNA library, generating 'SF-link'. Gradual enrichment of inhibitory DNAs was attained in the course of selection. One molecule, SFR-6-3, showed an IC(50) of around 30 nM, a K(d) of around 15 nM and high selectivity for CE. Sequence and structure analysis revealed a C-rich sequence without any guanine and possibly an i-motif structure, which must be novel to be found in in vitro-selected aptamers. SF-link technology, which is novel as the screening technology, provided a remarkable enrichment of specific protease inhibitors and has a potential to be further developed. |
| |
Keywords: | aptamers directed evolution substrate‐function link activity‐based selection proteases inhibitors |
本文献已被 PubMed 等数据库收录! |
|