title: Selection-by-function: efficient enrichment of cathepsin E inhibitors from a DNA library.
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.
authors.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
authors.forename: Mohammed
authors.initials: M
authors.lastname: Naimuddin
authors.forename: Koichirou
authors.initials: K
authors.lastname: Kitamura
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Kinoshita
authors.forename: Yoko
authors.initials: Y
authors.lastname: Honda-Takahashi
authors.forename: Marina
authors.initials: M
authors.lastname: Murakami
authors.forename: Masato
authors.initials: M
authors.lastname: Ito
authors.forename: Kenji
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Kazunori
authors.initials: K
authors.lastname: Hanada
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Husimi
authors.forename: Koichi
authors.initials: K
authors.lastname: Nishigaki
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D052158:Aptamers, Peptide / Q000737:chemistry; D001483:Base Sequence; D002402:Cathepsin D / Q000037:antagonists & inhibitors; D020059:Cathepsin E / Q000037:antagonists & inhibitors*; D004247:DNA / Q000737:chemistry / Q000235:genetics; D015723:Gene Library; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011480:Protease Inhibitors / Q000494:pharmacology*; D051381:Rats; D052156:SELEX Aptamer Technique / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D052158:Aptamers, Peptide; D011480:Protease Inhibitors; D004247:DNA; D020059:Cathepsin E; D002402:Cathepsin D