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Selection‐by‐function: efficient enrichment of cathepsin E inhibitors from a DNA library
Author(s) -
Naimuddin Mohammed,
Kitamura Koichirou,
Kinoshita Yasunori,
HondaTakahashi Yoko,
Murakami Marina,
Ito Masato,
Yamamoto Kenji,
Hanada Kazunori,
Husimi Yuzuru,
Nishigaki Koichi
Publication year - 2006
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/jmr.812
Subject(s) - protease , dna , biotinylation , aptamer , chemistry , peptide library , cathepsin , biochemistry , cathepsin d , systematic evolution of ligands by exponential enrichment , combinatorial chemistry , cathepsin b , cathepsin l , microbiology and biotechnology , biology , enzyme , peptide sequence , gene , rna
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. Copyright © 2006 John Wiley & Sons, Ltd.