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.

Development of Systemic in vitro Evolution and Its Application to Generation of Peptide-Aptamer-Based Inhibitors of Cathepsin E

“…1c and 2b), which enables selection-by-function (where those peptides that have succeeded to inhibit the protease can survive but those that have failed to inhibit it are removed during a cycle of screening as shown in Fig. 1c), a method that was initially devised for the selection of DNA aptamers, 27 which we have modified in this article for selecting peptide aptamers (for details, see Materials and Methods). Briefly, CE substrate sequence is linked to each inhibitory peptide sequence in a single molecule of the cDNA display construct.…”

“…In brief, the first technique is to generate a huge diversity (∼10 12 ) of molecules with a cDNA display construct 24,25 (i.e., an advanced version of mRNA display that displays a nascent protein linked to the mRNA-derived cDNA via puromycin), employing a shuffling technique, YLBS 26 (Y-ligationbased block shuffling), which generates combinatorial diversity of molecules (see Materials and Methods). The second technique, termed selectionby-function, 27 involves selection of peptides based on both affinity and function, in this case, inhibition of proteolytic activity. The third technique involves the generation of secondary libraries, which allows the generation and selection of clones with greater activity than in the first library, through the use of the block-shuffling method.…”

SELEX—A (r)evolutionary method to generate high-affinity nucleic acid ligands

“…Some add the biotin molecule to the unwanted strand and use the arising size difference in gel electrophoresis to distinguish between both strands (Fitzwater and Polisky, 1996). Others let the dsDNA (only one strand biotinylated) bind to streptavidin surfaces (beads or plates) and separate the strands after DNA denaturation (Naimuddin et al, 2007). Another possibility is to perform an asymmetric PCR which uses only one or a much bigger amount of one primer to obtain ssDNA products (Wu and Curran, 1999).…”

Inhibitors of Proteinases as Potential Anti-Cancer Agents

“…The new approach to the identification of inhibitors is appropriate selection of DNA aptamers strongly interacting with chosen enzyme. This methodology was used to identify the aptamer SF-6-3, which selectively and very strongly binds cathepsin E (Naimuddin et al, 2007).…”