Selection of an RNA molecule that specifically inhibits the protease activity of subtilisin

Hiroshi Takeno, Seiji Yamamoto, Terumichi Tanaka, Yoshiyuki Sakano, Yo Kikuchi

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

RNA ligands (RNA aptamers) to a protease subtilisin were selected from pools of random RNA by SELEX (systematic evolution of ligands by exponential enrichment) and by use of a subtilisin-immobilized Sepharose column. After eight rounds of selection, RNA aptamers were isolated by cloning to a plasmid vector. We characterized one of the selected RNA molecules. This RNA aptamer displayed specific inhibition toward the subtilisin activity, even when the assay for subtilisin was performed using the chromogenic small peptide as substrate, and almost no inhibitory activity toward trypsin and chymotrypsin, although these enzymes are serine proteases similar to subtilisin. These findings indicate that this RNA can differentially recognize the surfaces of similar proteases. Kinetic analysis of the RNA aptamer revealed that the inhibition constant (K(i)) toward subtilisin was 2.5 μM.

Original languageEnglish
Pages (from-to)1115-1119
Number of pages5
JournalJournal of Biochemistry
Volume125
Issue number6
Publication statusPublished - 1999
Externally publishedYes

Fingerprint

Subtilisin
Nucleotide Aptamers
Peptide Hydrolases
RNA
Molecules
SELEX Aptamer Technique
Chromogenics
Ligands
Cloning
Serine Proteases
Sepharose
Organism Cloning
Assays
Plasmids
Peptides
Kinetics
Substrates
Enzymes

Keywords

  • Aptamer
  • In vitro selection
  • Protease inhibitor
  • SELEX
  • Subtilisin

ASJC Scopus subject areas

  • Biochemistry

Cite this

Takeno, H., Yamamoto, S., Tanaka, T., Sakano, Y., & Kikuchi, Y. (1999). Selection of an RNA molecule that specifically inhibits the protease activity of subtilisin. Journal of Biochemistry, 125(6), 1115-1119.

Selection of an RNA molecule that specifically inhibits the protease activity of subtilisin. / Takeno, Hiroshi; Yamamoto, Seiji; Tanaka, Terumichi; Sakano, Yoshiyuki; Kikuchi, Yo.

In: Journal of Biochemistry, Vol. 125, No. 6, 1999, p. 1115-1119.

Research output: Contribution to journalArticle

Takeno, H, Yamamoto, S, Tanaka, T, Sakano, Y & Kikuchi, Y 1999, 'Selection of an RNA molecule that specifically inhibits the protease activity of subtilisin', Journal of Biochemistry, vol. 125, no. 6, pp. 1115-1119.
Takeno H, Yamamoto S, Tanaka T, Sakano Y, Kikuchi Y. Selection of an RNA molecule that specifically inhibits the protease activity of subtilisin. Journal of Biochemistry. 1999;125(6):1115-1119.
Takeno, Hiroshi ; Yamamoto, Seiji ; Tanaka, Terumichi ; Sakano, Yoshiyuki ; Kikuchi, Yo. / Selection of an RNA molecule that specifically inhibits the protease activity of subtilisin. In: Journal of Biochemistry. 1999 ; Vol. 125, No. 6. pp. 1115-1119.
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