Nuclease resistance of an extraordinarily thermostable mini-hairpin DNA fragment, d(GCGAAGC) and its application to in vitro protein synthesis

Satoko Yoshizawa, Takuya Ueda, Yoshiharu Ishido, Kin ichiro Miura, Kimitsuna Watanabe, Ichiro Hirao

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

The nuclease resistance of a short, thermostable minihairpin, d(GCGAAGC), and other related hairpins was examined. Hairpins possessing a purine-rich (GAA) or (GAAA) loop appeared to be more resistant against nucleases than those with a pyrimidine-rich loop or single-stranded oligomers. Among 8 kinds of oligodeoxyribonucleotides examined, the fragment most resistant against nucleases was a hairpin with the sequence of d(CGCGAAGCG). This hairpin was then utilized for the stabilization of mRNA in an in vitro translation system; the 3′-terminal region of an mRNA was hybridized with an oligodeoxyribonucleotide including the sequence complementary to the 3′-terminus of the mRNA tagged with the nuclease-resistant d(CGCGAAGCG) hairpin sequence. By using this method, dihydrofolate reductase (DHFR) mRNA was stabilized against nucleases contaminating a cell-free translation system of E.coli, with a consequent increase in protein synthesis efficiency of 200%.

Original languageEnglish
Pages (from-to)2217-2221
Number of pages5
JournalNucleic acids research
Volume22
Issue number12
DOIs
Publication statusPublished - 1994 Jun 25
Externally publishedYes

ASJC Scopus subject areas

  • Genetics

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