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

Nucleic Acids Res. 1994 Jun 25;22(12):2217-21. doi: 10.1093/nar/22.12.2217.

Abstract

The nuclease resistance of a short, thermostable mini-hairpin, 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%.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • DNA / chemistry*
  • DNA / metabolism
  • Escherichia coli
  • Molecular Sequence Data
  • Nucleic Acid Conformation*
  • Protein Biosynthesis
  • Single-Strand Specific DNA and RNA Endonucleases / metabolism*
  • Temperature
  • Tetrahydrofolate Dehydrogenase / biosynthesis
  • Tetrahydrofolate Dehydrogenase / genetics*

Substances

  • DNA
  • Tetrahydrofolate Dehydrogenase
  • Single-Strand Specific DNA and RNA Endonucleases