Open AccessNuclease resistance of an extraordinarily thermostable mini-hairpin DNA fragment, d(GCGAAGC) and its application toin vitroprotein synthesis
Author(s) -
Satoko Yoshizawa,
Takuya Ueda,
Yoshiharu Ishido,
Koryo Miura,
Kimitsuna Watanabe,
Ichiro Hirao
Publication year - 1994
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/22.12.2217
Subject(s) - nuclease , biology , dna , microbiology and biotechnology , dihydrofolate reductase , messenger rna , translation (biology) , in vitro , rna , biochemistry , gene
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%.