TY - JOUR
T1 - Nuclease resistance of an extraordinarily thermostable mini-hairpin DNA fragment, d(GCGAAGC) and its application to in vitro protein synthesis
AU - Yoshizawa, Satoko
AU - Ueda, Takuya
AU - Ishido, Yoshiharu
AU - Miura, Kin ichiro
AU - Watanabe, Kimitsuna
AU - Hirao, Ichiro
N1 - Funding Information:
The authors are grateful to Drs Masahiro Iwakura and Shinichi Ohashi of the Institute of Polymers and Textiles, the Japanese Ministry of International Trade and Industry for providing plasmid pBBKlOMM carrying the DHFR gene. The present work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (04226104) from the Japanese Ministry of Education, Science and Culture. S.Y. is supported by a fellowship from Nippon Zeon Co., Ltd.
PY - 1994/6/25
Y1 - 1994/6/25
N2 - 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%.
AB - 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%.
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U2 - 10.1093/nar/22.12.2217
DO - 10.1093/nar/22.12.2217
M3 - Article
C2 - 8036147
AN - SCOPUS:0028309010
SN - 0305-1048
VL - 22
SP - 2217
EP - 2221
JO - Nucleic acids research
JF - Nucleic acids research
IS - 12
ER -