Nuclease resistance of an extraordinarily thermostable mini-hairpin DNA fragment, d(GCGAAGC) and its application to in vitro protein synthesis.
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%.
Department of Chemistry and Biotechnology, Faculty of Engineering, University of Tokyo, Japan.
S
S
Yoshizawa
T
T
Ueda
Y
Y
Ishido
K
K
Miura
K
K
Watanabe
I
I
Hirao
D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism*; D013696:Temperature; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*
D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D015719:Single-Strand Specific DNA and RNA Endonucleases