Yoshizawa S, Ueda T, Ishido Y, Miura K, Watanabe K, Hirao I
Department of Chemistry and Biotechnology, Faculty of Engineering, University of Tokyo, Japan.
Nucleic Acids Res. 1994 Jun 25;22(12):2217-21. doi: 10.1093/nar/22.12.2217.
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%.
研究了短的、热稳定的小发夹d(GCGAAGC)及其他相关发夹的核酸酶抗性。具有富含嘌呤（GAA）或（GAAA）环的发夹似乎比具有富含嘧啶环或单链寡聚物的发夹对核酸酶更具抗性。在所研究的8种寡脱氧核糖核苷酸中，对核酸酶最具抗性的片段是具有d(CGCGAAGCG)序列的发夹。然后将该发夹用于体外翻译系统中mRNA的稳定化；mRNA的3'末端区域与包含与用核酸酶抗性d(CGCGAAGCG)发夹序列标记的mRNA的3'末端互补序列的寡脱氧核糖核苷酸杂交。通过使用这种方法，二氢叶酸还原酶（DHFR）mRNA对污染大肠杆菌无细胞翻译系统的核酸酶具有抗性，从而使蛋白质合成效率提高了200%。
