Translation of 2'-modified mRNA in vitro and in vivo.
Translation of 2'-modified mRNA in vitro and in vivo.
复制标题
2-修饰 mRNA 的体外和体内翻译。
作者：
H. Aurup;A. Siebert;F. Benseler;D. Williams;F. Eckstein
2'-氟-和2'-氨基-2'-脱氧核苷三磷酸已用于2'-修饰的荧光素酶mRNA 的体外转录。测试了 2'-修饰的含有脱氧核苷的转录物在 X.Laevis 卵母细胞以及兔网织红细胞裂解物中的荧光素酶表达。如 SDS 凝胶以及体内和体外酶活性测量所示，只有 2'-氟-2'-脱氧腺苷修饰的 mRNA 产生荧光素酶。 2'-氟-2'-脱氧嘧啶核苷修饰的 mRNA 不会产生荧光素酶活性。然而，他们将 35S 标记的蛋氨酸掺入兔网织红细胞裂解物的肽片段中，表明翻译过早终止。 2'-氨基修饰的转录本无法检测到或仅检测到极少的此类掺入。
2'-Fluoro- and 2'-amino-2'-deoxynucleoside triphosphates have been used for in vitro transcription of 2'-modified luciferase mRNA. The 2'-modified deoxynucleoside-containing transcripts were tested for the expression of luciferase in X.Laevis oocytes as well as in rabbit reticulocyte lysate. Only 2'-fluoro-2'-deoxy-adenosine-modified mRNA gave rise to luciferase as shown by SDS gel as well as by enzyme activity measurements in vivo as well as in vitro. 2'-Fluoro-2'-deoxy-pyrimidine nucleoside-modified mRNA did not give rise to luciferase activity. However, they directed incorporation of 35S-labeled methionine into peptide fragments in rabbit reticulocyte lysate indicating premature termination of translation. No or only extremely little of such incorporation could be detected with 2'-amino modified transcripts.