Aurup H, Siebert A, Benseler F, Williams D, Eckstein F
Max-Planck-Institut für experimentelle Medizin, Göttingen, Germany.
Nucleic Acids Res. 1994 Nov 25;22(23):4963-8. doi: 10.1093/nar/22.23.4963.
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.
2'-氟代和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。对含2'-修饰脱氧核苷的转录本进行了检测,以观察其在非洲爪蟾卵母细胞以及兔网织红细胞裂解物中荧光素酶的表达情况。如SDS凝胶所示,以及体内和体外的酶活性测量结果表明,只有2'-氟-2'-脱氧腺苷修饰的mRNA产生了荧光素酶。2'-氟-2'-脱氧嘧啶核苷修饰的mRNA未产生荧光素酶活性。然而,它们在兔网织红细胞裂解物中引导35S标记的甲硫氨酸掺入肽片段,表明翻译提前终止。对于2'-氨基修饰的转录本,未检测到或仅检测到极少的这种掺入。