Hüsken Dieter, Asselbergs Fred, Kinzel Bernd, Natt Francois, Weiler Jan, Martin Pierre, Häner Robert, Hall Jonathan
Department of Functional Genomics, Novartis Pharma AG, Lichtstrasse 35, CH-4002 Basel, Switzerland.
Nucleic Acids Res. 2003 Sep 1;31(17):e102. doi: 10.1093/nar/gng103.
A cellular assay has been developed to allow measurement of the inhibitory activity of large numbers of oligonucleotides at the protein level. The assay is centred on an mRNA fusion transcript construct comprising of a full-length reporter gene with a target region of interest inserted into the 3'-untranslated region. Luciferase and fluorescent reporter genes were used in the constructs. The insert can be from multiple sources (uncharacterised ESTs, partial or full-length genes, genes from alternate species, etc.). Large numbers of oligonucleotides were screened for antisense activity against a number of such constructs bearing different reporters, in different cell lines and the inhibitory profiles obtained were compared with those observed through screening the oligonucleotides against the corresponding endogenous genes assayed at the mRNA level. A high degree of similarity in the profiles was obtained indicating that the fusion constructs are suitable surrogates for the endogenous messages for characterisation of antisense oligonucleotides (ASOs). Furthermore, the results support the hypothesis that the secondary structure of mRNAs are divided into domains, the nature of which is determined by primary nucleotide sequence. Oligonucleotides whose activity is dependent on the local structure of their target mRNAs (e.g. ASOs, short interfering RNAs) can be characterised via such fusion RNA constructs.
已经开发出一种细胞测定法，用于在蛋白质水平上测量大量寡核苷酸的抑制活性。该测定法以一种mRNA融合转录本构建体为核心，该构建体由一个全长报告基因组成，在其3'-非翻译区插入了感兴趣的靶区域。构建体中使用了荧光素酶和荧光报告基因。插入片段可以来自多种来源（未表征的EST、部分或全长基因、其他物种的基因等）。针对许多带有不同报告基因的此类构建体，在不同细胞系中筛选了大量寡核苷酸的反义活性，并将获得的抑制谱与通过针对在mRNA水平测定的相应内源基因筛选寡核苷酸所观察到的抑制谱进行了比较。获得了高度相似的谱，表明融合构建体是用于表征反义寡核苷酸（ASO）的内源信息的合适替代物。此外，结果支持以下假设：mRNA的二级结构被分为不同结构域，其性质由一级核苷酸序列决定。其活性取决于靶mRNA局部结构的寡核苷酸（例如ASO、短干扰RNA）可以通过此类融合RNA构建体进行表征。
