Sohail M, Southern E M
University of Oxford, Department of Biochemistry, South Parks Road, Oxford OX1 3QU, UK.
Curr Opin Mol Ther. 2000 Jun;2(3):264-71.
Despite the simplicity of the concept, almost every step in an antisense experiment poses difficulties. Finding a site that is accessible to intermolecular hybridization with complementary nucleic acids is a major problem and determines the success or failure of an antisense experiment. A major determinant of accessibility appears to be the intramolecular folding in mRNAs that renders much of the molecule inaccessible. However, owing to our poor understanding of RNA folding and the mechanisms of heteroduplex formation, theoretical methods have limited use in finding accessible sites. Such methods are unable to address two major considerations when designing an antisense reagent, i.e., which is the most accessible sequence in the target and what length of the reagent works best in terms of activity and specificity. Empirical approaches appear more successful. Of notable interest, and reviewed here, are 'global' methods based on DNA arrays and on mapping of transcripts with RNase H.
尽管这个概念很简单，但反义实验中的几乎每一步都存在困难。找到一个能与互补核酸进行分子间杂交的位点是一个主要问题，它决定了反义实验的成败。可及性的一个主要决定因素似乎是mRNA中的分子内折叠，这使得分子的大部分区域无法接近。然而，由于我们对RNA折叠和异源双链体形成机制的了解不足，理论方法在寻找可及位点方面的应用有限。在设计反义试剂时，这些方法无法解决两个主要问题，即目标中最易接近的序列是哪一个，以及试剂的多长长度在活性和特异性方面效果最佳。经验方法似乎更成功。这里值得关注并进行综述的是基于DNA阵列和用RNase H对转录本进行定位的“全局”方法。
