Ellis J, Rogers J
Department of Physiology, University of Cambridge, UK.
Nucleic Acids Res. 1993 Nov 11;21(22):5171-8. doi: 10.1093/nar/21.22.5171.
We obtained a partial sequence of mouse calretinin mRNA from cDNA clones, and designed hammerhead ribozymes to cleave positions within it. With a view to optimising hammerhead ribozymes for eliminating the mRNA in vivo, we varied the length and sequence of the three duplex 'arms' and measured the cleavage of long RNA substrates in vitro at 37 degrees C (as well as 50 degrees C). Precise cleavage occurred, but it could only go to completion with a large excess of ribozyme. The evidence suggests that the rate-limiting step with a large target is not the cleavage, but the formation of the active ribozyme: substrate complex. The efficiency varied unpredictably according to the target site, the length of the substrate RNA, and the length of the ribozyme; secondary structure in vitro may be responsible. We particularly investigated the degree of sequence-specificity. Some mismatches could be tolerated, but shortening of the total basepairing with the substrate to less than 14 bp drastically reduced activity, implying that interaction with weakly-matched RNAs is unlikely to be a serious problem in vivo. These results suggest that specific and complete cleavage of a mRNA in vivo should be possible, given high-level expression of a ribozyme against a favourable target site.
我们从cDNA克隆中获得了小鼠钙视网膜蛋白mRNA的部分序列，并设计了锤头状核酶以切割其中的特定位置。为了优化锤头状核酶以在体内消除mRNA，我们改变了三个双链“臂”的长度和序列，并在37℃（以及50℃）下体外测量了长RNA底物的切割情况。精确切割发生了，但只有在大量过量的核酶存在下才能完成。证据表明，对于大的靶标，限速步骤不是切割，而是活性核酶：底物复合物的形成。效率根据靶位点、底物RNA的长度和核酶的长度而不可预测地变化；体外二级结构可能是原因。我们特别研究了序列特异性程度。一些错配是可以容忍的，但与底物的总碱基配对缩短至小于14个碱基对会大大降低活性，这意味着与弱匹配RNA的相互作用在体内不太可能是一个严重问题。这些结果表明，鉴于针对有利靶位点的核酶的高水平表达，在体内对mRNA进行特异性和完全切割应该是可能的。
