Development of site-specific RNA cleaving method using modified DNA splints-RNase H system

建立了RNase H特异性切割RNA位点的方法；已知RNase H仅在RNA- dna杂交体中水解RNA。该方法涉及使用含有四脱氧核糖核苷酸和2'-0-甲基寡核苷酸的嵌合寡核苷酸作为DNA夹板。利用低聚核糖核苷酸（9和18mer）和嵌合夹板（9mer）与大肠杆菌RNase h的杂交体进行模型实验。我们发现核糖链在RNA DNA杂交区域的3'端被特异性地切割。该技术已成功应用于RNA 90mer环和茎区的定点切割。我们还用大肠杆菌tRNA （f Met）检验了该方法的适用性，发现切割发生在受体-茎区所需的位置。

The method for cleaving RNA site-specifically with RNase H was developed; RNase H is known to hydrolyze RNA only in RNA-DNA hybrids. The method invloves the use of the chimeric oligonucleotide containing a tetradeoxyribonucleotide and 2'-0-methyloligoribonucleotide(s) as a DNA splint.Model experiments were carried out using the hybrids of oligo-ribonucleotides (9 and 18mer) and chimeric splints (9mer), and E. coli RNase H. We found that the ribo-strands were cleaved specifically at the 3'-end of the RNA DNA hybrid regions. This technique was successfully applied to the site-directed cleavage of the loop and stem regions in RNA 90mer. We also examined applicability of the method using E. coli tRNA (f Met) and found that the cleavages occurred at desired positions in the acceptor-stem region.