Synthesis and Use of Modified Oligonucleotides for the Study on a Substrate Recognition Site of RNase H

用耐光性RNA-DNA杂化底物研究了大肠杆菌核糖核酸酶H底物识别位点的光亲和标记。在DNA齐聚物(5‘-GTCATCTCC-3’)的5‘端或内侧引入苯基二氮杂衍生物。在后一种情况下，二嗪基团连接到C2‘修饰的尿苷残基上，该残基具有氨基连接臂，而不是T。光活性杂交体的混合物(DNA被标记为^&lt；32&gt；P)和核糖核酸酶H得到交联物(S)。阳离子交换层析可以将该产物从RNaseH中分离出来，尽管它被起始的核酸污染。每次反应的亲和标记产物用赖氨酰内肽酶消化。用反相高效液相色谱分离得到的多肽。对标记的RNase HS的肽图分析表明，LEP2、3和4上发生了交联反应。有报道称，Asp10、Glu48和Asp70对大肠杆菌RNase H的活性起关键作用(这些氨基酸残基位于上述多肽片段上)。这些数据表明，LEP2、3和4构成了该酶的底物识别位点。

Photoaffinity labeling of a substrate recognition site of E. Coli RNase H has been studied with photolabile RNA-DNA hybrid substrates. A phenyldiazirine derivative was introduced to DNA oligomer (5'-GTCATCTCC-3') at the 5'-end or the inside. In the latter case, the diazirine group was attached to C2'-modified uridine residue having an amino-linker arm which was introduced instead of T.Irradiation of a mixture of the photoactive hybrid (the DNA was labeled with ^<32>P) and RNase H gave crosslinked product(s). The product could be separated from RNase H by cation-exchange chromatography, although it was contaminated with starting nucleic acids. The affinity-labeled product from each reaction was digested with lysyl endopeptidase. The resultant peptides were separated by reverse-phase HPLC. Peptide-mapping analysis of labeled RNase Hs showed that crosslinking reactions had occurred at peptide LEP 2, 3 and 4. It has been reported that Asp 10, Glu48 and Asp 70 were crucial for E. coli RNase H activity (these amino acid residues are located at the above peptide fragments). These data suggest that LEP 2, 3 and 4 constitute the substrate recognition site of the enzyme.