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

利用可光性RNA-DNA杂交底物研究了大肠杆菌RNase H底物识别位点的光亲和标记。在DNA低聚物（5'-GTCATCTCC-3‘）的5’端或内部引入苯基二氮嘧啶衍生物。在后一种情况下，重氮嘧啶基团连接到C2'修饰的尿嘧啶残基上，该残基具有氨基连接臂，而不是t。光活性杂交（DNA标记为^<32>P）和RNase H的混合物照射产生交联产物。虽然产物被起始核酸污染，但通过阳离子交换层析可以从RNase H中分离出来。每个反应的亲和标记产物用赖氨酸内肽酶消化。所得肽用反相高效液相色谱法分离。标记RNase Hs的肽定位分析表明，在肽lep2、3和4处发生了交联反应。据报道，Asp 10、Glu48和Asp 70对大肠杆菌RNase H活性至关重要（这些氨基酸残基位于上述肽片段上）。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.