HIGHLY EFFICIENT CHEMICAL SYNTHESIS OF 2'-O-METHYLOLIGORIBONUCLEOTIDES AND TETRABIOTINYLATED DERIVATIVES - NOVEL PROBES THAT ARE RESISTANT TO DEGRADATION BY RNA OR DNA SPECIFIC NUCLEASES
HIGHLY EFFICIENT CHEMICAL SYNTHESIS OF 2'-O-METHYLOLIGORIBONUCLEOTIDES AND TETRABIOTINYLATED DERIVATIVES - NOVEL PROBES THAT ARE RESISTANT TO DEGRADATION BY RNA OR DNA SPECIFIC NUCLEASES
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DOI：
10.1093/nar/17.9.3373
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发表时间：
1989-05-11
影响因子：
14.9
通讯作者：
RYDER, U
中科院分区：
文献类型：
--
作者：
SPROAT, BS;LAMOND, AI;RYDER, U
以5-（4-硝基苯基）-1H-四氮唑为活化剂，以碱保护的5“-O-二甲氧基三苯甲基-2”-O-甲基核糖核苷酸-3“-O-（2-氰乙基N，N-二异丙基亚磷酰胺）为原料，固相合成了2”-O-甲基寡核糖核苷酸。通过三苯甲基阳离子释放判断，偶联产率大于99%。还描述了带有N4-（5-三氟乙酰氨基戊基）间隔基的修饰的2 ″-脱氧胞苷结构单元的制备。后一种化合物能够化学合成携带几个5“-末端生物素化位点（通常使用四个修饰的残基）的2”-O-甲基寡核糖核苷酸探针，其可以方便地使用多核苷酸激酶酶促32 P末端标记。与含有链霉亲和素的衍生物结合使用，这种生物素化探针在RNA-蛋白质复合物的生化纯化和电子显微镜中具有重要的应用。2“-O-甲基寡核糖核苷酸对RNA或DNA特异性核酸酶的降解具有完全抗性。相比之下，具有双重RNA/DNA特异性的核酸酶显示出完整的切割速率谱。
2''-O-Methyloligoribonucleotides have been synthesized on solid phase from base protected 5''-O-dimethoxytrityl-2''-O-methylribonucleotide-3''-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) using 5-(4-nitrophenyl)-1H-tetrazole as activator. Coupling yields greater than 99% were achieved, as judged by trityl cation release. The preparation of a modified 2''-deoxycytidine building block bearing an N4-(5-trifluoroacetylaminopentyl) spacer is also described. The latter compound enabled the chemical synthesis of 2''-O-methyloligoribonucleotide probes carrying several 5''-terminal biotinylation sites (in general four modified residues were used), which can be conveniently 32P end-labelled enzymatically using polynucleotide kinase. Used in conjunction with streptavidin-containing derivatives, such biotinylated probes have important applications in biochemical purification and electron microscopy of RNA-protein complexes. The 2''-O-methyloligoribonucleotides are completely resistant to degradation by either RNA or DNA specific nucleases. In contrast, nucleases with dual RNA/DNA specificity show a complete spectrum of cleavage rates.