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具有稀有碱基的生物活性天然转运RNA的化学合成。

Chemical synthesis of a biologically active natural tRNA with its minor bases.

作者信息

Gasparutto D, Livache T, Bazin H, Duplaa A M, Guy A, Khorlin A, Molko D, Roget A, Téoule R

机构信息

Département de Recherche Fondamentale de la Matière Condensée, CIS Bio International, Centre d'Etudes Nucléaires de Grenoble, France.

出版信息

Nucleic Acids Res. 1992 Oct 11;20(19):5159-66. doi: 10.1093/nar/20.19.5159.

DOI:10.1093/nar/20.19.5159
PMID:1383941
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC334300/
Abstract

The complete chemical synthesis of an E. coli tRNA(Ala) with its specific minor nucleosides, dihydrouridine, ribothymidine and pseudouridine, is reported. The method makes use of protected 2'-O-tertiobutyldimethylsilyl-ribonucleoside-3'-O-(2-cyanoethyl-N- ethyl-N- methyl)phosphoramidites. The exocyclic amino functions of the bases were protected by the phenoxyacetyl group for purines and acetyl for cytosine. The assembling has been performed on a silica support with coupling yield better than 98% within 2 min of condensation. Triethylamine tris-hydrofluoride allowed a clean and complete deprotection of the tBDMS groups. The synthetic tRNA(Ala) has been transcribed into cDNA by reverse transcriptase and sequenced. With E. coli alanyl-tRNA synthetase the alanyl acceptance activity and kcat/Km were 672 pmol/A260 and 6 x 10(4)M-1s-1, respectively.

摘要

报道了具有其特定稀有核苷（二氢尿苷、核糖胸腺嘧啶核苷和假尿苷）的大肠杆菌丙氨酸转运核糖核酸（tRNAAla）的全化学合成。该方法使用了受保护的2'-O-叔丁基二甲基甲硅烷基核糖核苷-3'-O-（2-氰基乙基-N-乙基-N-甲基）亚磷酰胺。碱基的环外氨基官能团对于嘌呤用苯氧乙酰基保护，对于胞嘧啶用乙酰基保护。组装在硅胶载体上进行，缩合2分钟内偶联产率优于98%。三乙胺三氢氟化物能对叔丁基二甲基甲硅烷基（tBDMS）基团进行彻底且完全的脱保护。合成的tRNAAla已通过逆转录酶转录成互补DNA（cDNA）并进行了测序。对于大肠杆菌丙氨酰-tRNA合成酶，丙氨酰接纳活性和催化常数与米氏常数的比值（kcat/Km）分别为672皮摩尔/A260和6×104M-1s-1。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1acb/334300/b596dde01cd1/nar00230-0196-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1acb/334300/78fedd1c2972/nar00230-0195-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1acb/334300/b596dde01cd1/nar00230-0196-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1acb/334300/78fedd1c2972/nar00230-0195-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1acb/334300/b596dde01cd1/nar00230-0196-a.jpg

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Abasic and oxidized ribonucleotides embedded in DNA are processed by human APE1 and not by RNase H2.嵌入DNA中的无碱基和氧化核糖核苷酸由人类APE1处理，而非由核糖核酸酶H2处理。
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