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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >USING IN VITRO SELECTION TO DIRECT THE COVALENT ATTACHMENT OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 REV PROTEIN TO HIGH-AFFINITY RNA LIGANDS
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USING IN VITRO SELECTION TO DIRECT THE COVALENT ATTACHMENT OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 REV PROTEIN TO HIGH-AFFINITY RNA LIGANDS

机译：使用体外选择直接将人免疫缺陷病毒1型REV蛋白与高亲和力RNA配体直接结合

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摘要

We have used an in vitro selection procedure called crosslinking SELEX (SELEX = systematic evolution of ligands by exponential enrichment) to identify RNA sequences that bind with high affinity and crosslink to the Rev protein from human immunodeficiency virus type 1 (HIV-1). A randomized TC?IA library substituted with the photoreactive chromophore 5-iodouracil was irradiated with monochromatic UV light in the presence of Rev. Those sequences with the ability to photocrosslink to Rev were partitioned from the rest of the RNA pool, amplified, and used for the next round of selection, Rounds of photocrosslinking selection were alternated with rounds of selection for RNA sequences with high affinity to Rev. This iterative, dual-selection method yielded RNA molecules with subnanomolar dissociation constants and high efficiency photocrosslinking to Rer. Some of the RNA molecules isolated by this procedure form a stable complex with Rev that is resistant to denaturing gel electrophoresis in the absence of UV irradiation. In vitro selection of nucleic acids by using modified nucleotides allows the isolation of nucleic acid molecules with potentially limitless chemical capacities to covalently attack a target molecule.
机译：我们已经使用了一种称为交联SELEX（SELEX =通过指数富集的配体的系统进化）的体外选择程序，以鉴定与人免疫缺陷病毒1型（HIV-1）的Rev蛋白具有高亲和力并交联的RNA序列。在Rev的存在下，用单色紫外光照射被光反应性生色团5-碘尿嘧啶取代的TCαIA随机库。将那些具有与Rev进行光交联的能力的序列与其余RNA库分开，进行扩增，并用于在下一轮选择中，交替进行光交联选择和对Rev具有高亲和力的RNA序列的选择。这种迭代，双重选择方法产生具有亚纳摩尔解离常数和与Rer高效光交联的RNA分子。通过此程序分离的某些RNA分子与Rev形成稳定的复合物，在不存在UV辐射的情况下可抵抗变性凝胶电泳。通过使用修饰的核苷酸在体外选择核酸，可以分离具有潜在无限化学能力的核酸分子，以共价攻击靶分子。

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