Jensen K B, Atkinson B L, Willis M C, Koch T H, Gold L
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder 80309, USA.
Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12220-4. doi: 10.1073/pnas.92.26.12220.
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 RNA 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 Rev. 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.
我们采用了一种名为交联SELEX（SELEX = 通过指数富集进行配体的系统进化）的体外筛选程序，以鉴定与人类免疫缺陷病毒1型（HIV-1）的Rev蛋白具有高亲和力并能交联的RNA序列。用光敏发色团5-碘尿嘧啶取代的随机RNA文库在Rev存在的情况下用单色紫外光照射。那些能够与Rev进行光交联的序列从RNA文库的其余部分中分离出来，进行扩增，并用于下一轮筛选。光交联筛选轮次与对Rev具有高亲和力的RNA序列的筛选轮次交替进行。这种迭代的双选方法产生了具有亚纳摩尔解离常数且能高效与Rev进行光交联的RNA分子。通过该程序分离出的一些RNA分子与Rev形成了稳定的复合物，在无紫外照射时能抵抗变性凝胶电泳。使用修饰核苷酸进行核酸的体外筛选能够分离出具有潜在无限化学能力以共价方式攻击靶分子的核酸分子。
