RNA aptamers that bind L-arginine with sub-micromolar dissociation constants and high enantioselectivity
RNA aptamers that bind L-arginine with sub-micromolar dissociation constants and high enantioselectivity
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DOI：
10.1093/nar/24.6.1029
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发表时间：
1996-03-15
影响因子：
14.9
通讯作者：
Famulok, M
中科院分区：
文献类型：
--
作者：
Geiger, A;Burgstaller, P;Famulok, M
使用完全随机化的RNA库以及由结合瓜氨酸的解离常数为60 μ M的RNA序列组成的简并库来选择紧密结合的精氨酸特异性RNA适体。应用基于亲和层析的改良的体外选择方案以允许富集高亲和性溶液结合剂，选择方案包括用非同源配体瓜氨酸进行阴性选择，以及在用过量的同源配体精氨酸进行亲和洗脱之前进行热变性步骤，20个循环后，即使在20 mM非同源氨基酸存在下变性/复性后，大多数库也特异性结合精氨酸基质。当在20 mM精氨酸存在下变性和洗脱时，对这些RNA适体进行克隆和测序。用所选序列中最丰富的克隆进行平衡透析，显示RNA/精氨酸亲和力的Kd值为330 nM，这比迄今为止已知的最紧密结合精氨酸的RNA提高了近200倍，这种RNA对精氨酸的识别是高度对映选择性的：L-精氨酸的结合比D-精氨酸好12000倍。化学修饰分析表明，适体的二级结构可能含有一个假结基序。我们的紧密结合精氨酸适体连接了许多天然的和体外选择的识别精氨酸的RNA，这里描述的RNA在它们的结合亲和力方面与其他体外选择实验中分离的低分子量分子的最紧密结合RNA适体进行了比较。
A completely randomized RNA pool as well as a degenerate pool comprised of an RNA sequence which binds citrulline with a dissociation constant of 60 mu M were used to select for tight binding arginine specific RNA aptamers, A modified in vitro selection scheme, based on affinity chromatography was applied to allow the enrichment of high affinity solution binders, The selection scheme included a negative selection with the noncognate ligand citrulline, and a heat denaturation step prior to affinity elution with an excess of the cognate ligand arginine, After 20 cycles the majority of the pools bound specifically to the arginine matrix even after denaturation/renaturation in the presence of 20 mM of a non-cognate amino acid, When denatured and eluted in the presence of 20 mM arginine, the selected RNAs quantitatively washed off the column, These RNA aptamers were cloned and sequenced, Equilibrium dialysis performed with the most abundant clone among the selected sequences revealed K-d values of 330 nM for the RNA/arginine affinity, which is nearly a 200-fold improvement over the tightest binding arginine binding RNAs known to date, Arginine recognition by this RNA is highly enantioselectice: L-arginine is bound 12 000-fold better than D-arginine. Chemical modification analysis revealed that the secondary structure of the aptamer might contain a pseudoknot motif, Our tight binding arginine aptamers join a number of natural and in vitro selected RNAs which recognize arginine, The RNAs described here compare in their binding affinity with the tightest binding RNA aptamers for low molecular weight molecules isolated in other in vitro selection experiments.