MOLECULAR RECOGNITION OF AMINO-ACIDS BY RNA-APTAMERS - AN L-CITRULLINE BINDING RNA MOTIF AND ITS EVOLUTION INTO AN L-ARGININE BINDER
MOLECULAR RECOGNITION OF AMINO-ACIDS BY RNA-APTAMERS - AN L-CITRULLINE BINDING RNA MOTIF AND ITS EVOLUTION INTO AN L-ARGININE BINDER
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DOI：
10.1021/ja00084a010
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发表时间：
1994-03-09
影响因子：
15
通讯作者：
FAMULOK, M
中科院分区：
文献类型：
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作者：
FAMULOK, M
通过在溶液中特异性结合L-瓜氨酸[L-（+）-2-氨基-5-脲基戊酸]的体外选择，富集了类似于10（15）种不同RNA分子的库。结合池的克隆和测序揭示了存在于所有选择的RNA中的两个高度保守的区域。这些共有序列的长度为10和6个碱基，并且是确定的二级结构的一部分。所有测序的RNA都有可能折叠成相同的结合基序。在此基础上，构建了一个44个核苷酸长的RNA。通过平衡凝胶过滤和分析柱色谱法测定44-mer RNA和L-瓜氨酸的解离常数，结果为62-68 μ M。通过用D-瓜氨酸、L-精氨酸[L-（+）-2-氨基-5-胍基戊酸]、L-合欢苷[L-（+）-2-氨基-3-脲基丙酸]、L-谷氨酰胺和尿素进行亲和洗脱，检测L-瓜氨酸-适体的特异性。与L-瓜氨酸的结合具有弱的对映选择性：D-瓜氨酸的Kd为180 μ M，对应的Δ Δ G为2.5 kJ mol（-1）（0.6 kcal mol（-1））。其他瓜氨酸类似物对RNA适体没有亲和力或亲和力很弱。其中一个L-瓜氨酸结合序列（克隆16）以每个碱基位置30%的突变率重新合成。对该掺杂池进行进一步的选择循环，并在用L-瓜氨酸、L-精氨酸、L-赖氨酸、L-合欢苷和L-谷氨酰胺衍生的琼脂糖上扩增。三个循环后，该池与L-瓜氨酸结合，四个循环后，L-精氨酸的适体出现。不能富集L-合欢苷、L-谷氨酰胺或L-赖氨酸的适体。在来自精氨酸库的22个序列中，11个有可能折叠成与L-瓜氨酸结合基序仅略有不同的基序，并且对其同源配体具有高度特异性。然而，结合库中也含有大量不同的和丰富得多的基序，没有相似性的“父母”的基序。最丰富的RNA对L-精氨酸的Kd值为56-76 μ M，对D-精氨酸的Kd值为410 μ M，这相当于Δ Δ G为4.6 kJ mol（-1）（1.1 kcal mol（-1））。这项研究提供了重新选择RNA适体以改变其结合特异性的第一个例子。
A pool of similar to 10(15) different RNA molecules was enriched by in vitro selection for specific binding to L-citrulline [L-(+)-2-amino-5-ureidovaleric acid] in solution. Cloning and sequencing of the binding pool revealed two highly conserved regions present in all of the selected RNAs. These consensus sequences are 10 and 6 bases in length and are part of a defined secondary structure. All of the sequenced RNAs had the potential to fold into the same binding motif. On the basis of this motif, a 44 nucleotide long RNA was constructed. The dissociation constant of the 44-mer RNA and L-citrulline was determined by equilibrium gel filtration and by analytical column chromatography and was found to be 62-68 mu M. Specificity of the L-citrulline-aptamer was tested by affinity elution with D-citrulline, L-arginine [L-(+)-2-amino-5-guanidinovaleric acid], L-albizziin [L-(+)-2-amino-3-ureidopropionic acid], L-glutamine, and urea. The binding to L-citrulline was weakly enantioselective: D-citrulline had a K-d of 180 mu M which corresponds to a Delta Delta G of 2.5 kJ mol(-1) (0.6 kcal mol(-1)). The other citrulline analogues had no or very weak affinity to the RNA-aptamer. One of the L-citrulline-binding sequences (clone 16) was resynthesized with a 30% mutation rate per base position. This doped pool was subjected to further cycles of selection and amplification on agaroses that were derivatized with L-citrulline, L-arginine, L-lysine, L-albizziin, and L-glutamine. After three cycles, the pool bound to L-citrulline and after four cycles, aptamers for L-arginine showed up. No aptamers to L-albizziin, L-glutamine, or L-lysine could be enriched. Of 22 sequences derived from the arginine pool, 11 had the potential to fold into a motif that was only slightly different from the L-citrulline-binding motif and was highly specific for its cognate ligand. However, the arginine-binding pool also contained a large number of different and much less abundant motifs with no similarity to the ''parent'' motif. The most abundant RNA had values of K-d of 56-76 mu M for L-arginine and a K-d Of 410 mu M for D-arginine, which corresponds to a Delta Delta G of 4.6 kJ mol(-1) (1.1 kcal mol(-1)). This study provides the first example of the reselection of an RNA-aptamer to change its binding specificity.