A simple ligand that selectively targets CUG trinucleotide repeats and inhibits MBNL protein binding
A simple ligand that selectively targets CUG trinucleotide repeats and inhibits MBNL protein binding
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DOI：
10.1073/pnas.0901824106
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发表时间：
2009-09-22
影响因子：
11.1
通讯作者：
Zimmerman, Steven C.
中科院分区：
文献类型：
--
作者：
Arambula, Jonathan F.;Ramisetty, Sreenivasa Rao;Zimmerman, Steven C.
这项工作描述了合理的设计，合成和研究的配体，选择性地配合CUG重复在RNA（和CTG重复在DNA中）具有高纳摩尔亲和力。该序列被认为是强直性肌营养不良1型（DM 1）的病原体，因为它能够隔离肌盲样（MBNL）蛋白。从市售化合物分两步合成配体1，并研究其与寡核苷酸中CTG和CUG重复序列的结合。对具有不同序列的1的等温滴定量热法研究显示出对T-T错配的偏好（Kd为390 +/- 80 nM），对单个C-C、A-A和G-G错配的亲和力分别降低13、169和85倍。1至多个CTG步骤序列的结合和Job分析揭示了与每隔一个T-T错配的高亲和力结合，对于近端T-T错配具有负协同性。1对（CUG）4步骤的亲和力提供430 nM的Kd，结合化学计量为1：1。RNA中对U-U的偏好保持不变，对单个C-C、A-A和G-G错配的亲和力分别降低6倍、>143倍和>143倍。配体1使MBNL 1 N与（CUG）（4）和（CUG）（12）之间形成的复合物不稳定，IC 50值分别为52 +/-20 μ M和46 +/-7 μ M，Ki值分别为6 +/-2 μ M和7 +/-1 μ M。这些值仅通过添加竞争性tRNA而发生最小程度的改变。配体1不使不相关的RNA-蛋白质复合物U1 A-SL 2 RNA复合物和性致死-tra RNA复合物不稳定。因此，配体1选择性地使MBNL 1 N-聚（CUG）复合物不稳定。
This work describes the rational design, synthesis, and study of a ligand that selectively complexes CUG repeats in RNA (and CTG repeats in DNA) with high nanomolar affinity. This sequence is considered a causative agent of myotonic dystrophy type 1 (DM1) because of its ability to sequester muscleblind-like (MBNL) proteins. Ligand 1 was synthesized in two steps from commercially available compounds, and its binding to CTG and CUG repeats in oligonucleotides studied. Isothermal titration calorimetry studies of 1 with various sequences showed a preference toward the T-T mismatch (Kd of 390 +/- 80 nM) with a 13-, 169-, and 85-fold reduction in affinity toward single C-C, A-A, and G-G mismatches, respectively. Binding and Job analysis of 1 to multiple CTG step sequences revealed high affinity binding to every other T-T mismatch with negative cooperativity for proximal T-T mismatches. The affinity of 1 for a (CUG) 4 step provided a Kd of 430 nM with a binding stoichiometry of 1:1. The preference for the U-U in RNA was maintained with a 6-, >143-, and >143-fold reduction in affinity toward single C-C, A-A, and G-G mismatches, respectively. Ligand 1 destabilized the complexes formed between MBNL1N and (CUG)(4) and (CUG)(12) with IC50 values of 52 +/- 20 mu M and 46 +/- 7 mu M, respectively, and K-i values of 6 +/- 2 mu M and 7 +/- 1 mu M, respectively. These values were only minimally altered by the addition of competitor tRNA. Ligand 1 does not destabilize the unrelated RNA-protein complexes the U1A-SL2 RNA complex and the Sex lethal-tra RNA complex. Thus, ligand 1 selectively destabilizes the MBNL1N-poly(CUG) complex.