Research Institute for Clinical Oncology, Saitama Cancer Center, Ina, Saitama 362-0806, Japan.
RNA. 2013 Jul;19(7):927-36. doi: 10.1261/rna.037879.112. Epub 2013 May 24.
AML1 (RUNX1) is a key transcription factor for hematopoiesis that binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. Aberrations in the AML1 gene are frequently found in human leukemia. To better understand AML1 and its potential utility for diagnosis and therapy, we obtained RNA aptamers that bind specifically to the AML1 Runt domain. Enzymatic probing and NMR analyses revealed that Apt1-S, which is a truncated variant of one of the aptamers, has a CACG tetraloop and two stem regions separated by an internal loop. All the isolated aptamers were found to contain the conserved sequence motif 5'-NNCCAC-3' and 5'-GCGMGN'N'-3' (M:A or C; N and N' form Watson-Crick base pairs). The motif contains one AC mismatch and one base bulged out. Mutational analysis of Apt1-S showed that three guanines of the motif are important for Runt binding as are the three guanines of RDE, which are directly recognized by three arginine residues of the Runt domain. Mutational analyses of the Runt domain revealed that the amino acid residues used for Apt1-S binding were similar to those used for RDE binding. Furthermore, the aptamer competed with RDE for binding to the Runt domain in vitro. These results demonstrated that the Runt domain of the AML1 protein binds to the motif of the aptamer that mimics DNA. Our findings should provide new insights into RNA function and utility in both basic and applied sciences.
AML1（RUNX1）是造血的关键转录因子，通过其 N 端 Runt 结构域与靶基因的 Runt 结合双链 DNA 元件（RDE）结合。AML1 基因的异常在人类白血病中经常发现。为了更好地理解 AML1 及其在诊断和治疗中的潜在用途，我们获得了特异性结合 AML1 Runt 结构域的 RNA 适体。酶探测和 NMR 分析表明，Apt1-S 是其中一个适体的截断变体，具有 CACG 四链体和由内部环分隔的两个茎区。所有分离的适体都被发现含有保守序列基序 5'-NNCCAC-3'和 5'-GCGMGN'N'-3'（M：A 或 C；N 和 N'形成 Watson-Crick 碱基对）。该基序包含一个 AC 错配和一个碱基突出。Apt1-S 的突变分析表明， motif 中的三个鸟嘌呤对于 Runt 结合很重要，正如 RDE 中的三个鸟嘌呤一样，它们直接被 Runt 结构域的三个精氨酸残基识别。对 Runt 结构域的突变分析表明，用于 Apt1-S 结合的氨基酸残基与用于 RDE 结合的氨基酸残基相似。此外，该适体在体外与 RDE 竞争与 Runt 结构域的结合。这些结果表明，AML1 蛋白的 Runt 结构域结合模拟 DNA 的适体的 motif。我们的研究结果应该为 RNA 的功能和在基础和应用科学中的应用提供新的见解。
