Frommer Jennifer, Hieronymus Robert, Selvi Arunachalam Tamil, Heeren Sabine, Jenckel Maria, Strahl Anne, Appel Bettina, Müller Sabine
Ernst Moritz Arndt University Greifswald; Institute for Biochemistry; Greifswald, Germany.
Ernst Moritz Arndt University Greifswald; Institute for Biochemistry; Greifswald, Germany; PG and Research Department of Chemistry; Thiagarajar College (Autonomous); Madurai, India.
RNA Biol. 2014;11(5):609-23. doi: 10.4161/rna.28526. Epub 2014 Mar 26.
In recent years, RNA has been shown to fulfil a number of cellular functions. This has led to much interest in elucidation of the structure of functional RNA molecules, and thus, in the preparation of suitably functionalized RNAs. The chemical synthesis of RNAs allows for the site-specific modification; however, is limited to sequences of about 60-70 nucleotides in length. At the example of the flavine mononucleotide (FMN) responsive aptamer of the ypaA riboswitch from B. subtilis, we demonstrate the highly efficient preparation of site-specifically modified long-mer RNAs. Our strategy consists of the chemical synthesis of fragments followed by enzymatic or chemical ligation. Splint ligation with T4 RNA ligase turned out to be most successful among the enyzymatic protocols. Highly efficient chemical ligation was performed by azide-alkyne cycloaddition of suitably modified RNA fragments. Wild-type and 2-aminopurine (2-AP)-modified variants of the ypaA aptamer were prepared. FMN binding to all synthesized ypaA aptamer variants is demonstrated. However, dissociation of FMN from its binding site by reduction of the isoalloxazin unit as demonstrated before for a small-hairpin-derived aptazyme could not be shown. This implies that either FMN is less accessible to reduction when it is bound to its natural aptamer; that reduced FMN remains bound to the aptamer; or that FMN upon reduction indeed is released from its binding site, without the aptamer folding back in the natural ligand-free state. The results of this study are of general interest to the preparation of site-specifically modified RNAs for investigation into structure and function.
近年来，RNA已被证明具有多种细胞功能。这引发了人们对阐明功能性RNA分子结构的浓厚兴趣，进而也对制备功能适当的RNA产生了兴趣。RNA的化学合成允许进行位点特异性修饰；然而，其长度限制在约60 - 70个核苷酸的序列。以枯草芽孢杆菌ypaA核糖开关的黄素单核苷酸（FMN）响应适体为例，我们展示了高效制备位点特异性修饰的长链RNA的方法。我们的策略包括片段的化学合成，随后进行酶促或化学连接。在酶促方法中，用T4 RNA连接酶进行夹板连接最为成功。通过对适当修饰的RNA片段进行叠氮化物 - 炔烃环加成反应进行高效化学连接。制备了ypaA适体的野生型和2 - 氨基嘌呤（2 - AP）修饰变体。证明了FMN与所有合成的ypaA适体变体的结合。然而，如之前在一个小发夹衍生的适体酶中所证明的，通过异咯嗪单元的还原使FMN从其结合位点解离的情况并未出现。这意味着要么FMN与其天然适体结合时难以被还原；要么还原后的FMN仍与适体结合；要么FMN还原后确实从其结合位点释放，但适体不会折叠回无天然配体的状态。这项研究的结果对于制备用于结构和功能研究的位点特异性修饰RNA具有普遍意义。
