Improved aptazyme design and in vivo screening enable riboswitching in bacteria
Improved aptazyme design and in vivo screening enable riboswitching in bacteria
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DOI：
10.1002/anie.200703700
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发表时间：
2008-01-01
影响因子：
16.6
通讯作者：
Hartig, Joerg S.
中科院分区：
文献类型：
--
作者：
Wieland, Markus;Hartig, Joerg S.
基于人工RNA的基因表达开关的发展受到了大量新近发现的自然存在的核糖开关的启发。[1]其中只有一个例子，GLMS核糖开关，在细菌中被一种小的代谢物激活后，通过核酶介导的RNA信息的切割来操作。[2]另一方面，已经产生了几个基于锤头状核酶(HHR)的所谓的aptazyme的例子，这些例子可以通过与引入的适配子结构域相互作用的配体来控制。[3，4]最近的结果表明，自然发生的锤头包括茎I和茎II之间的第三级相互作用，因此稳定了催化活性构象。[5-7]这种延伸的锤头状核酶显示出更高的活性，即使在细胞内存在低浓度的镁时也能进行有效的切割。[5]在这里，我们提出了一种新的配体控制的锤头状核酶的设计策略，它使得能够构建在细菌中起作用的人造核糖开关。包含茎I/茎II相互作用的快速切割锤头状核酶在哺乳动物细胞培养中以及在动物中被穆利根及其同事用于下调基因表达。[8，9]当HHR插入到mRNA中时，它可以有效地关闭基因表达。此外，核苷类似物被证明通过结合到细胞RNA中从而进入编码的核酶来抑制核酶。这些修饰导致了失活的锤头，从而导致受影响基因的表达水平上升。[8，9]除了这一有趣的发现外，寻找可用于各种生物体(如细菌)的无毒化合物需要通过更特定的机制产生aptazyme。为此，通过向mRNAs中引入适体而不是aptazyme来构建RNA开关。在添加适体特异性配体后，基因表达减少。[10]最近，一个与黄嘌呤类似物茶碱[11]特异结合的适体被插入细菌的mRNA中，使基因表达受到八倍的抑制。[12]使用一种先进的方法，Gallivan和他的同事筛选含有茶碱适配子的mRNAs变体，使基因表达能够有效地上调。[13-15]选定的模块在茶碱结合时触发核糖体结合位点(RBS)的释放。RBS内可访问的Shine-Dalgarno(SD)序列对于有效地启动翻译是必不可少的，[16]而依赖配体的SD掩蔽是自然发生的核糖开关中的一种常见机制。[17]为了构建在体内操作的基于HHR的基因表达开关，我们选择了一种核酶，它包含茎I和茎II的三级接触，能够实现快速切割动力学。此外，我们对核酶进行了改造，使RBS在mRNA切割时释放。这种新的设计是必要的，因为位于5о非翻译区的核酶对细菌基因的切割并不能有效地影响信息的翻译。我们扩展了HHR的茎I，因为它掩盖了SD序列(见SD/抗SD区域，图1A)。二级结构导致有效地抑制基因表达，如在核酶核心引入单点突变使HHR无效所显示的那样(见无效的HHR，图1B)。活性的HHR切割与SD配对的链，导致报告基因表达(参见图1B，HHR)。结合配体…阻断核糖体结合部位可及性策略的研究进展
The development of artificial RNA-based switches of gene expression is inspired by a wealth of recently discovered, naturally occurring riboswitches.[1] Among these, only one example, the glmS riboswitch, operates by ribozyme-mediated cleavage of the RNA message upon activation by a small metabolite in bacteria.[2] On the other hand, several examples of so-called aptazymes based on the hammerhead ribozyme (HHR) have been generated that can be controlled by ligands interacting with introduced aptamer domains.[3, 4] Recent results have shown that naturally occurring hammerheads comprise a tertiary interaction between stemsI and II, thereby stabilizing the catalytically active conformation.[5–7] Such extended hammerhead ribozymes display much higher activities, enabling efficient cleavage even when low magnesium concentrations are present such as inside cells.[5] Here, we present a novel design strategy for ligand-controlled hammerhead ribozymes that enables the construction of artificial riboswitches that function in bacteria. Fast-cleaving hammerhead ribozymes containing stem I/stem II interactions have been used before to down-regulate gene expression in mammalian cell culture as well as in animals by Mulligan and co-workers.[8, 9] The HHR efficiently switches off gene expression when inserted into an mRNA. In addition, nucleoside analogues were shown to inhibit the ribozyme by getting incorporated into cellular RNA and hence into the encoded ribozyme. The modifications result in inactivated hammerheads which lead to elevated expression levels of the affected genes.[8, 9] Besides this interesting finding, the quest for nontoxic compounds that can be used in a variety of organisms such as bacteria requires the generation of aptazymes operating by more specific mechanisms. For this purpose, RNA switches were constructed by introducing aptamers instead of aptazymes into mRNAs. Upon addition of the aptamer-specific ligand, gene expression was reduced.[10] More recently, an aptamer specifically binding the xanthine analogue theophylline [11] was inserted into the mRNA of bacteria allowing eightfold inhibition of gene expression.[12] Using an advanced approach, Gallivan and coworkers screened for variants of mRNAs containing the theophylline aptamer enabling efficient up-regulation of gene expression.[13–15] The selected modules trigger liberation of the ribosomal binding site (RBS) upon theophylline binding. An accessible Shine–Dalgarno (SD) sequence within the RBS is essential for efficient initiation of translation,[16] and liganddependent masking of the SD is a common mechanism in naturally occurring riboswitches.[17] To construct a HHR-based switch of gene expression that operates in vivo, we have chosen a ribozyme that comprises tertiary contacts of stemsI and II enabling fast cleavage kinetics. In addition, we engineered the ribozyme such that liberation of the RBS occurs upon mRNA cleavage. This novel design was necessary since cleavage of bacterial mRNA by a ribozyme positioned in the 5о-untranslated region does not efficiently affect translation of the message. We extended stem I of the HHR such as it masks the SD sequence (see SD/anti-SD region, Figure 1A). Secondary structure results in efficient inhibition of gene expression, as was shown by introducing a single point mutation to the ribozyme core that renders the HHR inactive (see inactive HHR, Figure 1 B). An active HHR cleaves off the strand that pairs with the SD, resulting in reporter gene expression (see Figure 1B, HHR). The developed strategy of blocking accessibility of the ribosome binding site in combination with ligand …