Data Sheet 1_Hammerhead ribozymes directed against mRNA of an essential gene inhibit Escherichia coli growth and enhance tetracycline efficacy.pdf

Aiming to find novel ways to inhibit bacterial growth, we tested hammerhead ribozymes targeting the mRNAacpP transcript, which encodes the essential acyl carrier protein in Escherichia coli. We engineered ribozymes with varying catalytic cores and arm lengths, finding that while short-armed ribozymes showed higher activity in vitro, long-armed variants demonstrated superior growth inhibition in vivo. Isothermal titration calorimetry confirmed tight binding between the ribozymes and the mRNA substrate, with association constants between 107 and 108 M−1, and gel electrophoresis verified substrate cleavage. Ribozymes were incorporated into bacterial plasmids, introduced via transformation into E. coli, and were expressed in a controlled manner, inhibiting bacterial growth by up to 70% over 24 h. Notably, ribozymes embedded within tRNA structures, a strategy intended to protect them from intracellular degradation, showed differential effectiveness compared to standalone variants; tRNA scaffolding preserved activity in long-armed but abolished it in short-armed constructs. Growth inhibition resulted from both mRNA cleavage and translational blocking, as demonstrated by comparing active ribozymes with their catalytically inactive variants. Furthermore, tetracycline efficacy was enhanced 2- to 4-fold in cells expressing ribozymes, indicating potential for synergy. This study demonstrates the first successful targeting of an essential gene in E. coli using hammerhead ribozymes, achieving growth inhibition through combined mechanisms of mRNA blocking and cleavage, and highlighting the potential of ribozymes as antibacterial strategies.

为探索抑制细菌生长的全新策略，本研究针对编码大肠杆菌（Escherichia coli）必需酰基载体蛋白的acpP基因mRNA转录本，测试了靶向该序列的锤头状核酶（hammerhead ribozyme）。我们构建了催化核心与臂长各不相同的核酶变体，研究发现：尽管短臂核酶在体外（in vitro）中表现出更高的催化活性，但长臂变体在体内（in vivo）中展现出更优异的生长抑制效果。等温滴定量热法（Isothermal titration calorimetry）证实核酶与mRNA底物间存在紧密结合，结合常数介于10⁷至10⁸ M⁻¹之间；凝胶电泳（gel electrophoresis）验证了底物剪切现象的发生。研究人员将核酶整合至细菌质粒（plasmid）中，通过转化法导入大肠杆菌，并实现了可控表达，在24小时内可将细菌生长抑制最高达70%。值得注意的是，嵌入转运RNA（transfer RNA，tRNA）结构中的核酶——这一策略旨在保护核酶免受胞内降解——与游离变体相比表现出差异化的作用效果：tRNA支架可保留长臂核酶的活性，却会使短臂核酶的活性完全丧失。生长抑制效应同时源于mRNA剪切与翻译阻断（translational blocking），这一点通过对比活性核酶与催化失活变体的实验结果得到了证实。此外，在表达核酶的大肠杆菌中，四环素（tetracycline）的抗菌效能提升了2至4倍，表明二者具备协同应用的潜力。本研究首次实现了利用锤头状核酶靶向大肠杆菌必需基因的成功案例，通过mRNA阻断与剪切的联合机制实现细菌生长抑制，凸显了核酶作为抗菌策略的应用潜力。