School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China.
Key Laboratory of Industrial Biotechnology, MOE, Jiangnan University, Wuxi, Jiangsu 214122, China.
ACS Synth Biol. 2023 Dec 15;12(12):3716-3729. doi: 10.1021/acssynbio.3c00519. Epub 2023 Dec 5.
Riboswitches are noncoding RNA switches that are largely utilized in bacteria and play a significant role in synthetic biology. Nonetheless, their natural counterparts possess lengthy sequences and intricate structures, posing challenges for their modular integration into complex gene circuits. Consequently, it is imperative to develop simplified synthetic riboswitches that can be effortlessly incorporated into gene circuits. The conventional approach to generate synthetic riboswitches entails tedious library construction and extensive screening, which frequently yields suboptimal performance. To overcome this obstacle, alternative methods are urgently needed. In this study, we created a novel approach to designing a diverse set of transcription-activating riboswitches that exhibit high performance and broad compatibility. The strategy involved starting with a synthetic theophylline RNA aptamer and designing an expression platform that forms a transcriptional terminator in its inactive state but switches to an antiterminator when it is activated. Several sequences were designed, constructed, and subjected to virtual screening, resulting in the identification of two transcription-activating riboswitches. These riboswitches were then engineered to reduce the basal leakage and increase the activation level through extending the hairpin region using a screened random sequence. These architecturally minimal synthetic riboswitches were highly adapted to different constitutive promoters in a modular manner, generating a differentially responsive output to theophylline. As a proof-of-principle, the synthetic riboswitches were applied to rewire a synthetic quorum-sensing circuit (QSC). The reprogrammed QSC successfully modulated the temporal responsive profile against the activation. This strategy is expected to expand the variety of high-performance riboswitches that are responsive to different ligands, thereby further facilitating the design of complex genetic circuits.
核糖开关是非编码 RNA 开关，主要在细菌中被广泛应用，并在合成生物学中发挥着重要作用。然而，它们的天然对应物具有长序列和复杂的结构，这对它们在复杂基因电路中的模块化集成构成了挑战。因此，开发能够轻松集成到基因电路中的简化合成核糖开关是至关重要的。生成合成核糖开关的传统方法需要繁琐的文库构建和广泛的筛选，这通常会导致性能不佳。为了克服这一障碍，迫切需要替代方法。在本研究中，我们创建了一种设计具有高性能和广泛兼容性的多样化转录激活核糖开关的新方法。该策略涉及从合成茶碱 RNA 适体开始，并设计一个表达平台，该平台在其非激活状态下形成转录终止子，但在被激活时转换为抗终止子。设计、构建了多个序列，并进行了虚拟筛选，从而鉴定出两种转录激活核糖开关。然后，通过使用筛选出的随机序列扩展发夹区域，对这些核糖开关进行工程改造，以降低基础泄漏并提高激活水平。这些结构最小的合成核糖开关以模块化的方式高度适应不同的组成型启动子，从而对茶碱产生不同响应的输出。作为原理验证，合成核糖开关被应用于重新布线合成群体感应电路 (QSC)。经重新编程的 QSC 成功地调节了对激活的时间响应特性。该策略有望扩展对不同配体有响应的高性能核糖开关的种类，从而进一步促进复杂遗传电路的设计。
