Rational Design\nof High-Efficiency Synthetic Small\nRegulatory RNAs and Their Application in Robust Genetic Circuit Performance\nThrough Tight Control of Leaky Gene Expression

Synthetic sRNAs show promise as tools\nfor targeted and programmable\ngene expression manipulation. However, the design of high-efficiency\nsynthetic sRNAs is a challenging task that necessitates careful consideration\nof multiple factors. Therefore, this study aims to investigate rational\ndesign strategies that significantly and robustly enhance the efficiency\nof synthetic sRNAs. This is achieved by optimizing the following parameters:\nthe sRNA scaffold, mRNA binding affinity, Hfq protein expression level,\nand mRNA secondary structure. By utilizing optimized synthetic sRNAs\nwithin a positive feedback circuit, we effectively addressed the issue\nof gene expression leakagean enduring challenge in synthetic\nbiology that undermines the reliability of genetic circuits in bacteria.\nOur designed synthetic sRNAs successfully prevented gene expression\nleakage, thus averting unintended circuit activation caused by initial\nexpression noise, even in the absence of signal molecules. This result\nshows that high-efficiency synthetic sRNAs not only enable precise\ngene knockdown for metabolic engineering but also ensure the robust\nperformance of synthetic circuits. The strategies developed here hold\nsignificant promise for broad applications across diverse biotechnological\nfields, establishing synthetic sRNAs as pivotal tools in advancing\nsynthetic biology and gene regulation.