(1) Introduction

Synthetic biology is a rapidly advancing field that deals with the development and construction of novel biological systems with specific functions. One of the emerging areas within synthetic biology is the use of synthetic small RNAs. These short RNA molecules have a regulatory function. Bacteria use small regulatory RNAs (sRNAs) to regulate mRNA translation. Typical sRNAs consist of a seed and a scaffold region, which are different modules that can be designed and recombined to generate synthetic sRNAs. The seed region is a crucial component in the regulation of gene expression. The scaffold serves as a structural backbone that supports the stability of the sRNA and promotes its interaction with proteins, such as the RNA chaperone Hfq. Synthetic sRNAs offer promising potential for various applications.

(2) Goals

The aim of this study was to test different synthetic seed regions and scaffolds for efficient regulation of gene expression in Escherichia coli.

(3) Material & methods

Golden Gate cloning was used for assembly of seed regions and scaffolds to construct synthetic sRNA expression plasmids. Synthetic seed regions and scaffolds were predicted by computational tools. The functionality of synthetic sRNAs was tested using phenotypic screens.

(4) Results

It is assumed that the regulatory activity of synthetic sRNAs depends on the seed and scaffold regions selected during construction. Since the seed region plays an important role in binding of the target mRNA, selection of the seed region is the crucial step in synthetic sRNA design. However, we observe that some scaffolds are more effective than others in regulating certain targets. We will present computational tools for prediction and design of both seed regions and scaffolds.

(5) Summary

It may be necessary to predict and test various seed regions and sRNA scaffold variants for different mRNA targets to achieve the desired regulatory effect.