Engineering Small Transcription Activating RNAs (STARs)

Efficient engineering of synthetic cellular functions is ultimately dependent on our ability to control gene expression in a precise, predictable and scalable manor. In bacteria, small RNA (sRNA) regulators of gene expression are an increasingly attractive substrate for this. As sRNAs regulate gene expression by formation of RNA structures, they are highly designable and characterizable – with a wealth of computational and experimental tools that can rapidly unlock functional design principles. Moreover, sRNAs are compact and place low-burden on cellular resources, meaning they are potentially highly scalable for multiplexing regulation in a single cell. Finally, sRNA transcriptional regulators can be utilized for higher-order regulation such as logic gates and networks, that process signals rapidly due to fast degradation rates of RNAs.

However, a fundamental gap in both natural and synthetic sRNA regulatory capabilities has been the lack of transcriptional activators – critically limiting their application for higher-order regulation. Recently we have addressed this gap by creating the first Small Transcription Activating RNAs, or STARs. The STAR system involves placing an RNA transcriptional terminator upstream of a target gene that acts as a blockade and prevents the cell from transcribing the gene. When the STAR is present, it removes this blockade, turning ON the downstream gene by allowing transcription to take place. The simplicity of this strategy enabled libraries of highly orthogonal STARs to be created that can be used to control expression of diverse genes, combinatorially combined with libraries of other regulators to allow for tunable and precise control of gene expression and combined to construct RNA-only logic gates. Finally, we present recent work harnessing computational RNA design tools to develop second-generation libraries of highly functional STARs with near digital dynamic ranges that can further expand the utility of this novel class of regulator.