Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies

ABSTRACT Regulatory RNAs have become integral components of the synthetic biology and bioengineering toolbox for controlling gene expression. We recently expanded this toolbox by creating small transcription activating RNAs (STARs) that act by disrupting the formation of a target transcriptional terminator hairpin placed upstream of a gene. While STARs are a promising addition to the repertoire of RNA regulators, much work remains to be done to optimize the fold activation of these systems. Here we apply rational RNA engineering strategies to improve the fold activation of two STAR regulators. We demonstrate that a combination of promoter strength tuning and multiple RNA engineering strategies can improve fold activation from 5.4‐fold to 13.4‐fold for a STAR regulator derived from the pbuE riboswitch terminator. We then validate the generality of our approach and show that these same strategies improve fold activation from 2.1‐fold to 14.6‐fold for an unrelated STAR regulator, opening the door to creating a range of additional STARs to use in a broad array of biotechnologies. We also establish that the optimizations preserve the orthogonality of these STARs between themselves and a set of RNA transcriptional repressors, enabling these optimized STARs to be used in sophisticated circuits. Biotechnol. Bioeng. 2016;113: 216–225. © 2015 Wiley Periodicals, Inc. Small transcription activating RNAs (STARs) activate transcription by interfering in trans with the formation of an intrinsic terminator hairpin upstream of a gene of interest. The work of Meyer et al. demonstrates the optimization of fold activation of STARs using several strategies. These strategies are applied to create improved STARs that are orthogonal to one another and to previously developed regulators.