title: 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.
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853.
authors.forename: Sarai
authors.initials: S
authors.lastname: Meyer
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853.
authors.forename: James
authors.initials: J
authors.lastname: Chappell
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853.
authors.forename: Sitara
authors.initials: S
authors.lastname: Sankar
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Chew
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853. jblucks@cornell.edu.
authors.forename: Julius B
authors.initials: JB
authors.lastname: Lucks
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005470:Fluorometry; D017930:Genes, Reporter / Q000235:genetics; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D009690:Nucleic Acid Conformation; D058727:RNA, Small Untranslated / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: D058727:RNA, Small Untranslated; D049452:Green Fluorescent Proteins
keywords: RNA engineering; small transcription activating RNA (STAR); synthetic biology; transcriptional activation; transcriptional regulator