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

BioRxiv : the Preprint Server for Biology
Sarai MeyerJulius B Lucks

Abstract

Engineered 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 stabilization 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. We also establish that the optimizations preserve the orthogonality of these STARs between themselves and a set of antisense RNA transcriptional repressors, enabling these optimized STARs to be used in more sophisticated circuits. These optimization strategies o...Continue Reading

Related Concepts

Biotechnology
Gene Expression
Genes
Genetic Engineering
RNA
Promoter
Tissue Engineering
Stabilization
Transcription Repressor/Corepressor
Array

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