Engineering Riboswitches in Vivo Using Dual Genetic Selection and Fluorescence-Activated Cell Sorting

Abstract

Riboswitches, noncoding RNAs that bind a small molecule effector to control gene expression at the level of transcription or translation, are uniquely suited to meet challenges in synthetic biology. To expand the limited set of existing riboswitches, we developed a riboswitch discovery platform that couples dual genetic selection and fluorescence-activated cell sorting to identify novel riboswitches from a 108 random-sequence library in which the aptamer domain of the ThiM#2 riboswitch was replaced with an N40 sequence. In a proof-of-principle validation, we identified novel riboswitches for the small molecule theophylline. Our best riboswitch (Hit 3–5) displays 2.3-fold activation of downstream gene expression in the presence of theophylline. Random mutagenesis of Hit 3–5, coupled with selections and screens, afforded improved riboswitches displaying nearly 3-fold activation. To the best of our knowledge, this is the first report of in vivo directed evolution of an aptamer domain to generate a functional riboswitch.

Attempted use of PACE for riboswitch discovery generates three new translational theophylline riboswitch side products

“…The paper from which we chose RS-C includes several RSs that show higher levels of reporter gene expression than J100377, J100381, and J100382, and one with a larger induction ratio [ 12 ]. Translational RSs with similar characteristics can also be found in other publications [ 14 – 16 ]. We have not measured the theophylline dose–response or explored their modularity with regard to various promoter strengths and reporter genes.…”

Evolving methods for rational de novo design of functional RNA molecules

“…Of course, it is possible to completely skip computational design and instead utilize high-throughput screening or selection approaches to obtain complete RNA devices with the advantage that no model is required [83,36,119]. In such studies, the measurement directly indicates the functionality of the individual devices from the variable pool.…”

Strategies for Improving Small-Molecule Biosensors in Bacteria

“…Similarly, directed evolution has also been used to improve the dynamic and operational range of biosensors, including the muconic acid biosensor BenM [ 96 ], the aromatic aldehyde biosensor PcaV [ 97 , 98 ], and the vanillic acid biosensor VanR [ 98 ]. This approach has also been used to improve RNA-based riboswitches [ 99 ], including sensors for theophylline [ 100 ] and thiamine pyrophosphate [ 101 ]. In contrast to rational engineering methods (discussed below), directed evolution can be performed without the need for significant prior knowledge regarding a biosensor of interest.…”