A high-throughput, quantitative cell-based screen for efficient tailoring of RNA device activity

Abstract

Recent advances have demonstrated the use of RNA-based control devices to program sophisticated cellular functions; however, the efficiency with which these devices can be quantitatively tailored has limited their broader implementation in cellular networks. Here, we developed a high-efficiency, high-throughput and quantitative two-color fluorescence-activated cell sorting-based screening strategy to support the rapid generation of ribozyme-based control devices with user-specified regulatory activities. The high-efficiency of this screening strategy enabled the isolation of a single functional sequence from a library of over 106 variants within two sorting cycles. We demonstrated the versatility of our approach by screening large libraries generated from randomizing individual components within the ribozyme device platform to efficiently isolate new device sequences that exhibit increased in vitro cleavage rates up to 10.5-fold and increased in vivo activation ratios up to 2-fold. We also identified a titratable window within which in vitro cleavage rates and in vivo gene-regulatory activities are correlated, supporting the importance of optimizing RNA device activity directly in the cellular environment. Our two-color fluorescence-activated cell sorting-based screen provides a generalizable strategy for quantitatively tailoring genetic control elements for broader integration within biological networks.

Control of alphavirus-based gene expression using engineered riboswitches

“…The improvement achieved by increasing the copy number of the switch was due to a very low background of expression in the absence of theophylline. The L2b8-a1 switch was identified by highthroughput screening of a randomized library of riboswitch sequences with mutations in the actuator domain of the ribozyme (Liang et al, 2012). Specific mutations in loop I of the ribozyme structure were shown to lower basal expression due to faster cleavage rates compared to the parent ribozyme sequence (Liang et al, 2012).…”

“…The L2b8-a1 switch was identified by highthroughput screening of a randomized library of riboswitch sequences with mutations in the actuator domain of the ribozyme (Liang et al, 2012). Specific mutations in loop I of the ribozyme structure were shown to lower basal expression due to faster cleavage rates compared to the parent ribozyme sequence (Liang et al, 2012). This enhanced ribozyme ability proved to be critical for our replicon system, as it likely overcame the robust RNA replication rate characteristic of these replicons.…”

“…However, the replicon containing the ON switch L2b8-a1 (described in Ref. (Liang et al, 2012)) showed a 3.1-fold increase in gene expression at 24 h with theophylline treatment. This enhanced switching of expression relative to the L2b8 switch was due to a decrease in the background expression observed without theophylline treatment.…”

“…1B). Numerous different ON and OFF switches have been developed and optimized by modifying the structure of the aptamer and actuator domains using both rational tuning strategies (Win and Smolke, 2007) and high-throughput screening (Liang et al, 2012). In addition to the replicons shown in Fig.…”

“…These riboswitches were developed from the hammerhead ribozyme from the satellite RNA of tobacco ringspot virus (sTRSV) conjugated to a theophylline aptamer (Win and Smolke, 2007). Multiple different variants of this switch have been designed to facilitate activation or inactivation of the ribozyme upon addition of theophylline (Liang et al, 2012;Win and Smolke, 2007). By insertion of these riboswitches into the TC-83 replicon, we were able to temporally control antigen expression, as well as modulate the type I IFN response induced by these replicons.…”