High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells

Abstract

Synthetic riboswitches mediating ligand-dependent RNA cleavage or splicing-modulation represent elegant tools to control gene expression in various applications, including nextgeneration gene therapy. However, due to the limited understanding of context-dependent structure-function relationships, the identification of functional riboswitches requires largescale-screening of aptamer-effector-domain designs, which is hampered by the lack of suitable cellular high-throughput methods. Here we describe a fast and broadly applicable method to functionally screen complex riboswitch libraries (~1.8 × 10 4 constructs) by cDNAamplicon-sequencing in transiently transfected and stimulated human cells. The selfbarcoding nature of each construct enables quantification of differential mRNA levels without additional pre-selection or cDNA-manipulation steps. We apply this method to engineer tetracycline-and guanine-responsive ON-and OFF-switches based on hammerhead, hepatitis-delta-virus and Twister ribozymes as well as U1-snRNP polyadenylation-dependent RNA devices. In summary, our method enables fast and efficient high-throughput riboswitch identification, thereby overcoming a major hurdle in the development cascade for therapeutically applicable gene switches.

A fluoride-responsive genetic circuit enables in vivo biofluorination in engineered Pseudomonas putida

“…Riboswitches have been adapted as biosensors, affording easy screening of high-producer microbial strains 27 . However, the widespread use of these regulatory devices is limited by their strong contextual effect on gene expression 28,29 . A fluoride ion (F − )responsive riboswitch (FRS) has been described in bacteria and archaea, where it regulates the expression of genes involved in ion detoxification-typically controlling F − transporters 30 .…”

A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors

“…We demonstrated that mutational analysis of individual parent sensor sequences can be performed to improve the in vivo gene regulatory activity of the original sensor sequence. The aptamer regions can also be isolated from DRIVER-selected biosensors and integrated into alternative RNA gene-regulatory platforms which may exhibit improved activities in specific cell types, including microRNAs 31 and ribosome binding sites 38 , or other ribozymes from that used here, including hammerhead, twister, or hepatitis delta virus ribozymes 39 . Finally, a number of high-throughput cell-based assays have been described, including RNA-seq 40 and FACS-seq 27 , that efficiently optimize the gene-regulatory activity of an RNA switch built on the ribozyme platform used here given an identified aptamer sequence.…”

Fundamental studies of functional nucleic acids: aptamers, riboswitches, ribozymes and DNAzymes

“…The authors used cDNA-ampliconsequencing to count conditionally expressed mRNAs in transiently transfected and ligand-stimulated human cells. 131 In contrast to other methods, the new self-barcoding strategy of each riboswitch library member avoided the need for additional cDNA-manipulation steps to introduce external sequencing barcodes. This method was demonstrated for engineering of guanine-and tetracycline-responsive off-and on-switches utilizing twister, hepatitis delta virus, and hammerhead ribozymes as well as U1-snRNP polyadenylation-dependent RNA devices.…”