Reprogramming Gene Expression by Targeting RNA-Based Interactions: A Novel Pipeline Utilizing RNA Array Technology

Abstract

Regulatory RNA-based interactions are critical for coordinating gene expression and are increasingly being targeted in synthetic biology, antimicrobial, and therapeutic fields. Bacterial trans-encoded small RNAs (sRNAs) regulate the translation and/or stability of mRNA targets through base-pairing interactions. These interactions are often integral to complex gene circuits which coordinate critical bacterial processes. The ability to predictably modulate these gene circuits has potential for reprogramming gene expression for synthetic biology and antibacterial purposes. Here, we present a novel pipeline for targeting such RNA-based interactions with antisense oligonucleotides (ASOs) in order to reprogram gene expression. As proof-of-concept, we selected sRNA–mRNA interactions that are central to the Vibrio cholerae quorum sensing pathway, required for V. cholerae pathogenesis, as a regulatory RNA-based interaction input. We rationally designed anti-sRNA ASOs to target the sRNAs and synthesized them as peptide nucleic acids (PNAs). Next, we devised an RNA array-based interaction assay to allow screening of the anti-sRNA ASOs in vitro. Finally, an Escherichia coli-based gene expression reporter assay was developed and used to validate anti-sRNA ASO regulatory activity in a cellular environment. The output from the pipeline was an anti-sRNA ASO that targets sRNAs to inhibit sRNA–mRNA interactions and modulate gene expression. This anti-sRNA ASO has potential for reprogramming gene expression for synthetic biology and/or antibacterial purposes. We anticipate that this pipeline will find widespread use in fields targeting RNA-based interactions as modulators of gene expression.

Identifying antisense oligonucleotides to disrupt small RNA regulated antibiotic resistance via a cell-free transcription-translation platform

“…Theoretically, this is due to the increased influx of antibiotics through continued OmpF porin expression caused by the PNAs and thus will help prolong the usability of existing antibiotics. Our work, and recent work that used ASOs to target the Qrr sRNA of Vibrio cholerae, 13 highlight sRNAs as potential new targets for antimicrobial therapeutics and the use of synthetic biology tools to identify ASOs. The in vitro interaction assay developed by Henderson et al allows for identification of ASOs that interfere with sRNA-mRNA binding.…”

“…Antisense oligonucleotides (ASOs) have emerged as an effective method to target both mRNAs and non-coding RNAs in several bacteria species. [10][11][12][13][14] ASOs are easily designed to target RNAs through complementary Watson-Crick base pairing and several nucleic acid analogues have been developed that are resistant to nuclease activity. 15 In particular, peptide nucleic acids (PNAs) provide a neutral, peptide-like backbone resistant to both nucleases and proteases.…”

Identifying Antisense Oligonucleotides to Disrupt Small RNA Regulated Antibiotic Resistance via a Cell-Free Transcription–Translation Platform

“…Theoretically, this is due to the increased influx of antibiotics through continued expression of the OmpF porin caused by the PNAs and thus will help prolong the usability of existing antibiotics. Our work and recent work that used ASOs to target the Qrr sRNA of Vibrio cholerae highlight sRNAs as potential new targets for antimicrobial therapeutics and the use of synthetic biology tools to identify ASOs. The in vitro interaction assay developed by Henderson et al allows for identification of ASOs that interfere with sRNA–mRNA binding.…”

“…There are several examples of antisense RNAs (asRNA) designed to modulate the activity of other short RNAs, including sRNAs, small transcription-activating RNAs (STARs), , and small guide RNAs (sgRNAs) . Furthermore, in nature there are sponge RNAs that play a critical role in post-transcriptional regulation involving sRNAs. , Antisense oligonucleotides (ASOs) have emerged as an effective method to target both mRNAs and noncoding RNAs in several bacteria species. ,− ASOs are easily designed to target RNAs through complementary Watson–Crick base pairing, and several nucleic acid analogues have been developed that are resistant to nuclease activity . In particular, peptide nucleic acids (PNAs) provide a neutral peptide-like backbone resistant to both nucleases and proteases .…”

MicroRNAs Encoded by Virus and Small RNAs Encoded by Bacteria Associated with Oncogenic Processes

“…Thereby, these tools continue to be updated, allowing the identification of new small RNAs codified by bacteria to move toward a more complete understanding of the functioning of bacterial regulatory networks [109]. These regulatory interactions should enable potential targets for synthetic biology, antimicrobials and therapeutics and this is because knowing these networks in the future could help to modulate these circuits in a predictable way [110]. Furthermore, one of the long term goals of synthetic biology is to develop designable genetic parts with predictable behaviors that can be used to implement various cellular functions, hence the discovery of noncoding RNAs is a natural target of synthetic biology [111].…”