loading page

Single-stranded RNA oligonucleotides that recruit endogenous hnRNPA1 enable the targeted reduction of gene expression
  • +1
  • Justin Melendez,
  • Ananya Pal,
  • Sidharth Puram,
  • Robi Mitra
Justin Melendez
Washington University School of Medicine in Saint Louis
Author Profile
Ananya Pal
Washington University School of Medicine in Saint Louis
Author Profile
Sidharth Puram
Washington University School of Medicine in Saint Louis
Author Profile
Robi Mitra
Washington University School of Medicine in Saint Louis
Author Profile

Abstract

Targeted gene knockdown has become one of the most powerful tools in molecular biology and holds substantial promise in therapeutic applications. While existing technologies such as siRNAs, CRISPRi, and ASOs effectively and specifically reduce gene expression, few can be used to first discover the genes that influence a particular phenotype and then directly transition to being used as oligonucleotide therapeutics. Thus, a tool that could help bridge the gap between target discovery and the development of therapeutic leads would benefit the scientific community. Here, we present hnRNPA1 recruiting oligonucleotides, or AROs, as single-stranded RNA (ssRNA) molecules that knockdown transcript levels of target genes. AROs target specific pre-mRNA transcripts via sequence homology and leverage the ubiquitous and abundant endogenous RNA-binding protein hnRNPA1 to degrade target transcripts. Using RT-qPCR, we show that AROs effectively knock down target genes when delivered via a plasmid and expressed using a Pol II promoter or when delivered directly as single-strand RNAs. Additionally, as proof of principle, we use a ssRNA ARO to knockdown KRT14 in squamous cell carcinoma and show reduced invasive potential. We believe AROs fill an important niche in the scientific toolbox by taking advantage of endogenous RNA binding machinery for RNA knockdowns.

Peer review status:IN REVISION

02 Jul 2021Submitted to Biotechnology and Bioengineering
03 Jul 2021Assigned to Editor
03 Jul 2021Submission Checks Completed
12 Jul 2021Reviewer(s) Assigned
29 Jul 2021Review(s) Completed, Editorial Evaluation Pending
29 Jul 2021Editorial Decision: Revise Major