Single-stranded RNA oligonucleotides that recruit endogenous hnRNPA1
enable the targeted reduction of gene expression
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.