Poster abstracts
Poster number 27 submitted by Shiqin Miao
Bifacial PNAs destabilize MALAT1 by 3′ poly(A) tail displacement from the U-rich Internal Loop
Shiqin Miao (Department of Chemistry and Biochemistry), Debmalya Bhunia (Department of Chemistry and Biochemistry), Shekar Devari (Department of Chemistry and Biochemistry), Yufeng Liang (Department of Chemistry and Biochemistry), Oliver Munyaradzi (Department of Chemistry and Biochemistry), Sarah Rundell (Department of Chemistry and Biochemistry)
Abstract:
We report herein a new class of synthetic reagents for targeting the element for nuclear expression (ENE) in MALAT1, a long noncoding RNA upregulated in many cancers. The cis-acting ENE contains a U-rich internal loop (URIL) that forms an 11 base UAU-rich triplex stem with the truncated 3′ poly-A terminus of MALAT1, protecting the terminus from exonuclease digestion and greatly extending transcript lifetime. Bifacial peptide nucleic acids (bPNAs) similarly bind URILs via base triple formation between two uracil bases and a synthetic base, melamine. We synthesized a set of low molecular weight bPNAs comprised of α-linked peptide, isodipeptide and diketopiperazine backbones and evaluated their ENE binding efficacy in vitro via oligo-A strand displacement and consequent exonuclease sensitivity. Degradation was greatly enhanced by bPNA treatment in the presence of exonucleases, with ENE halflife plunging to 6 min from >24h. RNA digestion kinetics could clearly distinguish between bPNAs with similar URIL affinity, highlighting the utility of functional assays for evaluating synthetic RNA binders. In vitro activity was mirrored by a 50% knockdown of MALAT1 expression in pancreatic cancer (PANC-1) cells upon treatment with bPNAs, consistent with intracellular digestion triggered by a similar ENE A-tail displacement mechanism. Pulldown from PANC-1 total RNA with biotinylated bPNA enriched MALAT1 >4500X, supportive of bPNA-URIL selectivity. Together, these experiments establish the feasibility of native transcript targeting by bPNA in both in vitro and intracellular contexts. Reagents such as bPNAs may be useful tools for investigation of transcripts stabilized by cis-acting poly(A) binding RNA elements.
Keywords: MALAT1, PNA, RNA targeting