Bifacial PNAs Destabilize MALAT1 by 3' A-Tail Displacement from the U-Rich Internal Loop

Area of Science:

• Molecular Biology
• RNA Biology
• Medicinal Chemistry

Background:

• MALAT1 is a long noncoding RNA frequently upregulated in various cancers.
• The 3' oligo-A tail and a U-rich internal loop (URIL) form a structure that protects MALAT1 from degradation, extending its transcript lifetime.
• Targeting RNA stability mechanisms is a promising strategy for therapeutic intervention.

Purpose of the Study:

• To develop and evaluate novel synthetic reagents, specifically bifacial peptide nucleic acids (bPNAs), for targeting the element for nuclear expression (ENE) in MALAT1.
• To assess the efficacy of bPNAs in promoting MALAT1 RNA degradation both *in vitro* and in cancer cells.
• To investigate the potential of bPNAs as tools for studying RNA regulation and as therapeutic agents.

Main Methods:

• Synthesis of low molecular weight bPNAs with varying backbones (α-linked peptide, isodipeptide, diketopiperazine).
• Evaluation of *in vitro* ENE binding efficacy using oligo-A strand displacement assays and subsequent exonuclease sensitivity assays.
• Assessment of MALAT1 expression knockdown in pancreatic cancer (PANC-1) cells treated with bPNAs.
• RNA pulldown assays using biotinylated bPNAs to determine target selectivity.

Main Results:

• bPNAs effectively bind to the URIL of MALAT1 via base triple formation, displacing the oligo-A tail.
• bPNA treatment significantly enhanced RNA degradation, reducing MALAT1 ENE half-life from over 24 hours to 6 minutes.
• A 50% knockdown of MALAT1 expression was observed in PANC-1 cells treated with bPNAs, indicating intracellular efficacy.
• Pulldown assays demonstrated a >4000-fold enrichment of MALAT1, confirming bPNA selectivity for the target RNA.

Conclusions:

• bPNAs represent a new class of synthetic reagents capable of targeting specific RNA structures like the MALAT1 ENE.
• These bPNAs can trigger the degradation of native RNA transcripts both *in vitro* and within living cells.
• bPNAs show promise as valuable tools for investigating RNA biology and potentially for developing RNA-targeted cancer therapies.