# Structural Characterization of DDX23 5′ UTR Regulatory Elements and Their Targeting by LNA-Modified Antisense Oligonucleotides

## TL;DR

This study explores RNA structures in the DDX23 mRNA and how antisense oligonucleotides can inhibit its translation for cancer therapy.

## Contribution

The study identifies a functional hairpin structure in DDX23 mRNA and establishes design principles for effective antisense oligonucleotides.

## Key findings

- Most predicted RNA G-quadruplex structures in DDX23 mRNA are not stable.
- A stable hairpin structure in DDX23 mRNA is a potential regulatory element.
- LNA-modified ASOs targeting the cap-proximal region inhibited translation by up to 80%.

## Abstract

Translation of mRNAs is a tightly regulated process in gene expression. In mRNA, the 5′ untranslated region (5′ UTR) controls ribosome recruitment and frequently contains structured elements that modulate translation efficacy. This study investigates stable structural motifs within the 5′ UTR of DDX23 mRNA, encoding a protein relevant for anticancer therapy, as potential regulators and targets for antisense oligonucleotides (ASOs). Despite bioinformatic predictions and transcriptomic validations suggesting RNA G-quadruplex (rG4) formation, comprehensive structural analysis using a light-up assay and CD, UV, and NMR spectroscopy revealed that most putative rG4-forming sequences do not fold into stable rG4 structures, although one of them exists in an equilibrium between rG4 and an alternative, likely hairpin, conformation. Reporter assays using a robust G4 stabilizer also argue against the significant regulatory role of rG4s in DDX23 mRNA translation. Instead, we identified and characterized a stable hairpin structure with potential regulatory function. Based on these findings, we designed fully locked nucleic acid (LNA)-modified ASOs to target this hairpin and regions flanking the upstream open reading frame (uORF) and start codon of the coding sequence. A reporter assay demonstrated that cap-proximal targeting achieved robust translation inhibition up to 80%. In contrast, targeting the efficiently translated uORF was ineffective, presumably due to steric hindrances from the ribosomal complex. The study yields crucial design principles for translation-regulating ASOs: avoid targeting regions shielded by efficient uORF translation and carefully tune ASO-RNA duplex stability to surpass endogenous structures without disrupting regulatory mechanisms. These findings provide insights into the regulation of DDX23 expression and establish a framework for developing ASO-based therapeutics with broad implications for mRNA targeting in anticancer applications.