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

Kamzeeva, Polina; Shepelev, Nikita; Zabbarova, Veronika; Brylev, Vladimir; Chistov, Alexey; Ryazantsev, Dmitriy; Kot, Erik; Novopashina, Darya; Rubtsova, Maria; Aralov, Andrey

doi:10.3390/ijms262211047

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

by

Polina Kamzeeva

^1,†,

Nikita Shepelev

^1,2,†

,

Veronika Zabbarova

²,

Vladimir Brylev

¹

,

Alexey Chistov

¹

,

Dmitriy Ryazantsev

¹

,

Erik Kot

¹

,

Darya Novopashina

³

,

Maria Rubtsova

^1,2,* and

Andrey Aralov

^1,4,5,*

¹

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, The Russian Academy of Sciences, 117437 Moscow, Russia

²

Chemistry Department, Lomonosov Moscow State University, 119234 Moscow, Russia

³

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia

⁴

Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine, Federal Medical Biological Agency, 119435 Moscow, Russia

⁵

Educational Resource Center for Cellular Technologies, RUDN University, 117198 Moscow, Russia

^*

Authors to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Int. J. Mol. Sci. 2025, 26(22), 11047; https://doi.org/10.3390/ijms262211047

Submission received: 7 October 2025 / Revised: 6 November 2025 / Accepted: 11 November 2025 / Published: 14 November 2025

(This article belongs to the Special Issue Advanced Research on Nucleic Acids: Therapeutic Potential and Applications, 2nd Edition)

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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.

Keywords: DDX23; hairpin; LNA; antisense oligonucleotides; translation regulation; uORF

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MDPI and ACS Style

Kamzeeva, P.; Shepelev, N.; Zabbarova, V.; Brylev, V.; Chistov, A.; Ryazantsev, D.; Kot, E.; Novopashina, D.; Rubtsova, M.; Aralov, A. Structural Characterization of DDX23 5′ UTR Regulatory Elements and Their Targeting by LNA-Modified Antisense Oligonucleotides. Int. J. Mol. Sci. 2025, 26, 11047. https://doi.org/10.3390/ijms262211047

AMA Style

Kamzeeva P, Shepelev N, Zabbarova V, Brylev V, Chistov A, Ryazantsev D, Kot E, Novopashina D, Rubtsova M, Aralov A. Structural Characterization of DDX23 5′ UTR Regulatory Elements and Their Targeting by LNA-Modified Antisense Oligonucleotides. International Journal of Molecular Sciences. 2025; 26(22):11047. https://doi.org/10.3390/ijms262211047

Chicago/Turabian Style

Kamzeeva, Polina, Nikita Shepelev, Veronika Zabbarova, Vladimir Brylev, Alexey Chistov, Dmitriy Ryazantsev, Erik Kot, Darya Novopashina, Maria Rubtsova, and Andrey Aralov. 2025. "Structural Characterization of DDX23 5′ UTR Regulatory Elements and Their Targeting by LNA-Modified Antisense Oligonucleotides" International Journal of Molecular Sciences 26, no. 22: 11047. https://doi.org/10.3390/ijms262211047

APA Style

Kamzeeva, P., Shepelev, N., Zabbarova, V., Brylev, V., Chistov, A., Ryazantsev, D., Kot, E., Novopashina, D., Rubtsova, M., & Aralov, A. (2025). Structural Characterization of DDX23 5′ UTR Regulatory Elements and Their Targeting by LNA-Modified Antisense Oligonucleotides. International Journal of Molecular Sciences, 26(22), 11047. https://doi.org/10.3390/ijms262211047

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

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