Hydrogen peroxide-triggered gene silencing in mammalian cells through boronated antisense oligonucleotides

Abstract

Hydrogen peroxide (H₂O₂) is a reactive oxygen species (ROS) involved in various diseases, including neurodegeneration, diabetes, and cancer. Here, we introduce a new approach to use H₂O₂ to modulate specific gene expression in mammalian cells. H₂O₂-responsive nucleoside analogues, in which the Watson-Crick faces of the nucleobases are caged by arylboronate moieties, were synthesized. One of these analogues, boronated thymidine (dT^B ), was incorporated into oligodeoxynucleotides (ODNs) using an automated DNA synthesizer. The hybridization ability of this boronated ODN to complementary RNA was clearly switched in the off-to-on direction upon H₂O₂ addition. Furthermore, we demonstrated H₂O₂-triggered gene silencing in mammalian cells using antisense oligonucleotides (ASOs) modified with dT^B . Our approach can be used for the regulation of any gene of interest by the sequence design of boronated ASOs and will contribute to the development of targeted disease therapeutics.

Fig. 1. (a) Chemical structure of boronated 2′-deoxyribonucleoside analogues synthesized in this study. (b) Schematic of H₂O₂-triggered molecule activation. (c) Gene silencing triggered by H₂O₂ through inactive boronated ASOs.

Scheme 1. Synthesis of boronated nucleoside analogues. Conditions: (a) triphosgene, Na₂CO₃, toluene, rt; (b) imidazole, toluene, rt, 87% over 2 steps; (c) Et₃OBF₄, DCM, rt; (d) 1, DBU, MeCN, rt, 98%; (e) TBAF, THF, 0 °C, 72%; (f) 1, DIAD, PPh₃, 1,4-dioxane, rt, then TBAF, 10%; (g) 2, DCM, 0 °C to rt, 91%; (h) TASF, DMF, 0 °C, 62%; (i) 2, DCM, 0 °C to rt, 92%; (j) HF-pyridine, 60 °C, 52%.

Fig. 2. (a) HPLC chromatograms of dT^Bpin after H₂O₂ addition at different time points. (b) Conversion rate of each boronated nucleoside analogue to the corresponding natural nucleoside. (c) H₂O₂-selectivity of dT^Bpin toward other ROS.

Fig. 3. (a) Synthetic scheme of boronated ODN. tac = tert-butylphenoxyacetyl. (b) HPLC chromatograms of ODN14 after H₂O₂ addition at different time points. (c) Thermal melting curves of the duplex formed between ODN14 and complementary RNA in the presence or absence of H₂O₂.

Fig. 4. (a) Sequences of ASOs. n = DNA, N = LNA (C^m = LNA-5-Me-cytidine), all internucleosidic linkages are phosphorothioated. (b) Intercellular gene silencing triggered by H₂O₂ using boronate ASOs. Three independent experiments were averaged and the error bars represent standard deviation.