Synthesis and physical and biological properties of 1,3-diaza-2-oxophenoxazine-conjugated oligonucleotides

https://doi.org/10.1016/j.bmc.2022.116972Get rights and content

Abstract

The artificial nucleobase 1,3-diaza-2-oxophenoxazine (tCO) and its derivative G-clamp strongly bind to guanine and, when incorporated into double-stranded DNA, significantly increase the stability of the latter. As the phenoxazine skeleton is a constituent of major pharmaceuticals, we hypothesized that oligonucleotides (ONs) containing phenoxazine bases would induce property changes related to intracellular uptake and migration in tissues. In this study, we designed and synthesized a novel G-clamp-linker antisense oligonucleotide (ASO) in which a G-clamp base with a flexible linker was introduced into the 5′-end of an ASO targeting mouse long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (mMALAT1). Compared to unconjugated ASO, the G-clamp-linker ASO induced significantly more effective knockdown of mMALAT1 in mouse skeletal muscle. The ASOs conjugated with 2′-deoxyribonucleotide(s) bearing a tCO nucleobase at the 5′-end exhibited a similar knockdown effect in skeletal muscle. Thus, it may be possible to improve therapeutic effects against skeletal muscle diseases, such as muscular dystrophy, by using ONs with incorporated phenoxazine nucleobases.

Introduction

The development of artificial nucleic acids and strategies for their delivery is essential for nucleic acid-based therapeutics, including antisense, siRNA, antigene, and decoy methods.1, 2 In the antisense strategy, artificial nucleic acids must bind strongly to complementary RNA and be nuclease-resistant. Typical artificial nucleic acids include sugar-modified 2′-O-Me-RNA, 2′,4′-bridged nucleic acid (2′,4′-BNA)/locked nucleic acid (LNA), phosphate-modified phosphorothioate (PS), and nucleobase-modified phenoxazines.3, 4 Effective delivery of oligonucleotides (ONs) is essential for the practical use of the antisense method because it improves cell membrane permeability and selective organ targeting in vivo. Liposomes and polymer carriers have been widely developed for the delivery of ONs.5, 6 Recently, ONs conjugated with ligands of cell membrane receptors have been developed for effective delivery into cells and targeted tissues in vivo.7, 8, 9, 10, 11, 12, 13, 14, 15, 16 ONs conjugated with N-acetyl-galactosamine (GalNAc), a ligand of the asialoglycoprotein receptor, are efficiently taken up by the liver.17, 18, 19, 20, 21, 22, 23, 24, 25 A recent promising study reported that the conjugation of ONs with glucagon-like peptide 1 improved uptake into pancreatic β-cells.26

The tricyclic cytosine derivative 1,3-diaza-2-oxophenoxazine (tCO) binds to guanine, and a duplex containing 2′-deoxyribonucleotide with tCO (dtCO) is stabilized by π-π stacking interactions between adjacent bases (Fig. 1a).27, 28, 29, 30 Furthermore, dtCO triphosphates have been incorporated into an ON using a polymerase.31, 32, 33 A G-clamp derivative with an aminoethoxy group introduced at the 9-position of tCO forms an additional fourth hydrogen bond at the O6 position of guanine (Fig. 1b).34, 35, 36, 37, 38 A duplex containing 2′-deoxyribonucleotide with a G-clamp (dG-clamp) is significantly more stable than that containing dtCO. The ON-containing dG-clamp exhibits excellent 3′-exonuclease resistance.34 This antisense oligonucleotide (ASO) containing a dG-clamp exhibits superior gene knockdown when compared to unmodified ASOs in the liver and kidney cells when administered through lipofection.39, 40

The phenoxazine skeleton is a major structural component of drugs,41, 42, 43, 44 and we were interested in the effects of phenoxazine bases in oligonucleotides (ONs) on intracellular uptake and migration in tissues. In this study, we designed and synthesized a novel G-clamp-linker ASO in which a G-clamp nucleobase was introduced into the 5′-end of the ASO via a flexible linker (Fig. 1c). G-clamp-linker-, dtCO-, and dG-clamp-conjugated ASOs targeting mouse long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (mMALAT1) were administered to mice, and gene expression in various organs was measured to evaluate the knockdown efficacies.

Section snippets

Design of a G-clamp-linker for ON delivery

The phenoxazine skeleton is a major structural component of drugs.41, 42, 43, 44 We hypothesized that tCO and G-clamp bases in ONs would affect properties such as migration in tissues and intracellular uptake and that the phenoxazine base may function as a ligand for receptors on the cell membrane.45, 46, 47, 48, 49, 50 We designed a novel G-clamp-linker ASO in which the 2′-deoxyribose was replaced with a branched triethylene glycol (bTEG) linker at the N3 position of the G-clamp base for

Discussion

dtCO and the dG-clamp are used in ONs to improve their duplex-forming ability through π-π stacking interactions between adjacent bases.,27, 28, 29, 30, 34, 35, 36, 37, 38 In this study, we investigated the knockdown activities of phenoxazine base conjugated ASOs on in each organ. We eliminated factors that allow tCO and G-clamp bases to improve duplex-forming abilities as much as possible to verify the organ-selective knockdown effects by quantifying mMALAT1 expression. The G-clamp-linker, dtCO

Conclusions

We designed a novel G-clamp-linker, and it was effectively synthesized and conjugated to the 5′-end of an ASO targeting mMALAT1. The G-clamp-linker-conjugated ASO showed superior knockdown efficacy in skeletal muscle. Similar effects were observed for dtCO-conjugated ASOs, especially when conjugated with PS linkages. This approach may be utilized to develop new ON-based therapies for skeletal muscle diseases, particularly, muscular dystrophy.

General information

Dehydrated acetonitrile, 1,4-dioxane, dichloromethane, N,N-dimethylformamide, ethanol, methanol, pyridine, and tetrahydrofuran were purchased from FUJIFILM Wako Chemicals. For column chromatography, Fuji Silysia PSQ-100B or PSQ-60B silica gel was used. 1H NMR, 13C NMR, 19F NMR, and 31P NMR spectra were recorded using JEOL JNM-ECS400 and JNM-ECA500 spectrometers. Chemical shift values are reported in parts per million (ppm) relative to internal tetramethylsilane (0.00 ppm) or residual methanol‑d4

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI under Grant Number 20K05706 and by the Japan Agency for Medical Research and Development (AMED) under Grant Numbers JP18am0301004 and JP19am0401003. We would like to thank Editage (www.editage.com) for English language editing.

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