Abstract
DNA/RNA heteroduplex oligonucleotide (HDO), composed of DNA/locked nucleic acid (LNA) antisense oligonucleotide (ASO) and complementary RNA, is a next-generation antisense therapeutic agent. HDO is superior to the parental ASO in delivering to target tissues, and it exerts a more potent gene-silencing effect. In this study, we aimed to elucidate the intracellular trafficking mechanism of HDO-dependent gene silencing. HDO was more preferably transferred to the nucleus after transfection compared to the parental ASO. To determine when and where HDO is separated into the antisense strand (AS) and complementary strand (CS), we performed live-cell time-lapse imaging and fluorescence resonance energy transfer (FRET) assays. These assays demonstrated that HDO had a different intracellular trafficking mechanism than ASO. After endocytosis, HDO was separated in the early endosomes, and both AS and CS were released into the cytosol. AS was more efficiently transported to the nucleus than CS. Separation, endosomal release, and initiation of nuclear transport were a series of time-locked events occurring at a median of 30 s. CS cleavage was associated with efficient nuclear distribution and gene silencing in the nucleus. Understanding the unique intracellular silencing mechanisms of HDO will help us design more efficient drugs and might also provide insight into innate DNA/RNA cellular biology.
Highlights
Antisense oligonucleotide (ASO) is a single-stranded therapeutic oligonucleotide that modulates RNA functions by binding to the targeted RNA through Watson-Crick base pairing.[1,2] ASO is typically designed as a “gapmer” structure, where wings of chemically modified nucleotides flank both sides of a central portion of DNA
heteroduplex oligonucleotide (HDO)-antimiR conjugated with GalNAc was more potent in the liver than the parent ASO conjugated with GalNAc, where delivery efficiency of HDO was comparable to that of ASO.[6]
We recently developed a novel highly efficient oligonucleotide, DNA/ RNA heteroduplex oligonucleotide (HDO), which is composed of an antisense gapmer (DNA nucleotides flanked by LNAs) and complementary RNA.[5,6,7]
Summary
Antisense oligonucleotide (ASO) is a single-stranded therapeutic oligonucleotide that modulates RNA functions by binding to the targeted RNA through Watson-Crick base pairing.[1,2] ASO is typically designed as a “gapmer” structure, where wings of chemically modified nucleotides flank both sides of a central portion of DNA.
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