Heteroduplex Oligonucleotide Research Articles

Separation-related rapid nuclear transport of DNA/RNA heteroduplex oligonucleotide: unveiling distinctive intracellular trafficking

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.

Highly efficient silencing of microRNA by heteroduplex oligonucleotides

AntimiR is an antisense oligonucleotide that has been developed to silence microRNA (miRNA) for the treatment of intractable diseases. Enhancement of its in vivo efficacy and improvement of its toxicity are highly desirable but remain challenging. We here design heteroduplex oligonucleotide (HDO)-antimiR as a new technology comprising an antimiR and its complementary RNA. HDO-antimiR binds targeted miRNA in vivo more efficiently by 12-fold than the parent single-stranded antimiR. HDO-antimiR also produced enhanced phenotypic effects in mice with upregulated expression of miRNA-targeting messenger RNAs. In addition, we demonstrated that the enhanced potency of HDO-antimiR was not explained by its bio-stability or delivery to the targeted cell, but reflected an improved intracellular potency. Our findings provide new insights into biology of miRNA silencing by double-stranded oligonucleotides and support the in vivo potential of this technology based on a new class of for the treatment of miRNA-related diseases.

Modulation of blood-brain barrier function by a heteroduplex oligonucleotide in vivo

The blood-brain barrier (BBB) is increasingly regarded as a dynamic interface that adapts to the needs of the brain, responds to physiological changes, and gets affected by and can even promote diseases. Modulation of BBB function at the molecular level in vivo is beneficial for a variety of basic and clinical studies. Here we show that our heteroduplex oligonucleotide (HDO), composed of an antisense oligonucleotide and its complementary RNA, conjugated to α-tocopherol as a delivery ligand, efficiently reduced the expression of organic anion transporter 3 (OAT3) gene in brain microvascular endothelial cells in mice. This proof-of-concept study demonstrates that intravenous administration of chemically synthesized HDO can remarkably silence OAT3 at the mRNA and protein levels. We also demonstrated modulation of the efflux transport function of OAT3 at the BBB in vivo. HDO will serve as a novel platform technology to advance the biology and pathophysiology of the BBB in vivo, and will also open a new therapeutic field of gene silencing at the BBB for the treatment of various intractable neurological disorders.

The effect of DNA/RNA heteroduplex oligonucleotides on muscle

Background: We recently have developed a new DNA/RNA heteroduplex oligonucleotide (HDO) with a structure of DNA/locked nucleotide acid gapmer antisense oligonucleotide (ASO) duplexed with a α-tocopherol conjugated complementary RNA strand (Toc-cRNA), and showed its high potency of gene silencing in liver compared to any kind of previously reported ASOs and siRNA at orders-of-magnitude. (Nishina K, et al. Nat Commun, 2015).

Heteroduplex oligonucleotide as a platform technology to modulate blood-brain barrier function in vivo

Heteroduplex oligonucleotide as a platform technology to modulate blood-brain barrier function in vivo

Silencing of malat1 long non-coding rna with DNA/RNA heteroduplex oligonucleotides exacerbates cerebral ischemia

Background: Malat1 long non-coding RNA is reported to regulate endothelial cells (ECs) and neuronal cell functions after ischemia. We previously developed an α-tocopherol-conjugated heteroduplex oligonucleotide (Toc-HDO), which strongly silenced its target gene in many organs.

Gene silencing effect of heteroduplex oligonucleotide and its biodistribution in neurons and brain endothelial cells after ischemia

Gene silencing effect of heteroduplex oligonucleotide and its biodistribution in neurons and brain endothelial cells after ischemia

Drug delivery system of therapeutic oligonucleotides.

Therapeutic oligonucleotides are promising technologies. Nevertheless, improvement of their efficacy is an important issue. Introducing this drug delivery system (DDS) makes for a great enhancement for delivery of oligonucleotides to targeted tissue or cells. The strategy of DDS for therapeutic oligonucleotides is divided into four categories, A) single piece of oligonucleotide, B) oligonucleotide-ligand conjugate, C) oligonucleotide-polymer conjugate, and D) nanoparticle. In this review we will describe those basic concepts, especially for the technology of conjugating ligand. In addition, we developed a new technology, heteroduplex oligonucleotide (HDO), binding ligand-molecule to antisense oligonucleotide indirectly. We also outline α-tocopherol (a natural isomer of vitamin E) conjugated HDO.

DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing.

Antisense oligonucleotides (ASOs) are recognized therapeutic agents for the modulation of specific genes at the post-transcriptional level. Similar to any medical drugs, there are opportunities to improve their efficacy and safety. Here we develop a short DNA/RNA heteroduplex oligonucleotide (HDO) with a structure different from double-stranded RNA used for short interfering RNA and single-stranded DNA used for ASO. A DNA/locked nucleotide acid gapmer duplex with an α-tocopherol-conjugated complementary RNA (Toc-HDO) is significantly more potent at reducing the expression of the targeted mRNA in liver compared with the parent single-stranded gapmer ASO. Toc-HDO also improves the phenotype in disease models more effectively. In addition, the high potency of Toc-HDO results in a reduction of liver dysfunction observed in the parent ASO at a similar silencing effect. HDO technology offers a novel concept of therapeutic oligonucleotides, and the development of this molecular design opens a new therapeutic field.

Sequence-specific cleavage of RNA by Type II restriction enzymes

The ability of 223 Type II restriction endonucleases to hydrolyze RNA–DNA heteroduplex oligonucleotide substrates was assessed. Despite the significant topological and sequence asymmetry introduced when one strand of a DNA duplex is substituted by RNA we find that six restriction enzymes (AvaII, AvrII, BanI, HaeIII, HinfI and TaqI), exclusively of the Type IIP class that recognize palindromic or interrupted-palindromic DNA sequences, catalyze robust and specific cleavage of both RNA and DNA strands of such a substrate. Time-course analyses indicate that some endonucleases hydrolyze phosphodiester bonds in both strands simultaneously whereas others appear to catalyze sequential reactions in which either the DNA or RNA product accumulates more rapidly. Such strand-specific variation in cleavage susceptibility is both significant (up to orders of magnitude difference) and somewhat sequence dependent, notably in relation to the presence or absence of uracil residues in the RNA strand. Hybridization to DNA oligonucleotides that contain endonuclease recognition sites can be used to achieve targeted hydrolysis of extended RNA substrates produced by in vitro transcription. The ability to ‘restrict’ an RNA–DNA hybrid, albeit with a limited number of restriction endonucleases, provides a method whereby individual RNA molecules can be targeted for site-specific cleavage in vitro.

A Dynamical Systems Model to Simulate the Perturbation Kinetics of Gene Expression by Antisense Oligonucleotides

Antisense oligonucleotides owe their efficacy to an ability to induce RNase H-dependent suppression of RNA translation, for sufficient time to allow physiological proteolysis. The magnitude and time delay preceding the protein nadir concentration determine the extent and timing of maximum antisense oligonucleotide activity. Antisense oligonucleotide degradation underlies reversal of RNA downregulation. The kinetics of protein downregulation is therefore determined by the complex interaction of both ligand chemistry (nuclease stability, affinity and RNase H activation), and gene expression kinetics. Optimization of antisense oligonucleotide efficacy and experimental design requires understanding of these interactions. The kinetics of protein and RNA downregulation have therefore been simulated by analysing a two-compartment kinetic model incorporating RNase H-dependent transcript degradation. The system of nonlinear differential equations describing this model was solved numerically using Runge–Kutte integration. The timecourse solutions corresponding to the four state variables (RNA, protein, antisense/RNA heteroduplex and antisense oligonucleotide), were determined simultaneously. This allowed systematic in silico examination of the consequences of altering variables such as oligonucleotide concentration, affinity, and stability, or the scheduling of multiple transfections on RNA and protein perturbations. By providing a tool for examining antisense oligonucleotide action theoretically, this heuristic model should facilitate both the rational design and interpretation of antisense experiments.