RESUMO
Recently, double-stranded oligonucleotide therapeutics with A-type duplex structures such as short interfering RNAs have gained considerable attention. We have reported the synthesis of cationic oligosaccharides that selectively bind to A-type oligonucleotide duplexes. In particular, oligodiaminogalactose (ODAGal) has a strong stabilizing effect on A-type oligonucleotide duplexes. However, an efficient synthetic method has not been established for ODAGals and the properties of ODAGals have been investigated only up to 4mer. The most crucial problem of the synthesis was side reactions on a p-methoxybenzyl (PMB) protecting group of a 3-hydroxy group. In this paper, the benzyl (Bn) group was chosen as a protecting group of the 3-hydroxy group to suppress the side reactions on protecting groups, and the yields of glycosylation reactions were significantly improved. Moreover, optimization of the conditions for the deprotection of the Bn groups allowed the efficient synthesis of fully deprotected ODAGals, and ODAGal 5mer and 6mer were synthesized for the first time. In addition, we systematically investigated the effects of these ODAGals on the properties of several oligonucleotide duplexes. It was found that ODAGal 4-6mers stabilized the A-type oligonucleotide duplexes thermally and biologically, typically without their structural changes and the effect was notable with longer ODAGals.
Assuntos
Oligonucleotídeos , RNA de Cadeia Dupla , Oligonucleotídeos/química , RNA Interferente Pequeno , Cátions/química , Oligossacarídeos/químicaRESUMO
Sequence-dependent off-target effects are a serious problem of antisense oligonucleotide-based drugs. Some of these side effects are induced by ribonuclease H (RNase H)-mediated cleavage of non-target RNAs with base sequences similar to that of the target RNA. We found that an artificial cationic oligosaccharide, ODAGal4, improved single-base discrimination for RNase H cleavage.
Assuntos
Ribonuclease HRESUMO
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.
Assuntos
DNA de Cadeia Simples/genética , Inativação Gênica , MicroRNAs/genética , Ácidos Nucleicos Heteroduplexes/genética , Oligonucleotídeos Antissenso/genética , Animais , Northern Blotting , DNA de Cadeia Simples/metabolismo , Feminino , Regulação da Expressão Gênica , Rim/metabolismo , Fígado/metabolismo , Camundongos Endogâmicos ICR , MicroRNAs/metabolismo , Ácidos Nucleicos Heteroduplexes/metabolismo , Ácidos Nucleicos Heteroduplexes/farmacocinética , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/farmacocinética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Baço/metabolismoRESUMO
The immunostimulatory activity of unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) could be improved via delivery to immune cells expressing Toll-like receptor 9 (TLR9). Previously, we showed that the polypod-like structured nucleic acid (polypodna), a nanostructured DNA comprised of three or more ODNs, was an efficient system for the delivery of CpG ODNs to immune cells. Because some TLR9-positive immune cells express mannose receptors (MR), the uptake of polypodna by immune cells can be further increased by its modification with mannose. In this study, we selected the phosphodiester CpG ODN, ODN1668, which has a sequence identical to CpG1668, and a hexapodna, a polypodna with six pods, to design a hexapodna that harbored ODN1668 or the mannosylated CpG ODN (Man-ODN1668) synthesized via modification of the 5'-terminal of ODN1668 with a synthesized mannose motif. By mixing ODN1668 or Man-ODN1668 with the hexapodna, ODN1668/hexapodna and Man-ODN1668/hexapodna were successfully formed with high yields. However, Man-ODN1668/hexapodna was found to induce a greater tumor necrosis factor-α release from TLR9- and MR-positive mouse peritoneal macrophages and macrophage-like J774.1 cells than Man-ODN1668 or ODN1668/hexapodna. These results indicate that the combination of mannose modification and incorporation into nanostructured DNA is a useful approach for enhancing the immunostimulatory activity of CpG ODN.
Assuntos
Adjuvantes Imunológicos/síntese química , DNA/química , Nanoestruturas/química , Oligodesoxirribonucleotídeos/farmacologia , Adjuvantes Imunológicos/farmacologia , Animais , Células Cultivadas , DNA/farmacocinética , Feminino , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Oligodesoxirribonucleotídeos/química , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Boranophosphate (PB) DNAs are promising antisense oligonucleotide candidates because of their attractive features, such as high nuclease resistance and low toxicity. However, a full boranophosphate backbone modification to antisense DNAs causes reduced duplex formation with complementary RNAs and reduced antisense activity. In this study, an efficient solid-phase synthesis of phosphate/boranophosphate (PO/PB) chimeric DNA was achieved by the combination of the H-phosphonate and H-boranophosphonate methods. The physiological and biological properties of the synthesized PO/PB chimeric DNAs were also evaluated. The strategy employed herein can facilitate the design and synthesis of PO/PB chimeric DNAs containing site-specific boranophosphate modifications.
Assuntos
Boranos/química , DNA/síntese química , Fosfatos/química , Técnicas de Síntese em Fase Sólida , Configuração de Carboidratos , DNA/químicaRESUMO
In this paper, we describe the first stereocontrolled synthesis and properties of boranophosphate DNA (PB-DNA), which contains all of the four nucleobases longer than 10mer. Synthesis was accomplished via an oxazaphospholidine approach combined with acid-labile protecting groups on nucleobases. It was demonstrated that there were significant differences between all-( Rp)- and all-( Sp)-PB-DNA in terms of the duplex-formation ability, nuclease resistance, and ribonuclease H (RNase H) activity. In particular, all-( Sp)-PB-DNA was demonstrated to show a duplex-formation ability with RNA and RNase H activity, both of which are necessary for antisense-type nucleic acid therapeutics.
Assuntos
Boranos/química , DNA/química , DNA/síntese química , Fosfatos/química , Técnicas de Química Sintética , Ribonuclease H/metabolismo , EstereoisomerismoRESUMO
Novel double-stranded RNA (dsRNA)-binding molecules were developed for the effective thermodynamic and biological stabilization of nucleic acids including short interfering RNAs (siRNAs). ß-(1â4)-Linked-2,6-diamino-2,6-dideoxy-d-galactopyranose oligomers (ODAGals) were synthesized for this purpose, and their binding ability with dsRNAs was evaluated. Fluorescence anisotropy measurements showed the 3mer and 4mer of ODAGals to be strongly bound (Kd < 0.02 µM). The UV melting experiments demonstrated that the binding of ODAGals to dsRNAs proceeded with significant thermodynamic stabilization of the duplexes. Furthermore, the 4mer of ODAGal was clearly revealed to almost completely protect siRNAs with a low N/P ratio (i.e., N in the oligocationic molecule to P in the siRNA ratio) from cleavage by RNase A. On the basis of these results, ODAGals can serve as promising stabilizers or carriers of dsRNA-based drugs such as RNAi drugs.
Assuntos
Oligossacarídeos/química , RNA de Cadeia Dupla/química , RNA Interferente Pequeno/química , Sítios de Ligação , Cátions/química , Estabilidade de RNARESUMO
Recently, we found DNA/RNA heteroduplex oligonucleotide-based antimiR (HDO-antimiR) can more efficiently inhibit the target miRNA than conventional antimiR after its cellular uptake. But the mechanism of HDO-antimiR about the target-silencing is unknown. We here tried to elucidate the interaction mechanism of HDO-antimiR to miRNA using molecular dynamics (MD) simulation. When interaction of the conventional antimiR or HDO-antimiR and the target miRNA was simulated, they combined with each other in various forms. In the hydrogen bond analyses, base site of the antimiR formed hydrogen bond with miRNA. On the other hand, phosphate site of the HDO-antimiR formed hydrogen bond with miRNA. These results suggested that there were differences about the binding mechanisms between antimiR and HDO-antimiR to the target miRNA. In particular, there was a difference in the binding site between antimiR and HDO-antimiR. Additionally, it was found that guanine in the miRNA is mainly involved in the binding to the antimiR or HDO-antimiR. MD simulation method is useful in understanding the mechanism of oligonucleotide therapeutics.
RESUMO
An increasing number of antisense oligonucleotides (ASOs) have been approved for clinical use. However, improvements of both efficacy and safety in the central nervous system (CNS) are crucial for the treatment with CNS diseases. We aimed to overcome the crucial issues by our development of various gapmer ASOs with a novel nucleoside derivative including a 2',4'-BNA/LNA with 9-(aminoethoxy)phenoxazine (BNAP-AEO). The various gapmer ASOs with BNAP-AEO were evaluated for thermal stability, in vitro and in vivo efficacy, and acute CNS toxicity. Thermal stability analysis of the duplexes with their complementary RNAs showed that ASOs with BNAP-AEO had a higher binding affinity than those without BNAP-AEO. In vitro assays, when transfected into neuroblastoma cell lines, demonstrated that ASOs with BNAP-AEO, had a more efficient gene silencing effect than those without BNAP-AEO. In vivo assays, involving intracerebroventricular injections into mice, revealed ASOs with BNAP-AEO potently suppressed gene expression in the brain. Surprisingly, the acute CNS toxicity in mice, as assessed through open field tests and scoring systems, was significantly lower for ASOs with BNAP-AEO than for those without BNAP-AEO. This study underscores the efficient gene-silencing effect and low acute CNS toxicity of ASOs incorporating BNAP-AEO, indicating the potential for future therapeutic applications.
RESUMO
[This corrects the article DOI: 10.1016/j.omtn.2024.102161.].
RESUMO
A critical strategy to improve the properties of oligonucleotide therapeutics is using cationic molecules as carriers. We developed artificial cationic molecules that bind to A-type oligonucleotide duplexes, such as siRNAs, in a stoichiometric ratio. In this study, we investigated the properties of oligo 2,6-diamino-D-galactoses (ODAGals) and L-2,4-diaminobutanoic acid oligomers (Dabs) and revealed their thermal and biological stabilization effects on A-type duplexes and their chemical stability. As a result, ODAGal and Dab with the same number of amino groups had the commensurate ability for the biological stabilization effect, whereas Dab enhanced the thermal stability of A-type duplexes more effectively than ODAGal.
Assuntos
Oligopeptídeos , RNA de Cadeia Dupla , RNA Interferente Pequeno/química , Oligonucleotídeos , Conformação de Ácido NucleicoRESUMO
Aggregation of the 43 kDa TAR DNA-binding protein (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RNA binding and TDP-43 N-terminal domain dimerisation has been suggested to ameliorate TDP-43 aggregation. However, the relationship between these factors and the solubility of TDP-43 is largely unknown. Therefore, we developed new oligonucleotides that can recruit two TDP-43 molecules and interfere with their intermolecular interactions via spatial separation. Using these oligonucleotides and TDP-43-preferable UG-repeats, we uncovered two distinct mechanisms for modulating TDP-43 solubility by RNA binding: One is N-terminal domain dimerisation, and the other is the spatial separation of two TDP-43 molecules. This study provides new molecular insights into the regulation of TDP-43 solubility.
Assuntos
Esclerose Lateral Amiotrófica , Degeneração Lobar Frontotemporal , Humanos , Proteínas de Ligação a DNA/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Degeneração Lobar Frontotemporal/metabolismo , Corpos de Inclusão/metabolismo , RNA/genética , RNA/metabolismoRESUMO
N-Trichloroacetyl analogs of N-acetylmannosamine 1-phosphate repeating units, which are found in capsular polysaccharides of Neisseria meningitidis serotype A, were successfully obtained via solid-phase synthesis using an oxazaphospholidine monomer. The introduction of the trichloroacetyl group into the amino group of mannosamine resulted in the stabilization of the reaction intermediates. Monosaccharide, disaccharide, and tetrasaccharide derivatives were obtained and isolated. This is the first example to demonstrate the synthesis of the N-acylated mannosamine 1-phosphate structure having no less than four phosphate linkages.
Assuntos
Neisseria meningitidis , Hexosaminas , Fosfatos , Polissacarídeos , Polissacarídeos Bacterianos/química , Sorogrupo , Técnicas de Síntese em Fase SólidaRESUMO
Recent advances in nucleic acid therapeutics increase the requirements for developing efficient methods for the chemical synthesis of oligodeoxyribonucleotides (ODNs). In this study, we report a new approach for the solution-phase synthesis of ODNs using the H-phosphonate method with N-unprotected 5'-phosphite monomers. The 5'-phosphite monomers are synthesized in a single step from unprotected 2'-deoxyribonucleosides using 5'-O-selective phosphitylation and can be applied to the synthetic cycle of the H-phosphonate method. We synthesized four kinds of 5'-phosphite monomers and then optimized the conditions for the condensation between the 3'-hydroxy groups of the 5'-phosphite monomers and the H-phosphonate monoesters. As a result of various investigations, solution-phase synthesis of trithymidine diphosphate (TTT) and tetramers containing four kinds of nucleobases was achieved according to the procedure consisting of repeated condensation, deprotection, and purification using simple extraction or precipitation.
RESUMO
Glycosyl phosphate repeating units can be found in the glycoconjugates of some bacteria and protozoa parasites. These structures and their P-modified analogs are attractive synthetic targets as antimicrobial, antiparasitic, and vaccine agents. However, P-modified glycosyl phosphates exist in different diastereomeric forms due to the chiral phosphorus atoms, whose configuration would highly affect their physiochemical and biochemical properties. In this study, a stereocontrolled method was developed for the synthesis of P-modified glycosyl phosphate repeating units derived from the lipophosphoglycan of Leishmania using the oxazaphospholidine approach. The solid-phase synthesis facilitated the elongation and purification of the glycosyl phosphate derivatives, while two P-modified glycosyl phosphates (boranophosphate and phosphorothioate) were successfully synthesized with up to three repeating units.
RESUMO
Achieving regulation of endogenous gene expression in the central nervous system (CNS) with antisense oligonucleotides (ASOs) administered systemically would facilitate the development of ASO-based therapies for neurological diseases. We demonstrate that DNA/RNA heteroduplex oligonucleotides (HDOs) conjugated to cholesterol or α-tocopherol at the 5' end of the RNA strand reach the CNS after subcutaneous or intravenous administration in mice and rats. The HDOs distribute throughout the brain, spinal cord and peripheral tissues and suppress the expression of four target genes by up to 90% in the CNS, whereas single-stranded ASOs conjugated to cholesterol have limited activity. Gene knockdown was observed in major CNS cell types and was greatest in neurons and microglial cells. Side effects, such as thrombocytopenia and focal brain necrosis, were limited by using subcutaneous delivery or by dividing intravenous injections. By crossing the blood-brain barrier more effectively, cholesterol-conjugated HDOs may overcome the limited efficacy of ASOs targeting the CNS without requiring intrathecal administration.
Assuntos
Barreira Hematoencefálica , RNA , Animais , Sistema Nervoso Central/metabolismo , Colesterol/metabolismo , DNA/metabolismo , Camundongos , Oligonucleotídeos/metabolismo , Oligonucleotídeos Antissenso/uso terapêutico , RNA/metabolismo , Ratos , RoedoresRESUMO
Heteroduplex oligonucleotides (HDOs) were a novel type of nucleic acid drugs based on an antisense oligonucleotide (ASO) strand and its complementary RNA (cRNA ) strand. HDOs were originally designed to improve the properties of RNase H-dependent ASOs and we reported in our first paper that HDOs conjugated with an α-tocopherol ligand (Toc-HDO ) based on a gapmer ASO showed 20 times higher silencing effect to liver apolipoprotein B (apoB) mRNA in vivo than the parent ASO. Thereafter the HDO strategy was found to be also effective for improving the properties of ASOs modulating blood-brain barrier function and ASO antimiRs which are RNase H-independent ASOs. Therefore, the HDO strategy has been shown to be versatile technology platform to develop effective nucleic acid drugs.
Assuntos
Inativação Gênica/efeitos dos fármacos , Ácidos Nucleicos Heteroduplexes/farmacologia , Oligonucleotídeos Antissenso/farmacologia , RNA/farmacologia , Animais , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Terapia Genética/métodos , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/uso terapêutico , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/uso terapêutico , RNA/química , RNA/uso terapêuticoRESUMO
Phosphorothioate (PS) modification, where a non-bridging oxygen atom in a phosphodiester linkage is replaced by a sulfur atom, is widely used to improve the properties of nucleic acid drugs. Each PS linkage can be found in two stereoisomers, Rp and Sp. Since one non-bridging oxygen or sulfur atom in Sp-PS or Rp-PS, respectively, is located close to the C-5 substituent of uracil in a DNA/RNA hybrid duplex, the combination of the stereochemistry of the PS linkages and the type of the C-5 modification of uracil bases is expected to affect the properties of the hybrid duplexes. Herein, DNA oligomers containing both stereopure phosphorohioate linkages and C-5 modified deoxyuridine derivatives were synthesized. The thermodynamic stability of the DNA/RNA and DNA/DNA duplexes and RNase H activity of the DNA/RNA duplexes were evaluated. The combination of 5-propynyluracil and (Rp)-PS linkages in a DNA strand could significantly increase the thermal stability of a DNA/RNA hybrid duplex without reducing its RNase H activity.
RESUMO
Small interfering RNAs (siRNAs) are potential tools for gene-silencing therapy, but their instability is one of the obstacles in the development of siRNA-based drugs. To improve siRNA stability, we synthesised a double-stranded RNA-binding cationic oligodiaminogalactose 4mer (ODAGal4) and investigated here its characteristics for siRNA stabilisation in vitro. ODAGal4 improved the resistance of various siRNAs against serum degradation. The effect of ODAGal4 on siRNA stabilisation was further amplified by introduction of modified nucleotides into the siRNA. In particular, a combination of ODAGal4 and incorporation of phosphorothioate linkages into the siRNA prominently prevented degradation by serum. The half-lives of fully phosphorothioate-modified RNA duplexes with ODAGal4 were more than 15 times longer than those of unmodified siRNAs without ODAGal4; this improvement in serum stability was superior to that observed for other chemical modifications. Serum degradation assays of RNAs with multiple chemical modifications showed that ODAGal4 preferentially improves the stability of RNAs with phosphorothioate modification among chemical modifications. Furthermore, melting temperature analysis showed that ODAGal4 greatly increases the thermal stability of phosphorothioate RNAs. Importantly, ODAGal4 did not interrupt gene-silencing activity of all the RNAs tested. Collectively, these findings demonstrate that ODAGal4 is a potent stabiliser of siRNAs, particularly nucleotides with phosphorothioate linkages, representing a promising tool in the development of gene-silencing therapies.
Assuntos
Oligossacarídeos/química , Estabilidade de RNA , RNA de Cadeia Dupla/química , RNA Interferente Pequeno/química , Soro/química , Animais , Inativação Gênica , Células HeLa , Humanos , Camundongos , Interferência de RNARESUMO
Correction for 'Enhancement in RNase H activity of a DNA/RNA hybrid duplex using artificial cationic oligopeptides' by Rintaro Iwata Hara et al., Chem. Commun., 2018, 54, 8526-8529.