RESUMO
We recently reported the antisense properties of a DNA/RNA heteroduplex oligonucleotide consisting of a phosphorothioate DNA-gapmer antisense oligonucleotide (ASO) strand and its complementary phosphodiester RNA/phosphorothioate 2'-O-methyl RNA strand. When α-tocopherol was conjugated with the complementary strand, the heteroduplex oligonucleotide silenced the target RNA more efficiently in vivo than did the parent single-stranded ASO. In this study, we designed a new type of the heteroduplex oligonucleotide, in which the RNA portion of the complementary strand was replaced with phosphodiester DNA, yielding an ASO/DNA double-stranded structure. The ASO/DNA heteroduplex oligonucleotide showed similar activity and liver accumulation as did the original ASO/RNA design. Structure-activity relationship studies of the complementary DNA showed that optimal increases in the potency and the accumulation were seen when the flanks of the phosphodiester DNA complement were protected using 2'-O-methyl RNA and phosphorothioate modifications. Furthermore, evaluation of the degradation kinetics of the complementary strands revealed that the DNA-complementary strand as well as the RNA strand were completely cleaved in vivo. Our results expand the repertoire of chemical modifications that can be used with the heteroduplex oligonucleotide technology, providing greater design flexibility for future therapeutic applications.
Assuntos
DNA/genética , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Oligodesoxirribonucleotídeos/genética , Células Cultivadas , DNA/administração & dosagem , Inativação Gênica , Oligodesoxirribonucleotídeos/administração & dosagem , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genéticaRESUMO
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
In this study, we conducted polymerase chain reaction (PCR) experiments using Escherichia coli (E. coli) and a Mars sand simulant (Mars Global Simulant MGS-1, Exolith Lab) to detect and analyze potential extraterrestrial life. The targeted DNA sequence is common among the bacterial kingdom on Earth. PCR experiments conducted after alkaline heat extraction, wherein samples with varying amounts of Mars sand simulant were compared, revealed that the simulant interfered with DNA detection. We then conducted PCR experiments following treatment with a sand DNA extraction kit on samples with various E. coli densities. DNA bands for a minimum E. coli density of 900 cells/(g sand) were confirmed, while no DNA bands were visible in the 90 cells/(g sand) sample with and without the Mars sand simulant. The total DNA mass contained in 900 cells was calculated to be 15.3 pg (i.e., 1.53 pg in 0.1 g sand sample we evaluated). We tested and compared the influence of the eluate of Mars sand simulant and DNA adsorption onto Mars sand simulant based on optical absorbance measurements. Our findings suggest that the mechanism by which the Mars sand simulant prevents PCR is through the adsorption of DNA onto the Mars sand simulant.
Assuntos
DNA Bacteriano , Escherichia coli , Exobiologia , Meio Ambiente Extraterreno , Marte , Reação em Cadeia da Polimerase , Areia , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Exobiologia/métodos , DNA Bacteriano/análise , DNA Bacteriano/genéticaRESUMO
Gapmer-type antisense oligonucleotides have not yet been approved for the treatment of central nervous system diseases, whereas steric-blocking-type antisense oligonucleotides have been well-developed for clinical use. We here characterize a new type of double-stranded oligonucleotides, overhanging-duplex oligonucleotides, which are composed of the parent gapmer and its extended complementary RNA. By intracerebroventricular injection, overhanging oligonucleotides show greater silencing potency with more efficient delivery into mouse brains than the parent single-stranded gapmer. Structure-activity relationship analyses reveal that the potency enhancement requires 13-mer or more overhanging oligonucleotides with a phosphorothioate backbone. Overhanging oligonucleotides provide a new platform of therapeutic oligonucleotides for gene modulation in the central nervous system.