Gene suppression via U1 small nuclear RNA interference (U1i) machinery using oligonucleotides containing 2′-modified-4′-thionucleosides

AbstractGene suppression via U1 small nuclear RNA interference (U1i) is considered to be one of the most attractive approaches, and takes the place of general antisense, RNA interference (RNAi), and anti-micro RNA machineries. Since the U1i can be induced by short oligonucleotides (ONs), namely U1 adaptors consisting of a ‘target domain’ and a ‘U1 domain’, we prepared adaptor ONs using 2′-modified-4′-thionucleosides developed by our group, and evaluated their U1i activity. As a result, the desired gene suppression via U1i was observed in ONs prepared as a combination of 2′-fluoro-4′-thionucleoside and 2′-fluoronucleoside units as well as only 2′-fluoronucleoside units, while those prepared as combination of 2′-OMe nucleoside/2′-OMe-4′-thionucleoside and 2′-fluoronucleoside units did not show significant activity. Measurement of Tm values indicated that a higher hybridization ability of adaptor ONs with complementary RNA is one of the important factors to show potent U1i activity

The AMPK/mTOR pathway is involved in D-dopachrome tautomerase gene transcription in adipocytes differentiated from SGBS cells, a human preadipocyte cell line

In adipose tissue, D-dopachrome tautomerase (DDT), a cytokine with structural similarity to macrophage migration inhibitory factor, is mainly expressed in adipocytes rather than preadipocytes and acts as an anti-obesity adipokine in an autocrine manner. However, its transcriptional regulation is largely unknown. In order to explore molecules affecting DDT transcription, a chemical library screening using HEK293 cells stably expressing a DDT promoter-reporter construct was performed. Several derivatives of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, were identified as transcriptional activators of the DDT gene. Furthermore, DDT mRNA levels were reduced in SGBS adipocytes treated with compound C, an AMPK inhibitor, suggesting involvement of AMPK in DDT transcription. Overexpression of the FOXO1 constitutive active form reduced transcriptional activity of the DDT gene in SGBS cells, but increased it in HEK293 cells. Cell-type specific effects were also observed in the DDT gene expression of cells treated with AS1842856, a FOXO1 inhibitor. Finally, involvement of the mammalian target of rapamycin (mTOR) signaling in DDT transcription in SGBS adipocytes was investigated. Rapamycin, an inhibitor of mTOR, increased DDT mRNA levels and attenuated the inhibitory effects of compound C on DDT mRNA levels in SGBS adipocytes. In conclusion, DDT transcription may be regulated in a cell-dependent manner, and were enhanced by AMPK activation in SGBS adipocytes through inhibiting the mTOR signaling

The novel functional nucleic acid iRed effectively regulates target genes following cytoplasmic delivery by faint electric treatment

An intelligent shRNA expression device (iRed) contains the minimum essential components needed for shRNA production in cells, and could be a novel tool to regulate target genes. However, general delivery carriers consisting of cationic polymers/lipids could impede function of a newly generated shRNA via electrostatic interaction in the cytoplasm. Recently, we found that faint electric treatment (fET) of cells enhanced delivery of siRNA and functional nucleic acids into the cytoplasm in the absence of delivery carriers. Here, we examined fET of cells stably expressing luciferase in the presence of iRed encoding anti-luciferase shRNA. Transfection of lipofectamine 2000 (LFN)/iRed lipoplexes showed an RNAi effect, but fET-mediated iRed transfection did not, likely because of the endosomal localization of iRed after delivery. However, fET in the presence of lysosomotropic agent chloroquine significantly improved the RNAi effect of iRed/fET to levels that were higher than those for the LFN/iRed lipoplexes. Furthermore, the amount of lipid droplets in adipocytes significantly decreased following fET with iRed against resistin in the presence of chloroquine. Thus, iRed could be a useful tool to regulate target genes following fET-mediated cytoplasmic delivery with endosomal escape devices

A Unique Gene-Silencing Approach, Using an Intelligent RNA Expression Device (iRed), Results in Minimal Immune Stimulation When Given by Local Intrapleural Injection in Malignant Pleural Mesothelioma

Background: We have recently introduced an intelligent RNA expression device (iRed), comprising the minimum essential components needed to transcribe short hairpin RNA (shRNA) in cells. Use of iRed efficiently produced shRNA molecules after transfection into cells and alleviated the innate immune stimulation following intravenous injection. Methods: To study the usefulness of iRed for local injection, the engineered iRed encoding luciferase shRNA (Luc iRed), complexed with cationic liposomes (Luc iRed/liposome-complexes), was intrapleurally injected into an orthotopic mesothelioma mouse model. Results: Luc iRed/liposome-complexes markedly suppressed the expression of a luciferase marker gene in pleurally disseminated mesothelioma cells. The suppressive efficiency was correlated with the expression level of shRNA within the mesothelioma cells. In addition, intrapleural injection of iRed/liposome-complexes did not induce IL-6 production in the pleural space and consequently in the blood compartment, although plasmid DNA (pDNA) or dsDNA (the natural construct for iRed) in the formulation did. Conclusion: Local delivery of iRed could augment the in vivo gene silencing effect without eliciting pronounced innate immune stimulation. Our results might hold promise for widespread utilization of iRed as an RNAi-based therapeutic for intracelial malignant cancers

Gene suppression via U1 small nuclear RNA interference (U1i) machinery using oligonucleotides containing 2'-modified-4'-thionucleosides

Gene suppression via U1 small nuclear RNA interference (U1i) is considered to be one of the most attractive approaches, and takes the place of general antisense, RNA interference (RNAi), and anti-micro RNA machineries. Since the U1i can be induced by short oligonucleotides (ONs), namely U1 adaptors consisting of a ‘target domain’ and a ‘U1 domain’, we prepared adaptor ONs using 2'-modified-4'-thionucleosides developed by our group, and evaluated their U1i activity. As a result, the desired gene suppression via U1i was observed in ONs prepared as a combination of 2'-fluoro-4'-thionucleoside and 2'-fluoronucleoside units as well as only 2'-fluoronucleoside units, while those prepared as combination of 2'-OMe nucleoside/2'-OMe-4'-thionucleoside and 2'-fluoronucleoside units did not show significant activity. Measurement of Tm values indicated that a higher hybridization ability of adaptor ONs with complementary RNA is one of the important factors to show potent U1i activity

Gene Silencing Using 4′-thioDNA as an Artificial Template to Synthesize Short Hairpin RNA Without Inducing a Detectable Innate Immune Response

The development of a versatile technique to induce RNA interference (RNAi) without immune stimulation in vivo is of interest as existing approaches to trigger RNAi, such as small interfering RNA (siRNA) and plasmid DNA (pDNA) expressing short hairpin RNA (shRNA), present drawbacks arising from innate immune stimulation. To overcome them, an intelligent shRNA expression device (iRed) designed to induce RNAi was developed. The minimum sequence of iRed encodes only the U6 promoter and shRNA. A series of iRed comprises a polymerase chain reaction (PCR)-amplified 4′-thioDNA in which any one type of adenine (A), guanine (G), cytosine (C), or thymine (T) nucleotide unit was substituted by each cognate 4′-thio derivatives, i.e., dSA iRed, dSG iRed, dSC iRed, and ST iRed respectively. Each modified iRed acted as a template to transcribe shRNA with RNAi activity. The highest shRNA yield was generated using dSC iRed that exerted gene silencing activity in an orthotopic mouse model of mesothelioma. Reducing the minimal structure required to transcribe shRNA and the presence of the 4′-thiomodification synergistically function to abrogate innate immune response induced by dsDNA. The iRed will introduce a new approach to induce RNAi without inducing a detectable innate immune response

New imidazopyridopyrimidine:naphthyridine base-pairing motif, ImN^[N]:NaO^[O], consisting of a DAAD:ADDA hydrogen bonding pattern, markedly stabilize DNA duplexes

The new imidazopyridopyrimidine:naphthyridine base-pairing motifs, ImO^[O]:NaN^[N] and ImN^[N]:NaO^[O], were designed. Among the base pairs examined, DNA duplexes containing ImN^[N]:NaO^[O] pair(s) consisting of a DAAD:ADDA hydrogen bonding pattern (D = donor, A = acceptor) were markedly stabilized thermally and thermodynamically

The Golgi-resident protein ACBD3 concentrates STING at ER-Golgi contact sites to drive export from the ER

STING, an endoplasmic reticulum (ER)-resident receptor for cyclic di-nucleotides (CDNs), is essential for innate immune responses. Upon CDN binding, STING moves from the ER to the Golgi, where it activates downstream type-I interferon (IFN) signaling. General cargo proteins exit from the ER via concentration at ER exit sites. However, the mechanism of STING concentration is poorly understood. Here, we visualize the ER exit sites of STING by blocking its transport at low temperature or by live-cell imaging with the cell-permeable ligand bis-pivSATE-2'F-c-di-dAMP, which we have developed. After ligand binding, STING forms punctate foci at non-canonical ER exit sites. Unbiased proteomic screens and super-resolution microscopy show that the Golgi-resident protein ACBD3/GCP60 recognizes and concentrates ligand-bound STING at specialized ER-Golgi contact sites. Depletion of ACBD3 impairs STING ER-to-Golgi trafficking and type-I IFN responses. Our results identify the ACBD3-mediated non-canonical cargo concentration system that drives the ER exit of STING

Synthesis and Behavior of DNA Oligomers Containing the Ambiguous Z-Nucleobase 5-Aminoimidazole-4-carboxamide

5-Amino-1-β-D-ribofuranosylimidazole-4-carboxamide 5′-monophosphate (ZMP) is a central intermediate in de novo purine nucleotide biosynthesis. Its nucleobase moiety, 5-aminoimidazole-4-carboxamide (Z-base), is considered an ambiguous base that can pair with any canonical base owing to the rotatable nature of its 5-carboxamide group. This idea of ambiguous base pairing due to free rotation of the carboxamide has been applied to designing mutagenic antiviral nucleosides, such as ribavirin and T-705. However, the ambiguous base-pairing ability of Z-base has not been elucidated, because the synthesis of Z-base-containing oligomers is problematic. Herein, we propose a practical method for the synthesis of Z-base-containing DNA oligomers based on the ring-opening reaction of an N1-dinitrophenylhypoxanthine (HxaDNP) base. Thermal denaturation studies of the resulting oligomers revealed that the Z-base behaves physiologically as an A-like nucleobase, preferentially forming pairs with T. We tested the behavior of Z-base-containing DNA oligomers in enzyme-catalyzed reactions: in single nucleotide insertion, Klenow fragment DNA polymerase recognized Z-base as an A-like analog and incorporated dTTP as a complementary nucleotide to Z-base in the DNA template; in PCR amplification, Taq DNA polymerase similarly incorporated dTTP as a complementary nucleotide to Z-base. Our findings will contribute to the development of new mutagenic antiviral nucleoside analogs