Synthesis of 4-Thiomodified c-di-AMP Analogs


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


Synthesis of oligonucleotides containing 5-homo-4-selenouridine derivative and its increased resistance against nuclease

As technologies using RNA or DNA have been developed, various chemical modifications of nucleosides have been attempted to increase the stability of oligonucleotides. Since it is known that 2'-OMe-modification greatly contributes to increasing the stability of oligonucleotides, we added 2'-OMe to our previously developed 4'-selenonucleoside and 5'-homo-4'-selenonucleoside as the modified monomers for oligonucleotide: 2'-methoxy-4'-selenouridine (2'-OMe-4'-Se-U) and 5'-homo-2'-methoxy-4'-selenouridine (5'-homo-2'-OMe-4'-Se-U). We synthesized oligonucleotides containing the chemically modified 4'-selenouridine and evaluated their thermal stability and nuclease resistance. In conclusion, the nuclease stability of the oligonucleotide containing 5'-homo-2'-OMe-4'-selenouridine increased while its thermal stability decreased.

Bioorganic & Medicinal Chemistry Letters


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-SATE-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 properties of fully-modified 4-selenoRNA, an endonuclease-resistant RNA analog

A large number of chemically modified oligonucleotides (ONs) have been developed for RNA-based technologies. In most modified RNAs, the characteristic 2'-hydroxyl (2'-OH) groups are removed to enhance both nuclease resistance and hybridization ability. However, the importance of the 2'-OH group in RNA structure and function is well known. Here, we report the synthesis and properties of 4'-selenoRNA in which all four nucleoside units retain the 2'-OH groups but contain a selenium atom instead of an oxygen atom at the 4'-position of the furanose ring. 4'-SelenoRNA has enhanced ability to form duplexes with RNA, and high endonuclease resistance despite the presence of the 2'-OH groups. X-ray crystallography analysis showed that the 4'-selenoRNA duplex adopts an A-conformation, similar to natural RNA, although one 4'-selenocytidine residue has unusual South-type sugar puckering. Furthermore, preliminary studies using 4'-seleno-modified siRNAs suggest that 4'-selenoRNA may be applicable to RNA interference technology. Collectively, our results raise the possibility of a new class of modified RNA in which 2'-OH groups do not need to be masked.


Cas9-mediated DNA cleavage guided by enzymatically prepared 4-thio-modified RNA

CRISPR-Cas9-mediated DNA editing relies on guide RNAs (gRNAs) that direct site-specific DNA cleavage by the Cas endonuclease. Because natural gRNA is susceptible to intracellular degradation, it is desirable to chemically protect it for efficient editing. Using 4'-thioribonucleoside 5'-triphosphates and T7 transcription, we have prepared 4'-thio-modified gRNAs that guide Cas9-mediated DNA cleavage. This approach is a simple way to obtain chemically modified RNA suitable for CRISPR-Cas9 DNA editing.


Rewriting the Central Dogma with Synthetic Genetic Polymers

DNA and RNA are ubiquitous molecules responsible for storage and transmission of genetic information and together comprise the central dogma of molecular biology. However, the recent emergence of synthetic genetic polymers is providing an opportunity to challenge the fundamental principles of life. Herein, we describe the ongoing attempts to rewrite the central dogma with 4'-thioDNA and 4'-thioRNA, which feature a sulfur instead of an oxygen atom in the furanose ring moiety. Using reconstituted Escherichia coli gene expression machinery, studies have shown that the genetic information conserved in 4'-thioDNA can be transcribed to 4'-thioRNA and eventually translated into protein, mirroring the processes that occur in nature. Such studies underscore the feasibility of controlling life by substances other than DNA and RNA.


Synthesis and Anti-dengue Virus Activity of 5-Ethynylimidazole-4-carboxamide (EICA) Nucleotide Prodrugs

We previously showed that 5-ethynyl-(1-β-D-ribofuranosyl)imidazole-4-carboxamide (1; EICAR) is a potent anti-dengue virus (DENV) compound but is cytotoxic to some cell lines, while its 4-thio derivative, 5-ethynyl-(4-thio-1-β-D-ribofuranosyl)imidazole-4-carboxamide (2; 4'-thioEICAR), has less cytotoxicity but also less anti-DENV activity. Based on the hypothesis that the lower anti-DENV activity of 2 is due to reduced susceptibility to phosphorylation by cellular kinase(s), we investigated whether a monophosphate prodrug of 2 can improve its activity. Here, we first prepared two types of prodrug of 1, which revealed that the S-acyl-2-thioethyl (SATE) prodrug had stronger anti-DENV activity than the aryloxyphosphoramidate (so-called ProTide) prodrug. Based on these findings, we next prepared the SATE prodrug of 4'-thioEICAR 18. As expected, the resulting 18 showed potent anti-DENV activity, which was comparable to that of 1; however, its cytotoxicity was also increased relative to 2. Our findings suggest that prodrugs of 4'-thioribonucleoside derivatives such as EICAR (1) represent an effective approach to developing potent biologically active compounds; however, the balance between antiviral activity and cytotoxicity remains to be addressed.


Convenient Synthesis of 3-Deazapurine Nucleosides (3-Deazainosine, 3-Deazaadenosine and 3-Deazaguanosine) Using Inosine as a Starting Material

A convenient synthetic method for preparing 3-deazapurine nucleosides (3-deazainosine, 3-deazaadenosine, and 3-deazaguanosine) from inosine via a 5-ethynyl-1-β-D-ribofuranosylimidazole-4-carboxamide (EICAR) derivative, which is a key intermediate, is described. A large-scale synthesis of an EICAR derivative starting from inosine was achieved in six steps via dinitrophenylation at the N position followed by ring opening, iodination of the resulting 5-amino group, and a palladium-catalyzed cross-coupling reaction. The resulting EICAR derivative was then converted into 3-deazainosine, 3-deazaadenosine, and 3-deazaguanosine. This route enabled us to synthesize 3-deazapurine nucleosides conveniently in good yields. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 5-ethynyl-1-β-D-ribofuranosylimidazole-4-carboxamide (EICAR) derivative 6 Basic Protocol 2: Preparation of 3-deazapurine nucleosides 8, 11, and 14.


5-Hydroxymethyltubercidin Exhibits Potent Antiviral Activity against Flaviviruses and Coronaviruses, including SARS-CoV-2

Newly emerging or re-emerging viral infections continue to cause significant morbidity and mortality every year worldwide, resulting in serious effects on both health and the global economy. Despite significant drug discovery research against dengue viruses (DENVs) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), no fully effective and specific drugs directed against these viruses have been discovered. Here, we examined the anti-DENV activity of tubercidin derivatives from a compound library from Hokkaido University and demonstrated that 5-hydroxymethyltubercidin (HMTU, HUP1108) possessed both potent anti-flavivirus and anti-coronavirus activities at submicromolar levels without significant cytotoxicity. Furthermore, HMTU inhibited viral RNA replication and specifically inhibited replication at the late stages of the SARS-CoV-2 infection process. Finally, we demonstrated that HMTU 5'-triphosphate inhibited RNA extension catalyzed by the viral RNA-dependent RNA polymerase. Our findings suggest that HMTU has the potential of serving as a lead compound for the development of a broad spectrum of antiviral agents, including SARS-CoV-2.


Synthesis of a cyclic dinucleotide analog with ambiguous bases, 5-aminoimidazole-4-carboxamide

Cyclic dinucleotides (CDNs) are second messengers composed of two purine nucleotides. In recent years, the structural diversity of CDNs and their functionality in biological processes are being intensely studied. Herein we report the chemical synthesis of cyclic di-5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl monophosphate (c-di-ZMP) (), which consists of two 5-amino-4-imidazolecarboxamide ribonucleotides (Z-ribonucleotides) linked via two phosphodiester linkages. Construction of the CDN skeleton with an -dinitrophenylhypoxanthine base (Hxa-base) by phosphoramidite chemistry and the subsequent ring-opening reaction of Hxa-base successfully yielded the desired .


Replacement of oxygen with sulfur on the furanose ring of cyclic dinucleotides enhances the immunostimulatory effect via STING activation

Cyclic dinucleotides (CDNs) are secondary messengers composed of two purine nucleotides linked two phosphodiester linkages: c-di-GMP, c-di-AMP, 3',3'-cGAMP, and 2',3'-cGAMP. CDNs activate the stimulator of interferon genes (STING) and trigger immune responses in mammalian species. CDNs are thus fascinating molecules as drug candidates, and chemically stable CDN analogues that act as STING agonists are highly desired at present. We herein report the practical synthesis of 4'-thiomodified c-di-AMP analogues, which have sulfur atoms at the 4'-position on the furanose ring instead of oxygen atoms, using simple phosphoramidite chemistry. The resulting 4'-thiomodified c-di-AMP analogues acted as potent STING agonists with long-term activity. Our results show that replacing O4' on CDNs with sulfur can lead to enhanced immunostimulatory effects STING activation.


Synthesis and Properties of 4-ThioLNA/BNA

To develop a new nucleoside analogue applicable to oligonucleotide therapeutics, we designed a 4'-thio analogue of an LNA/BNA monomer. Synthesis of 4'-hydroxymethyl-4'-thioribonucleoside was achieved by a tandem ring-contraction-aldol reaction of a 5-thiopyranose derivative and the subsequent Pummerer-type thioglycosylation reaction of the corresponding sulfoxide. Treatment of 4'-hydroxymethyl-4'-thiopyrimidine nucleosides with diphenyl carbonate in the presence of catalytic NaHCO gave the desired 4'-thioLNA/BNA monomers, which were introduced into oligonucleotides.


Gene Expression of 4'-Thioguanine DNA via 4'-Thiocytosine RNA.

DNA and RNA nucleotides are ubiquitous molecules that store and transmit genetic information. The emergence of synthetic elements that fulfill the function of DNA and RNA provides an alternative gene expression system. Herein, we demonstrate the gene expression of 4'-thioguanine DNA (dG DNA) via 4'-thiocytosine RNA (dC RNA) to give green fluorescent protein (GFPuv) in a single test tube. In replication, transcription, and translation, DNA/RNA polymerases and () ribosome can tolerate the replacement of O4' with S4' in the nucleotide, despite the fact that sulfur has a larger atomic radius than oxygen. Additionally, dG DNA and dC RNA acted as alternative genetic polymers to natural DNA and RNA for protein synthesis in artificial cells comprising a reconstituted gene expression machinery. This work involved simple experiments that are widely used in molecular biology, but which underscore the feasibility of life control by substances other than DNA/RNA nucleotides.

Journal of the American Chemical Society


Chemical Approaches for RNAi Drug Development.

RNA interference (RNAi) is the standard method of suppressing gene expression because of its target specificity, potency, and ability to silence the expression of virtually any gene. Using 21-mer small interfering RNA (siRNA) is the general approach for inducing RNAi, as siRNA can be easily prepared using a DNA/RNA synthesizer. Synthetic siRNA can be chemically modified to increase the potency of RNAi activity and abrogate innate immune stimulation. However, designing chemically modified siRNA requires substantial experimentation. A practical method for understanding the interaction of siRNA and RNAi-related proteins and how modifications affect RNA-protein interactions is therefore needed. Plasmid DNA (pDNA) expressing short hairpin RNA (shRNA) can also be used to induce RNAi. pDNA produces numerous shRNAs that induce RNAi with potent and longterm RNAi activity, even if only one pDNA molecule is delivered to the nucleus. However, this approach has some drawbacks with regard to its therapeutic application, such as a low pDNA transfection efficiency due to its huge molecular size and innate immune responses induced by extra genes, such as CpG motifs. To overcome these issues with RNAi inducers (siRNA and pDNA), our group developed some chemical approaches using chemically modified oligonucleotides. This article focuses on our two original approaches. The first involves the groove modification of siRNA duplexes to understand siRNA-protein interactions using 7-bromo-7-deazaadenosine and 3-bromo-3-deazaadenosine as chemical probes, while the second involves the generation of RNAi medicine using chemically modified DNA, known as an intelligent shRNA expression device (iRed).

Journal of the Pharmaceutical Society of Japan


フラノース環酸素原子を硫黄, セレン原子に置換した核酸誘導体の有機合成化学

フラノース環酸素原子を硫黄, セレン原子に置換した核酸誘導体の有機合成化学

Journal of Synthetic Organic Chemistry, Japan


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

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. 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. 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. 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.


Synthesis and properties of oligonucleotides containing a 2,6-diamino-3-deazapurine:furanopyrimidine base pair

Furanopyrimidine (FPy) and 2,6-diamino-3-deazapurine (DCPu) nucleosides with the ability to interact in DDD and AAA H-bonding patterns, respectively, were prepared. The N-1 p value of the DCPu nucleoside was estimated to be 6.4, which is due to the lack of a nitrogen atom at the 3-position, suggesting that DCPu acts as a base interacting in a DDD H-bonding pattern under near physiological conditions. As DCPu and FPy are expected to form a thermally stable DDD:AAA type of base pair in an oligodeoxynucleotide (ODN) duplex, they were incorporated into ODNs, and the value of the ODN duplex was determined. However, the ODN duplex containing a DCPu:FPy pair has a lower thermal stability than that containing a G:C pair does, although its thermal stability is equal to that of an ODN duplex with an A:T pair even under acidic conditions.

Nucleosides, Nucleotides & Nucleic Acids


Synthesis of nucleoside units possessing photoreactive diazirine groups on the major and minor groove faces.


Synthesis and biological evaluation of novel imidazole nucleosides as potential anti-dengue virus agents.

In this work, we developed imidazole nucleoside derivatives with anti-dengue virus (DENV) activity was examined. First, compounds in a nucleosides library were screened to find lead compounds which inhibit replication of DENV. As a result, 5-ethynyl-(1-β-d-ribofuranosyl)imidazole-4-carboxamide (1; EICAR) and its 4-carbonitrile derivative EICNR (2) were selected as promising antiviral compounds. However, both of them also exhibited cytotoxicity. In order to develop an effective and less toxic compound, 4'-thio and 4'-seleno derivatives of EICAR and EICNR 3-6 were prepared. The resulting 4'-thioEICAR and 4'-thioEICNR showed inhibitory effect on DENV replication without cytotoxicity as potent as ribavirin, a positive control.


四本の水素結合を介してペアを形成するImNN:NaOO塩基対の複製

四本の水素結合を介してペアを形成するImNN:NaOO塩基対の複製


Ribavirin-related compounds exert in vitro inhibitory effects toward rabies virus

Rabies remains an invariably fatal neurological disease despite the availability of a preventive vaccination and post-exposure prophylaxis that must be immediately administered to the exposed individual before symptom onset. There is no effective medication for treatment during the symptomatic phase. Ribavirin, a guanine nucleoside analog, is a potent inhibitor of rabies virus (RABV) replication in vitro but lacks clinical efficacy. Therefore, we attempted to identify potential ribavirin analogs with comparable or superior anti-RABV activity. Antiviral activity and cytotoxicity of the compounds were initially examined in human neuroblastoma cells. Among the tested compounds, two exhibited a 5- to 27-fold higher anti-RABV activity than ribavirin. Examination of the anti-RABV mechanisms of action of the compounds using time-of-addition and minigenome assays revealed that they inhibited viral genome replication and transcription. Addition of exogenous guanosine to RABV-infected cells diminished the antiviral activity of the compounds, suggesting that they are involved in guanosine triphosphate (GTP) pool depletion by inhibiting inosine monophosphate dehydrogenase (IMPDH). Taken together, our findings underline the potency of nucleoside analogs as a class of antiviral compounds for the development of novel agents against RABV.


Unnatural base pairs for synthetic biology

In this review, we have summarized the research effort into the development of unnatural base pairs beyond standard Watson-Crick (WC) base pairs for synthetic biology. Prior to introducing our research results, we present investigations by four outstanding groups in the field. Their research results demonstrate the importance of shape complementarity and stacking ability as well as hydrogen-bonding (H-bonding) patterns for unnatural base pairs. On the basis of this research background, we developed unnatural base pairs consisting of imidazo[5',4':4.5]pyrido[2,3-d]pyrimidines and 1,8-naphthyridines, i.e., Im : Na pairs. Since Im bases are recognized as ring-expanded purines and Na bases are recognized as ring-expanded pyrimidines, Im : Na pairs are expected to satisfy the criteria of shape complementarity and enhanced stacking ability. In addition, these pairs have four non-canonical H-bonds. Because of these preferable properties, ImN : NaO, one of the Im : Na pairs, is recognized as a complementary base pair in not only single nucleotide insertion, but also the PCR.


生物学的等価性に重点をおいた化学修飾核酸の創製-4'-セレノリボヌクレオシドの合成とオリゴマーへの導入-


A catch and release strategy towards HPLC-free purification of synthetic oligonucleotides using a phosphoramidite unit possessing a photocleavable azide linker

A convenient strategy to purify oligonucleotides (ONs) synthesized by solid phase synthesis on an automatic DNA/RNA synthesizer was described. By attaching a photocleavable azide linker as the last phosphoramidite unit in the ON synthesis, only the desired full-length sequence was 'caught' on a controlled pore glass (CPG) resin possessing an aza-dimethoxycyclooctyne (DIBAC) derivative. Washing the resulting CPG resin to remove all unbounded species, the subsequent photoirradiation allowed the pure ONs to be 'released' without leaving any chemical modifications on native ON structure or chemical reagents from the solid phase ON synthesis.


Synthesis and evaluation of c-di-4'-thioAMP as an artificial ligand for c-di-AMP riboswitch

Cyclic-di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger that binds to an RNA receptor called riboswitch and regulates its downstream genes involving cell wall metabolism, ion transport, and spore germination. Therefore, the c-di-AMP riboswitch can be a novel target of antibiotics. In this study, we synthesized c-di-4'-thioAMP (1), which possesses a sulfur atom instead of an oxygen atom in the furanose ring, as a candidate of a bioisoster for natural c-di-AMP. The resulting 1 bound to the c-di-AMP riboswitch with a micromolar affinity (34.8M), and the phosphodiesterase resistance of 1 was >12-times higher than that of c-di-AMP. Thus, 1 can be considered to be a stable ligand against a c-di-AMP riboswitch.


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--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.


Synthesis of 4'-selenoribonucleosides, the building blocks of 4'-selenoRNA, using a hypervalent iodine.

Herein is described a detailed protocol for the synthesis of 4'-selenoribonucleoside derivatives that involves the use of a hypervalent iodine species. These derivatives are versatile units for the preparation of 4'-selenoRNA. Large-scale synthesis of a 4-selenosugar starting from D-ribose is achieved in eight steps, including a final chromatographic purification. The resulting 4-selenosugar is then subjected to the one-pot Pummerer-like reaction using hypervalent iodine in the presence of silylated nucleobases. The reaction with silylated uracil affords the desired 4'-selenouridine derivatives with excellent -selectivity and in good yield. Conversely, when purine nucleobases are used in the Pummerer-like reaction, N7 4'-selenoribonucleoside isomers are obtained alongside the desired N9 isomers. However, the undesired N7 isomers can be converted to the desired N9 ones under acidic conditions. © 2017 by John Wiley & Sons, Inc.

Current Protocols in Nucleic Acid Chemistry


Practical synthesis of 4'-selenopurine nucleosides by combining chlorinated purines and 'armed' 4'-selenosugar


Groove modification of siRNA duplexes to elucidate siRNA-protein interactions using 7-bromo-7-deazaadenosine and 3-bromo-3-deazaadenosine as chemical probes

Elucidation of dynamic interactions between RNA and proteins is essential for understanding the biological processes regulated by RNA, such as RNA interference (RNAi). In this study, the logical chemical probes, comprising 7-bromo-7-deazaadenosine (Br(7)C(7)A) and 3-bromo-3-deazaadenosine (Br(3)C(3)A), to investigate small interfering RNA (siRNA)-RNAi related protein interactions, were developed. The bromo substituents of Br(7)C(7)A and Br(3)C(3)A are expected to be located in the major and the minor grooves, respectively, and to act as a steric hindrance in each groove when these chemical probes are incorporated into siRNAs. A comprehensive investigation using siRNAs containing these chemical probes revealed that (i) Br(3)C(3)A(s) at the 5'-end of the passenger strand enhanced their RNAi activity, and (ii) the direction of RISC assembly is determined by the interaction between Argonaute2, which is the main component of RISC, and siRNA in the minor groove near the 5'-end of the passenger strand. Utilization of these chemical probes enables the investigation of the dynamic interactions between RNA and proteins.


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.

Science and Technology of Advanced Materials


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.


Faithful PCR Amplification of an Unnatural Base-Pair Analogue with Four Hydrogen Bond

In vitro replication of an unnatural imidazopyridopyridine:naphthyridine base pair, (i.e., ImN(N):NaO(O)), having four hydrogen bonds was investigated. Kinetic studies of single-nucleotide insertion revealed that ImN(N) and NaO(O) were recognized as complementary bases by an exonuclease-deficient Klenow fragment with higher specificity and efficiency than two previously described pairs (ImN(O):NaO(N) and ImO(N):NaN(O)) because of higher thermal and thermodynamic stabilities and the DAAD:ADDA (D=donor, A=acceptor) hydrogen-bonding pattern of the ImN(N):NaO(O) pair. Faithful polymerase chain reaction (PCR) amplification of a DNA fragment containing the ImN(N):NaO(O) pair was achieved by using DNA polymerases possessing 3'5' exonuclease activity (99.5 % per doubling).


Synthesis of DNA fragments containing 2-deoxy-4-selenonucleoside units using DNA polymerases: comparison of dNTPs with O, S and Se at the 4-position in replication Org

4'-SelenoDNA fragments were synthesized for the first time using 4'-selenothymidine triphosphate (SeTTP) by taking advantage of its bioequivalence against DNA polymerases. DNA fragments each with a homologous element (O, S or Se) at the 4'-position of the thymidine units were effectively amplified using KOD Dash DNA polymerase.


Chemistry, properties, and in vitro and in vivo applications of 2'-O-methoxyethyl-4'-thioRNA, a novel hybrid type of chemically modified RNA.

We report the synthesis, properties, and in vitro and in vivo applications of 2'-O-methoxyethyl-4'-thioRNA (MOE-SRNA), a novel type of hybrid chemically modified RNA. In its hybridization with complementary RNA, MOE-SRNA showed a moderate improvement of Tm value (+3.4 °C relative to an RNA:RNA duplex). However, the results of a comprehensive comparison of the nuclease stability of MOE-SRNA relative to 2'-O-methoxyethylRNA (MOERNA), 2'-O-methyl-4'-thioRNA (Me-SRNA), 2'-O-methylRNA (MeRNA), 4'-thioRNA (SRNA), and natural RNA revealed that MOE-SRNA had the highest stability (t1/2 >48 h in human plasma). Because of the favorable properties of MOE-SRNA, we evaluated its in vitro and in vivo potencies as an anti-microRNA oligonucleotide against miR-21. Although the in vitro potency of MOE-SRNA was moderate, its in vivo potency was significant for the suppression of tumor growth (similar to that of MOERNA).


First Synthesis of Fully Modified 4-SelenoRNA and 2-OMe-4-selenoRNA Based on the Mechanistic Considerations of an Unexpected Strand Break

This study investigated oligonucleotide (ON) synthesis containing 4'-selenoribonucleoside(s) under standard phosphoramidite conditions. Careful operation using a manual ON synthetic system revealed that an unexpected strand break occurred to afford a C2-symmetric homodimer as a byproduct. In addition, this side reaction occurred during I2 oxidation. On the basis of these findings, the first synthesis of fully modified 4'-selenoRNA and 2'-OMe-4'-selenoRNA was achieved using tert-butyl hydroperoxide (TBHP) as the alternative oxidant.