Synthesis of 4-Thiomodified c-di-AMP Analogs

Cyclic diadenosine monophosphate (c-di-AMP) is a bacterial cyclic dinucleotide (CDN) comprising two adenosine monophosphates covalently linked by two 3',5'-phosphodiester bonds. c-di-AMP works as a second messenger, regulating many biological processes in bacteria such as cell wall homeostasis, DNA integrity, and sporulation via specific protein and/or RNA receptors. Moreover, c-di-AMP can function as an immunomodulatory agent in eukaryote cells via the stimulator of interferon genes (STING) signaling pathway. This protocol describes the chemical synthesis of two c-di-AMP analogs with a sulfur atom at the 4'-position of the furanose ring instead of an oxygen atom: c-di-4'-thioAMP (1) and cAMP-4'-thioAMP (2). Analogs 1 and 2 have resistance to phosphodiesterase-mediated degradation and are therefore useful for understanding the diverse biological phenomena regulated by c-di-AMP. In this protocol, two 4'-thioadenosine monomers are initially prepared via a Pummerer-like reaction assisted by hypervalent iodine. The CDN skeleton is then constructed through two key reactions based on phosphoramidite chemistry: dimerization of two appropriately protected nucleoside monomers to produce a linear dinucleotide, followed by macrocyclization of the resulting linear dinucleotide to form the CDN skeleton. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 4'-thioadenosine monomers 13 and 14 Basic Protocol 2: Preparation of c-di-4'-thioAMP (1) and cAMP-4'-thioAMP (2).


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 -dinitrophenylhypoxanthine (Hxa) 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.


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


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.


Use of modified U1 small nuclear RNA for rescue from exon 7 skipping caused by 5-splice site mutation of human cathepsin A gene

Cathepsin A (CTSA) is a multifunctional lysosomal enzyme, and its hereditary defect causes an autosomal recessive disorder called galactosialidosis. In a certain number of galactosialidosis patients, a base substitution from adenine to guanine is observed at the +3 position of the 7th intron (IVS7 +3a>g) of the CTSA gene. With this mutation, a splicing error occurs; and consequently mRNA lacking the 7th exon is produced. This skipping of exon 7 causes a frame shift of the transcripts, resulting in a non-functional CTSA protein and hence galactosialidosis. This mutation seems to make the interaction between the 5'-splice site of intron 7 of pre-mRNA and U1 small nuclear RNA (U1 snRNA) much weaker. In the present study, to produce properly spliced mRNA from the CTSA gene harboring this IVS7 +3a>g mutation, we examined the possible usefulness of modified U1 snRNA that could interact with the mutated 5'-splice site. Toward this goal, we first prepared a model system using a mutant CTSA mini gene plasmid for delivery into HeLa cells. Then, we examined the effectiveness of modified U1 snRNA on the formation of properly spliced mRNA from this mutant CTSA mini gene. As a result, we succeeded in obtaining improved formation of properly spliced CTSA mRNA. Our results suggest the usefulness of modified U1 snRNA for rescue from exon 7 skipping caused by the IVS7 +3a>g mutation of the CTSA gene.


Glycosylation reactions mediated by hypervalent iodine: application to the synthesis of nucleosides and carbohydrates

To synthesize nucleoside and oligosaccharide derivatives, we often use a glycosylation reaction to form a glycoside bond. Coupling reactions between a nucleobase and a sugar donor in the former case, and the reaction between an acceptor and a sugar donor of in the latter are carried out in the presence of an appropriate activator. As an activator of the glycosylation, a combination of a Lewis acid catalyst and a hypervalent iodine was developed for synthesizing 4'-thionucleosides, which could be applied for the synthesis of 4'-selenonucleosides as well. The extension of hypervalent iodine-mediated glycosylation allowed us to couple a nucleobase with cyclic allylsilanes and glycal derivatives to yield carbocyclic nucleosides and 2',3'-unsaturated nucleosides, respectively. In addition, the combination of hypervalent iodine and Lewis acid could be used for the glycosylation of glycals and thioglycosides to produce disaccharides. In this paper, we review the use of hypervalent iodine-mediated glycosylation reactions for the synthesis of nucleosides and oligosaccharide derivatives.


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.


Transcription of 4-thioDNA templates to natural RNA in vitro and in mammalian cells

Synthetic chemically modified nucleic acids, which are compatible with DNA/RNA polymerases, have great potential as a genetic material for synthetic biological studies. A comprehensive analysis of the transcription of 4'-thioDNA templates to natural RNA is herein reported. The modifications of DNA with 2'-deoxy-4'-thionucleosides, so-called 4'-thioDNA, were found to act as templates for transcription with RNA polymerases both in vitro and in mammalian cells.


Practical Syunthesis of 4'-Thioribonucleosides Starting from D-Ribose

A practical synthesis of 4'-thioribonucleosides, i.e., 4'-thiouridine, -cytidine, -adenosine, and -guanosine, which are versatile units for nucleic acids-based therapeutics, is described. Large-scale synthesis of 4-thiosugar starting from D-ribose was achieved (33%) in eight steps and with only three chromatographic purifications. After the appropriate chemical conversion of the 4-thiosugar, the resulting sulfoxide was subjected to the Pummerer reaction in the presence of silylated nucleobases. In reactions with silylated pyrimidine bases, the desired 4'-thioribonucleoside derivatives were obtained in good yield and -selectively. On the other hand, N-7 isomers were obtained mainly in the Pummerer reaction with purine bases under the same conditions. However, the desired N-9 isomers were obtained in moderate yields when the reaction mixtures were subsequently heated under reflux. As a result, effective synthesis of 4'-thioribonucleosides was accomplished. © 2014 by John Wiley & Sons, Inc.

Current Protocols in Nucleic Acid Chemistry


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.


Development of a new dumbbell-shaped decoy DNA using a combination of the unnatural base pair ImON:NaNO and a CuAAC reaction.

We describe the synthesis and potential application of a new dumbbell-shaped decoy DNA prepared using a combination of the base pair ImON:NaNO and a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. The CuAAC reaction between the azido group on 5'-end of oligodeoxynucleotide (ODN) and the ethynyl group on the NaNO base of the opposite strand did not proceed, whereas that between the azido group and the flexible hexynyl group on the NaNO base of the opposite strand proceeded smoothly to give a new dumbbell-shaped double-stranded ODN (dsODN). The resulting dsODN had extremely high thermal stability and exhibited exonuclease resistance. In addition, the terminal modification did not affect its helical structure, and thus, the dumbbell-shaped dsODN displayed promising in vitro activity in a competition assay with the NF-kB p50 transcription factor homodimer.


Allosteric control of a DNA-hydrolyzing deoxyribozyme with short oligonucleotides and its application in DNA logic gates.

Allosteric control of deoxyribozymes is useful for a broad range of practical applications, such as nucleic acid sensing and DNA-computing. We found that the catalytic activity of a DNA-hydrolyzing deoxyribozyme could be allosterically regulated by adding short oligonucleotides. We used this technique to construct deoxyribozyme-based logic gates.


The systemic administration of an anti-miRNA oligonucleotide encapsulated pH-sensitive liposome results in reduced level of hepatic microRNA-122 in mice.

Efficient delivery continues to be a challenge in microRNA (miRNA) therapeutics. We utilized a pH-sensitive multifunctional envelope-type nano device (MEND) containing a pH-sensitive lipid YSK05 (YSK05-MEND) to regulate liver specific miRNA-122 (miR-122). Anti-microRNA oligonucleotides including 2'-OMe and phosphorothioate modifications against miR-122 (AMO122) were encapsulated in the YSK05-MEND. Despite the lower uptake, the YSK05-MEND showed a higher activity in liver cancer cells than Lipofectamine2000 (LFN2k) due to efficient endosomal escape. Cytotoxicity was minimal at 100 nM of AMO122 in YSK05-MEND treated cells, but LFN2k showed toxicity at 50 nM. When mice were administrated with free AMO122, it was eliminated via the kidney due to its molecular weight, and lesser amounts were detected in the liver. Conversely, the YSK05-MEND delivered higher amounts of the AMO122 to the liver. Systemic administration of YSK05-MEND induced the knockdown of miR-122 and an increase in target genes in the liver, and a subsequent reduction in plasma cholesterol at a dose of 1 mg AMO/kg while free AMO122 showed no activity at the same dose. The effect of AMO122 delivered by YSK05-MEND persisted for over 2 weeks. These results suggest that YSK05-MEND is a promising system for delivering AMOs to the liver. (C) 2013 Elsevier B.V. All rights reserved.

Efficient delivery continues to be a challenge in microRNA (miRNA) therapeutics. We utilized a pH-sensitive multifunctional envelope-type nano device (MEND) containing a pH-sensitive lipid YSK05 (YSK05-MEND) to regulate liver specific miRNA-122 (miR-122). Anti-microRNA oligonucleotides including 2′-OMe and phosphorothioate modifications against miR-122 (AMO122) were encapsulated in the YSK05-MEND. Despite the lower uptake, the YSK05-MEND showed a higher activity in liver cancer cells than Lipofectamine2000 (LFN2k) due to efficient endosomal escape. Cytotoxicity was minimal at 100 nM of AMO122 in YSK05-MEND treated cells, but LFN2k showed toxicity at 50 nM. When mice were administrated with free AMO122, it was eliminated via the kidney due to its molecular weight, and lesser amounts were detected in the liver. Conversely, the YSK05-MEND delivered higher amounts of the AMO122 to the liver. Systemic administration of YSK05-MEND induced the knockdownofmiR-122andan increase in target genes inthe liver, and a subsequent reduction in plasma cholesterol at a dose of 1mgAMO/kgwhile free AMO122 showed no activity at the same dose. The effect ofAMO122 delivered by YSK05-MEND persisted for over 2 weeks. These results suggest that YSK05-MEND is a promising system for delivering AMOs to the liver.


Synthesis of a novel 1,2-dithianenucleoside via Pummerer-like reaction, followed by Vorbruggen glycosylation between a 1,2-dithiane derivative and uracil

The novel 1,2-dithianenucleoside was designed as a hybrid type of modification between 4'-thioribonucleoside and altritol nucleoside. The desired compound, i.e., 1-[(3R,4R,5S,6R)-4,5-dihydroxy-6-hydroxymethyl-1,2-dithianyl]uracil (20), was prepared via the Pummerer-like reaction, followed by Vorbruggen glycosylation between an appropriately protected 1,2-dithiane derivative and silylated uracil.


In vitro optimization of 2'-OMe-4'-thioribonucleoside-modified anti-microRNA oligonucleotides and its targeting delivery to mouse liver using a liposomal nanoparticle.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally. Previous studies, which characterized miRNA function, revealed their involvement in fundamental biological processes. Importantly, miRNA expression is deregulated in many human diseases. Specific inhibition of miRNAs using chemically modified anti-miRNA oligonucleotides (AMOs) can be a potential therapeutic strategy for diseases in which a specific miRNA is overexpressed. 2'-O-Methyl (2'-OMe)-4'-thioRNA is a hybrid type of chemically modified oligonucleotide, exhibiting high binding affinity to complementary RNAs and high resistance to nuclease degradation. Here, we evaluate 2'-OMe-4'-thioribonucleosides for chemical modification on AMOs. Optimization of the modification pattern using a variety of chemically modified AMOs that are perfectly complementary to mature miR-21 revealed that the uniformly 2'-OMe-4'-thioribonucleoside-modified AMO was most potent. Further investigation showed that phosphorothioate modification contributed to long-term miR-122 inhibition by the 2'-OMe-4'-thioribonucleoside-modified AMO. Moreover, systemically administrated AMOs to mouse using a liposomal delivery system, YSK05-MEND, showed delivery to the liver and efficient inhibition of miR-122 activity at a low dose in vivo.


PCR amplification of 4-thioDNA using 2-deoxy-4-thionucleoside 5-triphosphates

2'-Deoxy-4'-thioribonucleic acid (4'-thioDNA) having a sulfur atom instead of an oxygen atom in the furanose ring has a nuclease resistance and hybridization ability higher than that of natural DNA. Despite its great potential for various biological applications, a long 4'-thioDNA having all four kinds of 2'-deoxy-4'-thionucleosides has not been reported. In this study, we describe systematic analysis of the incorporation of 2'-deoxy-4'-thionucleoside 5'-triphosphates (dSNTPs) using various DNA polymerases. We found that family B DNA polymerases, which do not have 3'5' exonuclease activity, could efficiently incorporate dSNTPs via single nucleotide insertion and primer extension. Moreover, 104-mer PCR product was obtained even under the conditions in the presence of all four kinds of dSNTPs when KOD Dash DNA polymerase was used. The resulting PCR product was converted into a natural dsDNA by using PCR with dNTPs, and sequencing of the natural dsDNA revealed that the PCR cycle successfully proceeded without losing the sequence information of the template. To the best of our knowledge, this is the first example of accurate PCR amplification of highly modified DNA in the presence of only unnatural dNTPs.


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.


A practical post-modification synthesis of oligodeoxynucleotides containing 4,7-diaminoimidazo[5,4:4,5]pyrido[2,3-d]pyrimidine nucleosid

We describe herein the practical post-modification synthesis of oligodeoxynucleotides (ODNs) containing 4,7-diaminoimidazo[5',4':4,5]pyrido[2,3-d]pyrimidine nucleoside (ImN(N)). Since the ImN(N) nucleoside unit possessing tribenzoyl groups on its exocyclic amino groups as the protecting group was quite unstable under acidic conditions, cleavage of its glycosidic linkage in ODN has been suggested throughout the conditions of solid-phase synthesis. As an alternative approach, we investigated a post-modification synthesis of the desired ODNs containing the ImN(N) unit. Starting with protected 4-amino-7-chloro-1-(2-deoxy--D-ribofuranosyl)imidazo[5',4':4,5]pyrido[2,3-d]pyrimidine derivative 1, conversion into the corresponding phosphoramidite unit was examined. The p-bromobenzoyl group (p-BrBz) was the best protecting group of 4-amino group of 1 to give the phosphoramidite unit 9 for the post-modification synthesis. After carrying out the ODN synthesis linked to the controlled pore glass (CPG) support, the support was treated with ammonium hydroxide at 55 °C to remove the protecting groups, detach the ODN form the CPG support, and convert the 7-chloro group into a desired amino group. As a result, the desired ODNs containing ImN(N) were obtained in good yield.


Intracellular stability of 2-OMe-4-thioribonucleoside modified siRNA leads to long-term RNAi effect

Chemically modified siRNAs are expected to have resistance toward nuclease degradation and good thermal stability in duplex formation for in vivo applications. We have recently found that 2'-OMe-4'-thioRNA, a hybrid chemical modification based on 2'-OMeRNA and 4'-thioRNA, has high hybridization affinity for complementary RNA and significant resistance toward degradation in human plasma. These results prompted us to develop chemically modified siRNAs using 2'-OMe-4'-thioribonucleosides for therapeutic application. Effective modification patterns were screened with a luciferase reporter assay. The best modification pattern of siRNA, which conferred duration of the gene-silencing effect without loss of RNAi activity, was identified. Quantification of the remaining siRNA in HeLa-luc cells using a Heat-in-Triton (HIT) qRT-PCR revealed that the intracellular stability of the siRNA modified with 2'-OMe-4'-thioribonucleosides contributed significantly to the duration of its RNAi activity.


New imidazopyridopyrimidine:naphthyridine base-pairing motif, ImNN:NaOO, 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.


Highly efficient enzymatic synthesis of 30-deoxyapionucleic acid (apioNA) having the four natural nucleobases

The synthesis of the 3'-deoxyapionucleoside 3''-triphosphates (apioNTPs) having the four natural nucleobases and their enzymatic incorporation into a DNA-DNA primer-template have been tried. Therminator DNA polymerase was shown to incorporate these apioNTPs effectively giving 43mer DNA-apioNA chimera.


Mutagenicity of secondary oxidation products of 8-oxo-7,8-dihydro-2-deoxyguanosine 5-triphosphate (8-hydroxy-2- deoxyguanosine 5-triphosphate)

8-Oxo-7,8-dihydroguanine (8-hydroxyguanine) is oxidized more easily than normal nucleobases, which can produce spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). These secondary oxidation products of 8-oxo-7,8-dihydroguanine are highly mutagenic when formed within DNA. To evaluate the mutagenicity of the corresponding oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-hydroxy-2'- deoxyguanosine 5'-triphosphate) in the nucleotide pool, Escherichia coli cells deficient in the mutT gene were treated with H(2)O(2), and the induced mutations were analyzed. Moreover, the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh were also introduced into competent E. coli cells. The H(2)O(2) treatment of mutT E. coli cells resulted in increase of G:C T:A and A:T T:A mutations. However, the incorporation of exogenous Sp and Gh 2'-deoxyribonucleotides did not significantly increase the mutation frequency. These results suggested that the oxidation product(s) of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate induces G:C T:A and A:T T:A mutations, and that the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh exhibit quite weak mutagenicity, in contrast to the bases in DNA.


Practical synthesis of 4'-selenopyrimidine nucleosides using hypervalent iodine


Synthesis and characterization of oligodeoxynucleotides containing a novel tetraazabenzo[cd]azulene:naphthyridine base pair

The synthesis and thermal stability of oligodeoxynucleotides (ODNs) containing 4-amino-2,3,5,6-tetraazabenzo[cd]azulen-7-one nucleosides 5 (BaO(N)) with the aim of developing new base pairing motif is described. The tricyclic nucleoside 5 was prepared starting with the 7-deaza-7-iodopurine derivative 1 via a palladium catalyzed cross-coupling reaction with methyl acrylate, followed by an intramolecular cyclization. The resulting nucleoside was incorporated into ODNs, and the base pairing property of the BaO(N):NaN(O) (2-amino-7-hydroxy-1,8-naphthyridine nucleoside) pair in the duplex was evaluated by a thermal denaturation study. The melting temperature (T(m)) of the duplex containing the BaO(N):NaN(O) pair showed a higher value than that of the duplexes containing the adenine:thymine (A:T) and the guanine:cytosine (G:C) pairs, however it was lower than that of the ImO(N):NaN(O) (ImO(N)=7-amino-imidazo[5',4':4,5]pyrido[2,3-d]pyrimidin-4(5H)-one nucleoside) pair. A temperature-dependent (1)H NMR study revealed that the H-bonding ability of BaO(N) was lower than that of ImO(N), which would explain why the BaO(N):NaN(O) pair was less thermally stable than the ImO(N):NaN(O) pair.


Synthesis and properties of a novel nucleoside derivative possessing a 2,3,5,6-tetraazabenzo[cd]azulene skeleton


A convenient RNA synthesis using a phosphoramidite possessing a biotinylated photocleavable group

A convenient RNA synthesis using a biotin-streptavidin interaction and a photocleavable protecting group is described. The biotinylated photocleavable group was introduced at the 2'-position of the uridine derivative. Using the phosphoramidite 12, we attempted the synthesis of a 21mer RNA, which is pure enough to show potent RNAi activity compared with a conventionally prepared and HPLC-purified 21mer RNA with the same sequence.


NMR study of the DNA duplex containing a thermally stable base-pairing motif


RNAを標的とする低分子創薬の進展

RNAを標的とする低分子創薬の進展

RNAは，現在の生命科学研究の大きなターゲットとなっている生体分子である．触媒作用を持つRNA分子であるリボザイム，二本鎖RNAがmRNAを特異的に分解する現象であるRNA干渉(RNA interference:RNAi)，タンパク質に翻訳されない非コードRNA(non-coding RNA:ncRNA)の発見により，RNAが生命の起源であるというRNAワールド仮説までもが提唱されるようになっている．これらの様々な新機能の発見に伴い，RNAは新規創薬標的としての注目を集めている．RNAを標的とする創薬では，RNAiを利用するshort interference RNA(siRNA)などの研究開発が盛んであるが，本稿ではRNAを標的とした低分子創薬に焦点をあてて解説する．
RNAを標的とする低分子創薬の歴史は意外と古い．ストレプトマイシンやカナマイシンに代表されるアミノグリコシド系抗菌薬は，細菌のリボソームRNA中の40残基程度の小領域に結合し，翻訳を阻害することで細菌の増殖を抑える．またアミノグリコシド系抗菌薬は，エイズウイルスのtrans-acting responsive sequence(TAR)およびrev responsive sequence(RRE)領域のバルジ構造に結合することで，ウイルスゲノムの転写・翻訳を抑制することも知られている．
本稿では，主に細菌に存在し代謝産物などのmRNAへの結合によって遺伝子発現を制御する「リボスイッチ」と，ほ乳類細胞に存在し遺伝子発現を制御する短いRNA(23残基程度)である「マイクロRNA(microRNA:miRNA)」に焦点を絞り，これらを標的とする創薬展開の実例を紹介する．