•Phosphorotioate oligonucleotides are analyzed using size exclusion chromatography.•They interact with column packing material and thereby they elute late and broadly.•Single- and double-stranded phosphorothioate oligonucleotides are discriminated.We evaluated size exclusion chromatography (SEC) for the detection of high-order structure of phosphorothioate oligonucleotides (PS-oligo). Because of strong interaction between PS-oligo and column packing material, peaks were broader and elution time was longer than those of the corresponding natural DNA oligonucleotides. However, single- and double-stranded structures of PS-oligo were clearly separated and discriminated, while single-stranded with high-order structures such as G-quadruplex and hairpin structure were not distinguished from each other.

We designed and synthesized a novel artificial 2′-O,4′-C-methylene bridged nucleic acid (2′,4′-BNA/LNA) with a phenoxazine nucleobase and named this compound BNAP. Oligodeoxynucleotide (ODN) containing BNAP showed higher binding affinities toward complementary DNA and RNA as compared to ODNs bearing 2′,4′-BNA/LNA with 5-methylcytosine or 2′-deoxyribonucleoside with phenoxazine. Thermodynamic analysis revealed that BNAP exhibits properties associated with the phenoxazine moiety in DNA/DNA duplexes and characteristics associated with the 2′,4′-BNA/LNA moiety in DNA/RNA duplexes.

Xeno nucleic acids (XNAs) are a group of chemically modified nucleic acid analogues that have been applied to various biological technologies such as antisense oligonucleotides, siRNAs and aptamers.

Antisense oligonucleotides are attractive therapeutic agents for several types of disease. One of the most promising modifications of antisense oligonucleotides is the introduction of bridged nucleic acids. As we report here, we designed novel bridged nucleic acids, triazole-bridged nucleic acid (TrNA), and tetrazole-bridged nucleic acid (TeNA), whose sugar conformations are restricted to N-type by heteroaromatic ring-bridged structures. We then successfully synthesized TrNA and TeNA and introduced these monomers into oligonucleotides. In UV-melting experiments, TrNA-modified oligonucleotides exhibited increased binding affinity toward complementary RNA and decreased binding affinity toward complementary DNA, although TeNA-modified oligonucleotides were decomposed under the annealing conditions. Enzymatic degradation experiments demonstrated that introduction of TrNA at the 3′-terminus rendered oligonucleotides resistant to nuclease digestion. Furthermore, we tested the silencing potencies of TrNA-modified antisense oligonucleotides using in vitro and in vivo assays. These experiments revealed that TrNA-modified antisense oligonucleotides induced potent downregulation of gene expression in liver. In addition, TrNA-modified antisense oligonucleotides showed a tendency for increased liver biodistribution. Taken together, our findings indicate that TrNA is a good candidate for practical application in antisense methodology.

We synthesized thymidine derivatives of 2′-C,4′-C-ethyleneoxy-bridged 2′-deoxyribonucleic acids with an 8′-methyl group ((R)-Me-EoDNA and (S)-Me-EoDNA) and without any substituent (EoDNA). Oligonucleotides including these EoDNAs showed high hybridization abilities with complementary RNA and excellent enzymatic stabilities compared with natural DNA. Moreover, the in vitro antisense potency of oligonucleotides with these EoDNAs and our recently reported methylene-EoDNAs was investigated and compared with that of LNA, which is a practical chemical modification for oligonucleotide-therapeutic agents. The results showed that EoDNAs and methylene-EoDNAs could be promising candidates for antisense technology.

Conformationally restricted nucleoside analogues 2′,4′-BNA/LNA-7-deazaguanine (LNA-7cG) and 2′,4′-BNA/LNA-8-aza-7-deazaguanine (LNA-8n7cG), which avoid extra hydrogen bond formation at the 7-position of the guanine nucleobase, were successfully synthesized and incorporated into oligonucleotides. While the LNA-7cG-containing oligonucleotides show high duplex-forming ability with complementary DNA and RNA similar to LNA-G, the LNA-8n7cG-containing oligonucleotide has lower binding affinity than that of natural 2′-deoxyguanosine. This disparity in thermostability is also observed in 7-deazaadenosine analogues (LNA-7cA, LNA-8n7cA). Thermodynamic parameters and computational chemistry revealed that an inappropriate glycosidic torsion angle χ of 2′,4′-BNA/LNA-8-aza-7-deazapurine analogues destabilizes duplex formation in contrast to 2′,4′-BNA/LNA-7-deazapurine analogues. This result indicates that the nucleobase rotation angle plays an important role in duplex binding affinity. In addition, LNA-7cG-modified oligonucleotide effectively suppresses aggregation even in a guanine-rich sequence.

It has been confirmed by our previous studies that a 2′,4′-BNANC[N–Me]-modified antisense gapmer displays high affinity and selectivity to the target RNA strand, promising mRNA inhibitory activity and excellent nuclease resistance. Herein, we have obtained a crystal structure that provides insights into these excellent antisense properties.

We prepared an oligodeoxynucleotide (ODN) bearing two 4-hydroxy-2-mercaptobenzimidazole nucleobase analogues (SBNV and SBNB) modified with different photolabile groups. This ODN enabled a light-triggered strand exchange reaction in a wavelength-selective manner.

A sulfonamide-bridged nucleic acid without an N-substituent (SuNA[NH]) was successfully synthesized. A comparison of the SuNA[NMe]- and SuNA[NH]-modified oligonucleotides revealed that the duplex-forming abilities of the SuNA[NMe]-modified oligonucleotides with complementary DNA and RNA were higher than those of the SuNA[NH]-modified oligonucleotides. The crystal structures of DNA duplexes containing a SuNA[NR] revealed that the helical structures of the two duplexes and hydration patterns around the bridge moiety were different. These results provide insights into hydration patterns and rationale for the high RNA affinity of SuNA-modified oligonucleotides.

The targeting of abundant hepatic asialoglycoprotein receptors (ASGPR) with trivalent N-acetylgalactosamine (GalNAc) is a reliable strategy for efficiently delivering antisense oligonucleotides (ASOs) to the liver. We here experimentally demonstrate the high systemic potential of the synthetically-accessible, phosphodiester-linked monovalent GalNAc unit when tethered to the 5′-terminus of well-characterised 2′,4′-bridged nucleic acid (also known as locked nucleic acid)-modified apolipoprotein B-targeting ASO via a bio-labile linker. Quantitative analysis of the hepatic disposition of the ASOs revealed that phosphodiester is preferable to phosphorothioate as an interunit linkage in terms of ASGPR binding of the GalNAc moiety, as well as the subcellular behavior of the ASO. The flexibility of this monomeric unit was demonstrated by attaching up to 5 GalNAc units in a serial manner and showing that knockdown activity improves as the number of GalNAc units increases. Our study suggests the structural requirements for efficient hepatocellular targeting using monovalent GalNAc and could contribute to a new molecular design for suitably modifying ASO.

Recently, 7-substituted 7-deazapurine nucleoside triphosphates and 5-substituted pyrimidine nucleoside triphosphates (dNamTPs) were synthesized to extend enzymatically using commercially available polymerase. However, extension was limited when we attempted to incorporate the substrates consecutively. To address this, we have produced a mutant polymerase that can efficiently accept the modified nucleotide with amphiphilic groups as substrates. Here we show that the KOD polymerase mutant, KOD exo−/A485L, had the ability to incorporate dNamTP continuously over 50 nt, indicating that the mutant is sufficient for generating functional nucleic acid molecules.

•Novel fluorescent nucleoside, dCDAIN, was successfully synthesized.•The oligonucleotides containing dCDAIN could work as fluorescent probes.•Fluorescence intensity increased 4-fold when dCDAIN was opposite an abasic site.A novel fluorescent cytidine derivative (dCDAIN) bearing 4-(diarylmethylene)imidazolinones (DAINs) at C5-position was successfully synthesized and incorporated into oligonucleotides. The oligonucleotide bearing dCDAIN hybridized with complimentary strand in a sequence specific manner, and the florescence intensity was changed depending on the nucleobases opposite to dCDAIN in the double stranded DNA or RNA. Interestingly, the fluorescence intensity was higher when the dCDAIN-containing oligonucleotide hybridized with a mismatched sequence. Especially, when dCDAIN was located at the opposite position of an abasic site, the fluorescent intensity was four times higher than that observed in a single stranded state.

We previously reported the synthesis and evaluation of 2′-O,4′-C-spirocyclopropylene-bridged nucleic acid (scpBNA) bearing a thymine (T) nucleobase. Oligonucleotides (ONs) modified with scpBNA-T exhibited strong binding affinity to complementary single-stranded RNA (ssRNA) and high enzymatic stability. These biophysical properties suggest that scpBNAs are well suited for use in antisense strategies. Herein, we describe the synthesis of scpBNA monomers bearing 5-methylcytosine (mC), adenine (A), and guanine (G) nucleobases for use in a variety of sequences. The prepared scpBNA monomers were incorporated into ONs at various positions. The scpBNA-modified ONs exhibited excellent duplex-forming ability with the complementary ssRNA comparable to ONs modified with 2′-O,4′-C-methylene-bridged nucleic acid (2′,4′-BNA/LNA). Moreover, ON modified with scpBNA-mC, -A, and -G showed higher enzymatic stability than the corresponding 2′,4′-BNA/LNA-modified ON. These results demonstrated a promising role for the incorporation of scpBNA monomers into therapeutic antisense ONs.

2′-O,4′-C-Spirocyclopropylene bridged nucleic acid (scpBNA), an analogue of 2′-O,4′-C-methylene bridged nucleic acid (2′,4′-BNA/LNA) bearing a cyclopropane ring at the 6′-position, was synthesized and successfully incorporated into oligonucleotides. The scpBNA-modified oligonucleotides showed excellent duplex-forming ability with complementary single-stranded RNA and exhibited increased enzymatic stability as compared to the corresponding natural and 2′,4′-BNA/LNA-modified oligonucleotides. Our results demonstrate the potential of scpBNA for gene therapeutics, such as antisense technology.

Oligonucleotides (ONs) modified with a locked nucleic acid (LNA) are widely used in the fields of therapeutics, diagnosis, and nanotechnology. There have been significant efforts towards developing LNA analogues bearing modified bridges to improve their hybridization ability, nuclease resistance, and pharmacokinetic profiles. Moreover, nucleobase modifications of LNA are useful strategies for the functionalization of ONs. Modifications of the C5-position of pyrimidine nucleobases are particularly interesting because they enable predictable positioning of functional groups in the major groove of the duplex. Here we report the synthesis of C5-azobenzene-functionalized LNA uridine (LNA-UAz) and properties of LNA-UAz-modified ONs, including isomerization properties, hybridization ability, and enzyme stability. LNA-UAz in ON is photo-isomerized effectively and reversibly by irradiation at 365 nm (trans to cis) and 450 nm (cis to trans). LNA-UAz-modified ONs show RNA-selective hybridization ability despite the large hydrophobic azobenzene moiety extending into the major groove of the duplex. The enzymatic stability of LNA-UAz-modified ONs is higher than that of natural and LNA-modified ONs with or without photo-irradiation. Our results indicate that LNA-UAz holds promise for RNA targeting and photo-switchable technologies.

High scalability of a novel bicyclic nucleoside building block, amido-bridged nucleic acid (AmNA), to diversify pharmacokinetic properties of therapeutic antisense oligonucleotides is described. N2′-functionalization of AmNA with a variety of hydrophobic groups is straightforward. Combinations of these modules display similar antisense knockdown effects and improve cellular uptake, relative to sequence-matched conventional 2′,4′-bridged nucleic acid (2′,4′-BNA) in vivo.

A boat-shaped pyranosyl nucleic acid (BsNA) having an exocyclic methylene group in the sugar moiety was synthesized to investigate the possibility that the axial H3′ of original BsNA is the cause of its duplex destabilization. The synthesized BsNA analog was chemically stable against various nucleophiles. From the thermal stability of duplex oligonucleotides including the BsNA analog, it was found that the duplex-forming ability can be sensitive to the size of functional groups at the 3′-position.

Here, we describe the enzymatic construction of a new larger base pair formed between adenine (A) and a 4-hydroxy-2-mercaptobenzimidazole (SB) nucleobase analogue. We investigated the enzymatic incorporation of 2′-deoxynucleoside-5′-triphosphate bearing a SB nucleobase analogue (dSBTP) into oligonucleotides (ONs) by DNA polymerases. dSBTP could be effectively incorporated at the site opposite a dA in a DNA template by several B family DNA polymerases. These findings provide new insights into various aspects of biotechnology, including the design of non-natural base pairs.

A novel 2′-O,4′-C-bridged nucleic acid, 3,4-dihydro-2H-pyran bridge moiety (DpNA), with a dioxabicyclo[3.2.1]oct-3-ene ring was designed. Construction of the dihydropyran bridge was achieved by dehydration of a six-membered hemiacetal ring, and the DpNA monomer was synthesized in 10 steps from 5-methyluridine (total yield 9%). The synthesized DpNA monomer was incorporated into oligonucleotides to examine the properties of the modified oligonucleotides. The DpNA-modified oligonucleotides possessed high affinity toward ssRNA and were more resistant to nucleases compared to the corresponding natural oligonucleotide.

A light-triggered strand exchange reaction was developed using the change in the hydrogen-donor–acceptor pattern of a nucleobase analogue. We demonstrated that a new light-responsive nucleobase analogue derived from 4-hydroxy-2-mercaptobenzimidazole (SBNV) preferentially recognized guanine before photoirradiation and adenine after photoirradiation in duplexes. By using oligodeoxynucleotides modified with SBNV, a light-triggered strand exchange reaction targeting different mRNA fragment sequences was achieved. These results indicate that SBNV could be a powerful material for manipulating a nucleic acid assembly in a spatially and temporally controlled manner.

A novel 2′,4′-BNA/LNA analog bridged by guanidine, termed as guanidine bridged nucleic acid (GuNA), was synthesized and incorporated into oligonucleotides. Thermal stabilities and nuclease resistance of GuNA-modified oligonucleotides were investigated and compared with those of 2′,4′-BNA/LNA and natural DNA oligonucleotides. GuNA exhibited interestingly high binding affinity towards complementary ssDNA than 2′,4′-BNA/LNA.

2′-N,4′-C-(N-Methylamino)sulfonylmethylene-bridged thymidine (SuNA), which has a six-membered linkage including a sulfonamide moiety, was synthesized and introduced into oligonucleotides. The oligonucleotides containing SuNA exhibited excellent nuclease resistance, a high affinity toward single-stranded RNA, and a low affinity toward single-stranded DNA compared to the natural oligonucleotide.

•A set of diblock glycopolymers with different pendant carbohydrate moieties were synthesized.•The results indicated that the endocytic pathway affects the fate of the internalized polyplexes.•The galactose bearing diblock glycopolymers showed faster cellular uptake and good cell viability.Elevated low-density-lipoprotein cholesterol (LDL-C) level is a major risk factor leading to cardiovascular diseases. Therefore, since the proprotein convertase subtilisin kexin type 9 (PCSK9) regulates LDL-C receptors, it represents the appropriate target for cholesterol-lowering gene therapy. However, although delivery of antisense oligonucleotides (ODNs) is a promising therapeutic method for the treatment of several diseases, it is fundamental to develop new and efficacious carriers that transport the ODNs into the target cell in a nontoxic manner.This study reports on the synthesis, characterization and in vitro testing of a new liver specific carrier based on linear cationic diblock glycopolymer composed of galactosyl ureaethyl methacrylate (GAMA) and the primary amine-containing dimethylamino ethyl methacrylate (DMAEMA). Delivery experiments proved that the poly(galactosyl ureaethyl methacrylate -b-dimethylamino ethyl methacrylate) diblock copolymer (pGa4D47), internalized in a receptor-mediated manner, exhibited a much faster nuclear transportation than ODNs carried by pDMAEMA homopolymer or glycopolymer bearing glucose moieties.

Hydrogen bonds (H-bonds) formed between nucleobases play an important role in the construction of various nucleic acid structures. The H-donor and H-acceptor pattern of a nucleobase is responsible for selective and correct base pair formation. Herein, we describe an 8-thioadenine nucleobase analogue and an 8-thiohypoxanthine nucleobase analogue with a photolabile 6-nitroveratryl (NV) group on the sulfur atom (SANV and SHNV, respectively). Light-triggered removal of the NV group causes tautomerization and a change in the H-bonding pattern of SANV and SHNV. This change in the H-bonding pattern has a strong effect on base recognition by 8-thiopurine nucleobase analogues. In particular, base recognition by SHNV is clearly shifted from guanine to adenine upon photoirradiation. These results show that a photoinduced change in the H-bonding pattern is a unique strategy for manipulating nucleic acid assembly with spatiotemporal control.

A phosphoramidite of a 2′-O,4′-C-methylene-bridged nucleoside, bearing 4-(2,4,6-triisopropylbenzenesulfonyloxy)pyridin-2-one as a nucleobase precursor, was synthesized and introduced into an oligonucleotide. Treatment with various secondary amines after elongating the oligonucleotide on an automated DNA synthesizer enabled facile and mild conversion of the precursor into the corresponding N,N-disubstituted 3-deazacytosine nucleobases. The evaluation of the triplex-forming ability of the synthesized oligonucleotides with double-stranded DNA showed that the nucleobase possessing the (3S)-3-guanidinopyrrolidine moiety can recognize a CG base pair with high sequence-selectivity and binding-affinity.

Several triplex-forming oligonucleotides (TFOs) partially modified with 2′-O-(2-aminoethyl)- or 2′-O-(2-guanidinoethyl)-nucleotides were synthesized and their association rate constants (kon) with double-stranded DNA were estimated by UV spectrophotometry. Introduction of cationic modifications in the 5′-region of the TFOs significantly increased the kon values compared to that of natural TFO, while no enhancement in the rate of triplex DNA formation was observed when the modifications were in the middle and at the 3′-region. The kon value of a TFO with three adjacent cationic modifications at the 5′-region was found to be 3.4 times larger than that of a natural one. These results provide useful information for overcoming the inherent sluggishness of triplex DNA formation.

2′-O,4′-C-Ethyleneoxy bridged 5-methyluridine (EoNA-T), possessing a seven-membered linkage and an anomeric 4′-carbon, was synthesized and introduced into oligonucleotides by using an automated DNA synthesizer. The EoNA-modified oligonucleotides significantly stabilized the duplexes with single-stranded RNA and triplexes with double-stranded DNA relative to the natural oligonucleotide and oligonucleotides modified by another seven-membered bridged 5-methyluridine, 2′,4′-BNACOC-T. In addition, EoNA-T showed excellent nuclease resistance.

In order to expand the target sequence used in triplex DNA formation, seven novel nucleotide analogues were synthesized and incorporated into triplex-forming oligonucleotides by post-elongation modification approaches. Among them, GPB, equipped with a suitable restricted conformation of sugar and nucleobase moieties, was found to have the highest sequence-selectivity and affinity towards CG base pairs within double-stranded DNA.

Phosphoramidites containing 2-propynyloxy or 1-butyn-4-yl as nucleobase precursors were synthesized and introduced into oligonucleotides using an automated DNA synthesizer. Copper-catalyzed alkyne-azide 1,3-dipolar cycloaddition of the oligonucleotides with various azides gave the corresponding triazolylated oligonucleotides, triplex-forming ability of these synthetic oligonucleotides with double-stranded DNA targets was evaluated by UV melting experiments. It was found that nucleobases containing 2-(1-m-carbonylaminophenyl-1,2,3-triazol-4-yl)ethyl units likely interacted with A of a TA base pair in a parallel triplex DNA.

Previously, we reported that the 3,4-epoxypiperidine structure, whose design was based on the active site of DNA alkylating antitumor antibiotics, azinomycins A and B, possesses prominent DNA cleavage activity. In this report, novel caged DNA alkylating agents, which were designed to be activated by UV irradiation, were synthesized by the introduction of four photo-labile protecting groups to a 3,4-epoxypiperidine derivative. The DNA cleavage activity and cytotoxicity of the caged DNA alkylating agents were examined under UV irradiation. Four caged DNA alkylating agents showed various degrees of bioactivity depending on the photosensitivity of the protecting groups.

Oligonucleotides containing 4′-carboxy-, 4′-methoxycarbonyl-, 4′-carbamoyl-, and 4′-methylcarbamoyl-thymidines, and their 2′-methoxy, 2′-amino or 2′-acetamido analogs were prepared. Their duplex-forming ability with DNA and RNA complements was evaluated by UV melting experiments. Interestingly, 4′-carboxythymidine existing in the S-type sugar conformation was found to lead to an increase in the stability of the duplex formed with RNA complements compared to natural thymidine.

Various stereochemically pure cationic phosphorothioate oligonucleotides bearing aminoalkyl moieties were synthesized, and their duplex-forming ability against single-stranded DNA (ssDNA), single-stranded RNA (ssRNA) and triplex-forming ability against double-stranded DNA (dsDNA) were evaluated by UV melting experiments. The cationic Rp stereoisomers showed improved duplex-forming ability against ssDNA, triplex-forming ability against dsDNA and nuclease stability.

A boat-shaped glucopyranosyl nucleic acid (BsNA) was synthesized to investigate the possibility that the lean of a nucleobase is a factor affecting duplex-forming ability of oligonucleotides. From the crystal structure of a BsNA nucleoside and the thermal stability of duplex oligonucleotides, it was found that not only the lean of the base but also the rotation angle of the glycosidic bond axis were important factors in a stable duplex formation.

In order to expand target sequences in triplex DNA formation, the development of a nucleobase that recognizes a CG base pair in dsDNA was attempted. A 4-[(3R,4R)-dihydroxypyrrolidino]pyrimidin-2-one nucleobase was found to recognize a CG base pair with high sequence-selectivity.

The bridged nucleic acid (BNA) containing a thiol at the 6′-position in the bridged structure was synthesized from the disulfide-type BNA and conjugated with various functional molecules via the thioether or the disulfide linkage post-synthetically and efficiently in solution phase. The disulfide-linked conjugate was cleaved under reductive conditions derived from glutathione and an oligonucleotide bearing a free thiol was released smoothly. Conjugated functional molecules had great effects on duplex stability with the DNA complement. In contrast, the molecules little influenced the stability with the RNA complement. Moreover, the oligonucleotides with functional groups at the 6′-position had as high or higher resistances against 3′-exonuclease than phosphorothioate oligonucleotide (S-oligo).

Using the copper(I)-catalyzed alkyne-azide 1,3-dipolar cycloaddition, a post-elongation modification of 1-ethynyl substituted nucleobases has been employed to construct 18 variations of oligonucleotides from a common oligonucleotide precursor. The triplex-forming ability of each oligonucleotide with dsDNA was evaluated by the UV melting experiment. It was found that triazole nucleobases generally tend to exhibit binding affinities in the following order: CG > TA > AT, GC base pairs. Among the triazole nucleobases examined, a 1-(4-ureidophenyl)triazole provided the best result with regard to affinity and selectivity for the CG base pair.

A novel method for 18F-radiolabeling of oligodeoxynucleotides (ODNs) by a Cu-catalyzed Huisgen reaction has been developed by using the lowest possible amount of the precursor biomolecule for the realization of stoichiometry-oriented PET (positron emission tomography) chemistry. Under the optimized cyclization conditions of p- or m-azido([18F]fluoromethyl)benzene and alkyne-substituted ODN (20 nmol) at 40 °C for 15 min in the presence of CuSO4, TBTA [tris((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)amine], and sodium ascorbate (2:1:2), the synthesis of 18F-labeled ODNs with sufficiently high radioactivities of 2.1–2.5 GBq and specific radioactivities of 1800–2400 GBq/μmol have been accomplished for use in animal and human PET studies.

A novel derivative of 2′,4′-bridged nucleic acid, named hydroxamate-bridged nucleic acid (HxNA), containing a six-membered perhydro-1,2-oxazin-3-one ring, was designed and synthesized. The introduction of a carbonyl function along with an N–O linkage in the six-membered bridged structure is the unique structural feature of the novel 2′,4′-bridged nucleic acid analogue. The design was carried out to restrict the flexibility of the sugar moiety through the trigonal planarity of carbonyl function, which would improve the properties of the modification. The synthesized monomer was incorporated into oligonucleotides, and their properties were examined. The HxNA-modified oligonucleotides exhibited selectively high affinity toward complementary ssRNA. Furthermore, the nuclease resistance of the HxNA-modified oligonucleotide was found to be higher than that of the corresponding natural and 2′,4′-BNA/LNA-modified oligonucleotides. Interestingly, exposure of HxNA modified oligonucleotide to 3′-exonuclease resulted in gradual opening of the bridge, which stopped further digestion. Moreover, ring-opening of only one modification at the 3′-end of the oligonucleotides was observed, even if two or three HxNA modifications were present in the sequence. The results demonstrate the strong potential of the HxNA modification as a switch for the generation of highly nuclease-resistant RNA selective oligonucleotide in situ, which could have potential applications in antisense technology.

A novel nucleoside analog with a disulfide bridge structure at the sugar moiety, which shows redox-responsive reversibility of the sugar conformation due to formation and scission of the disulfide bond, was designed and synthesized.

A novel bridged nucleic acid bearing cyclic urea structure was successfully synthesized and introduced into oligonucleotide, displaying attractive characteristics of highly RNA selective hybridization ability and excellent resistance towards nuclease degradation.

We succeeded in the synthesis of triplex-forming oligonucleotides (TFOs) that contain a deoxyribonucleotide (Py) bearing a 2-pyridine nucleobase or the 2′,4′-BNA congener (PyB). By UV melting experiments, it was found that 2-pyridine was a very promising nucleobase for the sequence-selective recognition of a CG base pair within double-stranded DNA (dsDNA) in a parallel motif triplex. Moreover, PyB in TFOs showed stronger affinity to a CG base pair than Py with further increase in the selectivity. Using TFO including multiple PyB units, triplex formation with dsDNA containing three CG base pairs was observed.

In this study, a number of 2′,4′-BNA- and 2′,4′-BNANC-modified siRNAs were designed and synthesized. Their thermal stability, nuclease resistance and gene silencing properties against cultured mammalian cells were evaluated and compared with those of natural siRNAs. The 2′,4′-BNA- and 2′,4′-BNANC-modified siRNAs (named siBNA and siBNANC, respectively) showed very high Tm values, were remarkably stable in serum sample and showed promising RNAi properties equal to those exhibited by natural siRNAs. Thermally stable siBNAs composed of slightly modified sense and antisense strands were capable of suppressing gene expression equal to that of natural siRNA. A number of modifications on the sense strand by 2′,4′-BNA or 2′,4′-BNANC, either consecutively or separated by natural RNA nucleotides, is tolerable in RNAi machinery. Modifications at the Argonauate (Ago2) cleavage site of the sense strand (9–11th positions from the 5′-end of the sense strand) produced variable results depending on siRNA composition. Mostly, modification at the 10th position diminished siRNA activity. In moderately modified siRNAs, modification at the 11th position displayed usual RNAi activity, while modification at the 9th position showed variable results depending on siRNA composition.

Oligonucleotides including C-nucleotides having 1-substitued 1H-1,2,3-triazoles as artificial nucleobases were conveniently synthesized by the post-elongation modification method using the copper(I)-catalyzed alkyne–azide 1,3-dipolar cycloaddition (CuAAC) reaction. The base-pairing properties of the triazole nucleobase analogs in forming duplexes with oligonucleotides were investigated by the Tm experiments.

The synthesis and properties of a bridged nucleic acid analogue containing a N3′→P5′ phosphoramidate linkage, 3′-amino-2′,4′-BNA, is described. A heterodimer containing a 3′-amino-2′,4′-BNA thymine monomer, and thymine and methylcytosine monomers of 3′-amino-2′,4′-BNA and their 5′-phosphoramidites, were synthesized efficiently. The dimer and monomers were incorporated into oligonucleotides by conventional 3′→5′ assembly, and 5′→3′ reverse assembly phosphoramidite protocols, respectively. Compared to a natural DNA oligonucleotide, modified oligonucleotides containing the 3′-amino-2′,4′-BNA residue formed highly stable duplexes and triplexes with single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded DNA (dsDNA) targets, with the average increase in melting temperature (Tm) against ssDNA, ssRNA and dsDNA being +2.7 to +4.0 °C, +5.0 to +7.0 °C, and +5.0 to +11.0 °C, respectively. These increases are comparable to those observed for 2′,4′-BNA-modified oligonucleotides. In addition, an oligonucleotide modified with a single 3′-amino-2′,4′-BNA thymine residue showed extraordinarily high resistance to nuclease degradation, much higher than that of 2′,4′-BNA and substantially higher even than that of 3′-amino-DNA and phosphorothioate oligonucleotides. The above properties indicate that 3′-amino-2′,4′-BNA has significant potential for antisense and antigene applications.

In order to expand target sequences in triplex DNA formation, the development of a nucleobase that recognizes a CG base pair in dsDNA was attempted. A 4-[(3R,4R)-dihydroxypyrrolidino]pyrimidin-2-one nucleobase was found to recognize a CG base pair with high sequence-selectivity.

In order to expand the target sequence used in triplex DNA formation, seven novel nucleotide analogues were synthesized and incorporated into triplex-forming oligonucleotides by post-elongation modification approaches. Among them, GPB, equipped with a suitable restricted conformation of sugar and nucleobase moieties, was found to have the highest sequence-selectivity and affinity towards CG base pairs within double-stranded DNA.

Previously, we reported that the 3,4-epoxypiperidine structure, whose design was based on the active site of DNA alkylating antitumor antibiotics, azinomycins A and B, possesses prominent DNA cleavage activity. In this report, novel caged DNA alkylating agents, which were designed to be activated by UV irradiation, were synthesized by the introduction of four photo-labile protecting groups to a 3,4-epoxypiperidine derivative. The DNA cleavage activity and cytotoxicity of the caged DNA alkylating agents were examined under UV irradiation. Four caged DNA alkylating agents showed various degrees of bioactivity depending on the photosensitivity of the protecting groups.

It has been confirmed by our previous studies that a 2′,4′-BNANC[N–Me]-modified antisense gapmer displays high affinity and selectivity to the target RNA strand, promising mRNA inhibitory activity and excellent nuclease resistance. Herein, we have obtained a crystal structure that provides insights into these excellent antisense properties.

Oligonucleotides (ONs) modified with a locked nucleic acid (LNA) are widely used in the fields of therapeutics, diagnosis, and nanotechnology. There have been significant efforts towards developing LNA analogues bearing modified bridges to improve their hybridization ability, nuclease resistance, and pharmacokinetic profiles. Moreover, nucleobase modifications of LNA are useful strategies for the functionalization of ONs. Modifications of the C5-position of pyrimidine nucleobases are particularly interesting because they enable predictable positioning of functional groups in the major groove of the duplex. Here we report the synthesis of C5-azobenzene-functionalized LNA uridine (LNA-UAz) and properties of LNA-UAz-modified ONs, including isomerization properties, hybridization ability, and enzyme stability. LNA-UAz in ON is photo-isomerized effectively and reversibly by irradiation at 365 nm (trans to cis) and 450 nm (cis to trans). LNA-UAz-modified ONs show RNA-selective hybridization ability despite the large hydrophobic azobenzene moiety extending into the major groove of the duplex. The enzymatic stability of LNA-UAz-modified ONs is higher than that of natural and LNA-modified ONs with or without photo-irradiation. Our results indicate that LNA-UAz holds promise for RNA targeting and photo-switchable technologies.

2′-O,4′-C-Spirocyclopropylene bridged nucleic acid (scpBNA), an analogue of 2′-O,4′-C-methylene bridged nucleic acid (2′,4′-BNA/LNA) bearing a cyclopropane ring at the 6′-position, was synthesized and successfully incorporated into oligonucleotides. The scpBNA-modified oligonucleotides showed excellent duplex-forming ability with complementary single-stranded RNA and exhibited increased enzymatic stability as compared to the corresponding natural and 2′,4′-BNA/LNA-modified oligonucleotides. Our results demonstrate the potential of scpBNA for gene therapeutics, such as antisense technology.

A novel bridged nucleic acid bearing cyclic urea structure was successfully synthesized and introduced into oligonucleotide, displaying attractive characteristics of highly RNA selective hybridization ability and excellent resistance towards nuclease degradation.

A novel nucleoside analog with a disulfide bridge structure at the sugar moiety, which shows redox-responsive reversibility of the sugar conformation due to formation and scission of the disulfide bond, was designed and synthesized.

A novel 2′,4′-BNA/LNA analog bridged by guanidine, termed as guanidine bridged nucleic acid (GuNA), was synthesized and incorporated into oligonucleotides. Thermal stabilities and nuclease resistance of GuNA-modified oligonucleotides were investigated and compared with those of 2′,4′-BNA/LNA and natural DNA oligonucleotides. GuNA exhibited interestingly high binding affinity towards complementary ssDNA than 2′,4′-BNA/LNA.

The bridged nucleic acid (BNA) containing a thiol at the 6′-position in the bridged structure was synthesized from the disulfide-type BNA and conjugated with various functional molecules via the thioether or the disulfide linkage post-synthetically and efficiently in solution phase. The disulfide-linked conjugate was cleaved under reductive conditions derived from glutathione and an oligonucleotide bearing a free thiol was released smoothly. Conjugated functional molecules had great effects on duplex stability with the DNA complement. In contrast, the molecules little influenced the stability with the RNA complement. Moreover, the oligonucleotides with functional groups at the 6′-position had as high or higher resistances against 3′-exonuclease than phosphorothioate oligonucleotide (S-oligo).

High scalability of a novel bicyclic nucleoside building block, amido-bridged nucleic acid (AmNA), to diversify pharmacokinetic properties of therapeutic antisense oligonucleotides is described. N2′-functionalization of AmNA with a variety of hydrophobic groups is straightforward. Combinations of these modules display similar antisense knockdown effects and improve cellular uptake, relative to sequence-matched conventional 2′,4′-bridged nucleic acid (2′,4′-BNA) in vivo.

A sulfonamide-bridged nucleic acid without an N-substituent (SuNA[NH]) was successfully synthesized. A comparison of the SuNA[NMe]- and SuNA[NH]-modified oligonucleotides revealed that the duplex-forming abilities of the SuNA[NMe]-modified oligonucleotides with complementary DNA and RNA were higher than those of the SuNA[NH]-modified oligonucleotides. The crystal structures of DNA duplexes containing a SuNA[NR] revealed that the helical structures of the two duplexes and hydration patterns around the bridge moiety were different. These results provide insights into hydration patterns and rationale for the high RNA affinity of SuNA-modified oligonucleotides.

A light-triggered strand exchange reaction was developed using the change in the hydrogen-donor–acceptor pattern of a nucleobase analogue. We demonstrated that a new light-responsive nucleobase analogue derived from 4-hydroxy-2-mercaptobenzimidazole (SBNV) preferentially recognized guanine before photoirradiation and adenine after photoirradiation in duplexes. By using oligodeoxynucleotides modified with SBNV, a light-triggered strand exchange reaction targeting different mRNA fragment sequences was achieved. These results indicate that SBNV could be a powerful material for manipulating a nucleic acid assembly in a spatially and temporally controlled manner.

A phosphoramidite of a 2′-O,4′-C-methylene-bridged nucleoside, bearing 4-(2,4,6-triisopropylbenzenesulfonyloxy)pyridin-2-one as a nucleobase precursor, was synthesized and introduced into an oligonucleotide. Treatment with various secondary amines after elongating the oligonucleotide on an automated DNA synthesizer enabled facile and mild conversion of the precursor into the corresponding N,N-disubstituted 3-deazacytosine nucleobases. The evaluation of the triplex-forming ability of the synthesized oligonucleotides with double-stranded DNA showed that the nucleobase possessing the (3S)-3-guanidinopyrrolidine moiety can recognize a CG base pair with high sequence-selectivity and binding-affinity.

We succeeded in the synthesis of triplex-forming oligonucleotides (TFOs) that contain a deoxyribonucleotide (Py) bearing a 2-pyridine nucleobase or the 2′,4′-BNA congener (PyB). By UV melting experiments, it was found that 2-pyridine was a very promising nucleobase for the sequence-selective recognition of a CG base pair within double-stranded DNA (dsDNA) in a parallel motif triplex. Moreover, PyB in TFOs showed stronger affinity to a CG base pair than Py with further increase in the selectivity. Using TFO including multiple PyB units, triplex formation with dsDNA containing three CG base pairs was observed.

Oligonucleotides containing 4′-carboxy-, 4′-methoxycarbonyl-, 4′-carbamoyl-, and 4′-methylcarbamoyl-thymidines, and their 2′-methoxy, 2′-amino or 2′-acetamido analogs were prepared. Their duplex-forming ability with DNA and RNA complements was evaluated by UV melting experiments. Interestingly, 4′-carboxythymidine existing in the S-type sugar conformation was found to lead to an increase in the stability of the duplex formed with RNA complements compared to natural thymidine.

We prepared an oligodeoxynucleotide (ODN) bearing two 4-hydroxy-2-mercaptobenzimidazole nucleobase analogues (SBNV and SBNB) modified with different photolabile groups. This ODN enabled a light-triggered strand exchange reaction in a wavelength-selective manner.

We synthesized thymidine derivatives of 2′-C,4′-C-ethyleneoxy-bridged 2′-deoxyribonucleic acids with an 8′-methyl group ((R)-Me-EoDNA and (S)-Me-EoDNA) and without any substituent (EoDNA). Oligonucleotides including these EoDNAs showed high hybridization abilities with complementary RNA and excellent enzymatic stabilities compared with natural DNA. Moreover, the in vitro antisense potency of oligonucleotides with these EoDNAs and our recently reported methylene-EoDNAs was investigated and compared with that of LNA, which is a practical chemical modification for oligonucleotide-therapeutic agents. The results showed that EoDNAs and methylene-EoDNAs could be promising candidates for antisense technology.

Hydrogen bonds (H-bonds) formed between nucleobases play an important role in the construction of various nucleic acid structures. The H-donor and H-acceptor pattern of a nucleobase is responsible for selective and correct base pair formation. Herein, we describe an 8-thioadenine nucleobase analogue and an 8-thiohypoxanthine nucleobase analogue with a photolabile 6-nitroveratryl (NV) group on the sulfur atom (SANV and SHNV, respectively). Light-triggered removal of the NV group causes tautomerization and a change in the H-bonding pattern of SANV and SHNV. This change in the H-bonding pattern has a strong effect on base recognition by 8-thiopurine nucleobase analogues. In particular, base recognition by SHNV is clearly shifted from guanine to adenine upon photoirradiation. These results show that a photoinduced change in the H-bonding pattern is a unique strategy for manipulating nucleic acid assembly with spatiotemporal control.

Three 2′-C,4′-C-ethyleneoxy-bridged 2′-deoxyribonucleic acids possessing six-membered bridges with 6′-oxygen and 8′-exocyclic methylene groups (methylene-EoDNAs) were designed and synthesized in nine to ten steps from 5-methyluridine. The methylene-EoDNA-modified oligonucleotides showed excellent binding affinity with target ssRNA and extremely high nuclease resistance compared with natural oligonucleotides. These results proved the potential of methylene-EoDNAs for nucleic acid based technology.

Recent advances in RNA interference (RNAi)-based drug development have partially allowed systemic administration of these agents in vivo with promising therapeutic effects. However, before chemically modified small-interfering RNAs (siRNAs) can be applied clinically, their in vivo effects should be thoroughly assessed. And while many studies have assessed the effects of chemically modified siRNAs in vitro, there has been no comprehensive assessment of their effects in vivo. Here, we aimed to elucidate the effects of administering chemically modified siRNAs in vivo and to propose a 2′,4′-bridged nucleic acid (BNA)/locked nucleic acid (LNA)-based siRNA candidate for dyslipidemia. A potentially therapeutic siRNA, siL2PT-1M, was modified with phosphorothioate (PS) and 2′,4′-BNA/LNA in its sense strand and with 2′-methoxy (2′-OMe) nucleotides in its immunostimulatory motif; administration of siL2PT-1M resulted in sustained reductions in serum total cholesterol (TC) (24 days) and a concomitant apolipoprotein B (apoB) mRNA reduction in liver without adverse effects. The 2′,4′-BNA/LNA modification in the sense strand was greatly augmented the duration of the RNAi effect, whereas cholesterol conjugation shortened the duration. Cholesterol-conjugated immunostimulatory siRNA (isRNA) induced higher serum interferon-α (IFN-α) levels than did nonmodified isRNA, indicating that the immune reaction was facilitated by cholesterol conjugation. Our results indicated that modification of the adenosine residues complementary to the immunostimulatory motif and of central 5′-UG-3′ in the sense strand would ameliorate the negative immune response.

Recent findings in molecular biology implicate the involvement of proprotein convertase subtilisin/kexin type 9 (PCSK9) in low-density lipoprotein receptor (LDLR) protein regulation. The cholesterol-lowering potential of anti-PCSK9 antisense oligonucleotides (AONs) modified with bridged nucleic acids (BNA-AONs) including 2′,4′-BNA (also called as locked nucleic acid (LNA)) and 2′,4′-BNANC chemistries were demonstrated both in vitro and in vivo. An in vitro transfection study revealed that all of the BNA-AONs induce dose-dependent reductions in PCSK9 messenger RNA (mRNA) levels concomitantly with increases in LDLR protein levels. BNA-AONs were administered to atherogenic diet-fed C57BL/6J mice twice weekly for 6 weeks; 2′,4′-BNA-AON that targeted murine PCSK9 induced a dose-dependent reduction in hepatic PCSK9 mRNA and LDL cholesterol (LDL-C); the 43% reduction of serum LDL-C was achieved at a dose of 20 mg/kg/injection with only moderate increases in toxicological indicators. In addition, the serum high-density lipoprotein cholesterol (HDL-C) levels increased. These results support antisense inhibition of PCSK9 as a potential therapeutic approach. When compared with 2′,4′-BNA-AON, 2′,4′-BNANC-AON showed an earlier LDL-C–lowering effect and was more tolerable in mice. Our results validate the optimization of 2′,4′-BNANC-based anti-PCSK9 antisense molecules to produce a promising therapeutic agent for the treatment of hypercholesterolemia.