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Antisense oligonucleotides (ASOs) are synthetic single-stranded oligonucleotides that bind to RNAs through Watson–Crick base pairings. They are actively being developed as therapeutics for various human diseases. ASOs containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are known to trigger innate immune responses via interaction with toll-like receptor 9 (TLR9). However, the TLR9-stimulatory properties of ASOs, specifically those with lengths equal to or less than 20 nucleotides, phosphorothioate linkages, and the presence and arrangement of sugar-modified nucleotides—crucial elements for ASO therapeutics under development—have not been thoroughly investigated. In this study, we first established SY-ODN18, an 18-nucleotide phosphorothioate oligodeoxynucleotide with sufficient TLR9-stimulatory activity. We demonstrated that an unmethylated CpG motif near its 5′-end was indispensable for TLR9 activation. Moreover, by utilizing various sugar-modified nucleotides, we systematically generated model ASOs, including gapmer, mixmer, and fully modified designs, in accordance with the structures of ASO therapeutics. Our results illustrated that introducing sugar-modified nucleotides in such designs significantly reduces TLR9-stimulatory activity, even without methylation of CpG motifs. These findings would be useful for drug designs on several types of ASOs. [ABSTRACT FROM AUTHOR]

Recently, it was reported that the alkynyl modification of nucleobases mitigates the toxicity of antisense oligonucleotides (ASO) while maintaining the efficacy. However, the general effect of alkynyl modifications on the duplex‐forming ability of oligonucleotides (ONs) is unclear. In this study, post‐synthetic nucleobase modification by Sonogashira coupling in aqueous medium was carried out to efficiently evaluate the physiological properties of various ONs with alkynyl‐modified nucleobases. Although several undesired reactions, including nucleobase cyclization, were observed, various types of alkynyl‐modified ONs were successfully obtained via Sonogashira coupling of ONs containing iodinated nucleobases. Evaluation of the stability of the duplex formed by the synthesized alkynyl‐modified ONs showed that the alkynyl modification of pyrimidine was less tolerated than that of purine, although both the modifications occurred in the major groove of the duplex. These results can be attributed to the bond angle of the alkyne on the pyrimidine and the close proximity of the alkynyl substituents to the phosphodiester backbone. The synthetic method developed in this study may contribute to the screening of the optimal chemical modification of ASO because various alkynyl‐modified ONs that are effective in reducing the toxicity of ASO can be easily synthesized by this method. [ABSTRACT FROM AUTHOR]

Metal‐mediated base pairs have widespread applications, such as in DNA‐metal nanodevices and sensors. Here, we focused on their sugar conformation in duplexes and observed the crystallographic structure of the non‐canonical DNA/DNA duplex containing 2′‐O,4′‐C‐methylene bridged nucleic acid in the presence of AgI ions. The X‐ray crystallographic structure was successfully obtained at a resolution of 1.5 Å. A novel type of AgI‐mediated base pair between the N1 positions of anti‐conformation of adenines in the duplex was observed. In the central non‐canonical region, a hexad nucleobase structure containing AgI‐mediated base pairs between the N7 positions of guanines was formed. A highly bent non‐canonical structure was formed at the origin of AgI‐mediated base pairs in the central region. The bent duplex structure induced by the addition of AgI ions might become a powerful tool for dynamic structural changes in DNA nanotechnology applications. [ABSTRACT FROM AUTHOR]

Halogen-modified nucleic acid molecules, such as trifluorothymidine (FTD) and 5-fluorouracil, are widely used in medical science and clinical site. These compounds have a very similar nucleobase structure. It is reported that both of these compounds could be incorporated into DNA. The incorporation of FTD produces highly anti-tumor effect. However, it is not known whether to occur a significant effect by the incorporation of 5-fluorouracil. Nobody knows why such a difference will occur. To understand the reason why there is large differences between trifluorothymidine and 5-fluorouracil, we have performed the molecular dynamics simulations and molecular orbital calculations. Although the active interaction energy between Halogen-modified nucleic acids or and complementary adenine was increased, in only FTD incorporated DNA, more strongly dispersion force interactions with an adjacent base were detected in many thermodynamic DNA conformations. As the results, the conformational changes occur even if it is in internal body temperature. Then the break of hydrogen bonding between FTD and complementary adenine base occur more frequently. The double helix structural destabilization of DNA with FTD is resulted from autoagglutination caused by the bonding via halogen orbitals such as halogen bonding and the general van der Waals interactions such as CH– π , lone pair (LP)– π , and π – π interactions. Therefore, it is strongly speculated that such structural changes caused by trifluoromethyl group is important for the anti-tumor effect of FTD alone. [ABSTRACT FROM AUTHOR]

Metal‐mediated base pairs (MMBPs) formed by natural or artificial nucleobases have recently been developed. The metal ions can be aligned linearly in a duplex by MMBP formation. The development of a three‐ or more‐metal‐coordinated MMBPs has the potential to improve the conductivity and enable the design of metal ion architectures in a duplex. This study aimed to develop artificial self‐bases coordinated by three linearly aligned AgI ions within an MMBP. Thus, artificial nucleic acids with a 1,3,9‐triaza‐2‐oxophenoxazine (9‐TAP) nucleobase were designed and synthesized. In a DNA/DNA duplex, self‐base pairs of 9‐TAP could form highly stable MMBPs with three AgI ions. Nine equivalents of AgI led to the formation of three consecutive 9‐TAP self‐base pairs with extremely high stability. The complex structures of 9‐TAP MMBPs were determined by using electrospray ionization mass spectrometry and UV titration experiments. Highly stable self‐9‐TAP MMBPs with three AgI ions are expected to be applicable to new DNA nanotechnologies. [ABSTRACT FROM AUTHOR]

Nucleic acids are the principal materials for the preservation and flow of genetic information. The control of the properties of nucleic acids by using external stimuli is important for regulating biological processes and for the possibility of developing unique molecular machines. This account summarizes the development of external stimuli-responsive nucleic acids by our group. Our external stimuli-responsive nucleic acids are designed and synthesized through sugar, backbone, and nucleobase modifications. Various external stimuli, such as light, acids, or oxidants/reductants can trigger changes in nucleic acid structure. By means of such structural alterations, various properties of nucleic acid, including their hybridization ability, nuclease resistance, backbone degradability, and base-recognition ability, can be changed. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Graphical abstract Highlights • 2′- O ,4′- C -aminomethylene bridged nucleic acid (2′,4′-BNANC) monomers were prepared. • These monomers contained guanine, adenine, thymine, or 5-methylcytosine. • The key reaction was a transglycosylation reaction for the purine-bearing monomers. • An improved route was also reported toward the pyrimidine-bearing monomers. • Duplexes of the fully-2′,4′-BNANC-modified oligonucleotides were thermally stable. Abstract We efficiently synthesized 2′- O ,4′- C -aminomethylene-bridged nucleic acid (2′,4′-BNANC) monomers bearing the four nucleobases, guanine, adenine, thymine, and 5-methylcytosine and incorporated these monomers into oligonucleotides. Initially, we carried out the transglycosylation reaction on several 2′- O -substituted 5-methyluridines to evaluate the effects of 2′-substitutions on this reaction. Under the optimized conditions, purine nucleobases were successfully introduced, and 2′,4′-BNANC monomers bearing adenine or guanine were obtained over several steps. In addition, the improved synthesis of the 2′,4′-BNANC monomers bearing thymine or 5-methylcytosine was also achieved. The obtained 2′,4′-BNANC monomers were subsequently incorporated into oligonucleotides and the duplex-forming abilities of the modified oligonucleotides were investigated. Duplexes containing 2′,4′-BNANC monomers in both or either strands were found to possess excellent thermal stabilities. [ABSTRACT FROM AUTHOR]

• AEX for quality control of siRNA as non-denatured forms under alkaline conditions. • Optimized conditions showed potential to separate excess single strands adequately. • Non-optimal duplexes with truncated ends were eluted at different retention times. • Non-optimal duplexes with bulge structures were eluted at different retention times. • A retention model of short double-strand with overhang bases was proposed. Small interfering RNA (siRNA), consisting of two complementary single-stranded RNAs with overhanging bases, is being adopted as a potent and specific inhibitor of target gene expression. However, non-duplexed single strands and undesired double strands composed of impurities (e.g., n-1 mer) could be produced in addition to the target double strand in the siRNA manufacturing process. Compared to the liquid chromatography analysis of single strands, the analysis of the duplexes under non-denaturing conditions is challenging, since restricted chromatographic conditions are required to maintain the Watson-Crick hydrogen bonds. This study reports the analysis of double-stranded oligomers having approximately 20 base pairs with some overhanging bases as non-denatured forms by anion-exchange chromatography (AEX). Optimization of the chromatographic conditions could potentially achieve the adequate separation of excess single strands from the double strand. Non-optimal duplexes, such as duplexes with long overhangs or bulge structures, were prepared by intentionally deleting terminal or middle nucleotide(s) of either the sense or the antisense strand, and these non-optimal duplexes were eluted at different retention times in most of the cases. Interestingly, under alkaline chromatographic conditions (pH 9.0), non-optimal duplexes containing a shortmer tended to exhibit a stronger retention than their parent duplexes, although they possessed a less negative charge. This study demonstrated some retention behavior of double strands with overhangs by AEX under non-denaturing conditions. [ABSTRACT FROM AUTHOR]

[Display omitted] The artificial nucleobase 1,3-diaza-2-oxophenoxazine (tCO) and its derivative G-clamp strongly bind to guanine and, when incorporated into double-stranded DNA, significantly increase the stability of the latter. As the phenoxazine skeleton is a constituent of major pharmaceuticals, we hypothesized that oligonucleotides (ONs) containing phenoxazine bases would induce property changes related to intracellular uptake and migration in tissues. In this study, we designed and synthesized a novel G-clamp-linker antisense oligonucleotide (ASO) in which a G-clamp base with a flexible linker was introduced into the 5′-end of an ASO targeting mouse long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (m MALAT1). Compared to unconjugated ASO, the G-clamp-linker ASO induced significantly more effective knockdown of m MALAT1 in mouse skeletal muscle. The ASOs conjugated with 2′-deoxyribonucleotide(s) bearing a tCO nucleobase at the 5′-end exhibited a similar knockdown effect in skeletal muscle. Thus, it may be possible to improve therapeutic effects against skeletal muscle diseases, such as muscular dystrophy, by using ONs with incorporated phenoxazine nucleobases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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Abstract: Oligonucleotides containing 2′-deoxyribonucleotides bearing hydroxyphenyl nucleobases or their 2′,4′-BNA-modified analogs were synthesized, and the triplex-forming ability of their oligonucleotides with double-stranded DNA targets was evaluated by UV melting experiments. Results showed that 2′-deoxyribonucleotide bearing 2′-hydroxyphenyl nucleobase could be recognized by a dUA base pair while no affinity to a TA base pair was observed. The ,4′-BNA modification led to a further increase in the binding affinity to a dUA base pair. The ,4′-BNA bearing 3-hydroxyphenyl nucleobase showed moderate binding affinity to a TA base pair, but without selectivity. [Copyright &y& Elsevier]