Development of an Artificial Nucleic Acid Skeleton Allowing for Unnatural-Type Triplex DNA Formation with Duplex DNA Having a TA Inversion Site.

Triplex DNA formation has generated much interest as a genomic targeting tool that directly targets duplex DNA. However, fundamental limitations in the base pairs of target duplex DNA sequences that can form stable triplex DNA have limited the application. Recently, we have reported on the recognition of CG and 5mCG base pairs by artificial nucleic acid derivatives with a 2'-deoxynebularine skeleton. Therefore, we attempted to explore the basic skeleton that is important for the development of new artificial nucleic acids allowing for the recognition of TA base pairs. In this study, we focused on a benzimidazole skeleton and introduced a hydroxyl group to enable one-point hydrogen bonding. We have synthesized artificial nucleoside analogues with hydroxyl group on the benzimidazole and incorporated their amidite derivatives into triplex forming oligonucleotides (TFOs). The gel shift assay was performed to evaluate the triplex DNA formation ability of synthesized TFOs, and TFOs containing hydroxybenzimidazole were successfully recognized TA base pairs for all four different sequences. Moreover, compared to the results for the TFOs containing benzimidazole, which suggested hydrogen bonding formation at the hydroxyl group. Therefore, hydroxybenzimidazole would be an important artificial nucleic acid skeleton for TA base pair recognition.

Post-Synthetic Modification of Triplex-Forming Oligonucleotides Containing 2-Aminoethyl-2'-Deoxynebularine Derivatives.

This article describes the detailed synthetic protocol for the preparation of oligonucleotides containing 2-guanidinoethyl-2'-deoxynebularine and 2-ureidoethyl-2'-deoxynebularine nucleoside derivatives. These derivatives are obtained by a post-synthetic modification of triplex-forming oligonucleotides (TFOs) containing 2-aminoethyl-2'-deoxynebularine, which is useful for forming stable triplex DNA with duplex DNA sequences containing 5m CG and CG interrupting sites. The hydroxyl groups of the sugar moiety of commercially available 2'-deoxyguanosine are acetyl-protected, the 6-position is chlorinated and reduced to give a 2-substituted nebularine derivative, and then the sugar moiety is deprotected. The hydroxyl groups of the sugar moiety are silyl-protected and the amino group at the 2-position is iodinated before being coupled with diethyl malonate. The ethyl ester is reduced and the resulting alcohol converted to an amino group for protection. The compound is then converted to a phosphoramidite unit and incorporated into a TFO. Subsequent modification of the aminoethyl group on the TFO completes the synthesis of the oligonucleotides containing 2-guanidinoethyl-2'-deoxynebularine and 2-ureidoethyl-2'-deoxynebularine. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparation of the phosphoramidite unit of the 2-aminoethyl-2'-deoxynebularine derivative (14) Basic Protocol 2: Post-synthetic modification of oligonucleotides containing 2-aminoethyl-2'-deoxynebularine derivatives Basic Protocol 3: Determination of the triplex-forming ability of oligonucleotides containing 2-aminoethyl-2'-deoxynebularine derivatives.

Inhibition of transcription and antiproliferative effects in a cancer cell line using antigene oligonucleotides containing artificial nucleoside analogues.

Antigene methods are promising novel therapeutic approaches to suppress abnormal gene expression. One of these methods inhibits transcription by forming triplex DNA against duplex DNA. However, by using natural-type triplex-forming oligonucleotides (TFOs), stable triplex formation is limited to homopurine and homopyrimidine strands in targeted duplex DNA. We recently developed artificial nucleoside analogues with the ability to recognize CG and TA inversion sites. We successfully formed stable unnatural-type triplex DNA for duplex DNA containing a CG base pair and extended the target sequence using TFOs containing 2-amino-3-methylpyridinyl pseudo-dC (3MeAP-ΨdC). Therefore, this present study investigated triplex-forming regions and synthesized antigene TFOs containing 3MeAP-ΨdC. Some of the synthesized antigene TFOs reduced transcription products and inhibited cell proliferation in several types of cultured cancer cells. The antigene effects of antigene TFOs containing artificial nucleic acids were markedly stronger than those of natural-type TFOs, and these results clearly demonstrated the usefulness of incorporating artificial nucleic acids within TFOs.

The development of non-natural type nucleoside to stabilize triplex DNA formation against CG and TA inversion site.

Based on the sequence-specific recognition of target duplex DNA by triplex-forming oligonucleotides (TFOs) at the major groove side, the antigene strategy has been exploited as a gene-targeting tool with considerable attention. Triplex DNA is formed via the specific base triplets by the Hoogsteen or reverse Hoogsteen hydrogen bond interaction between TFOs and the homo-purine strand from the target duplex DNA, leading to the established sequence-specificity. However, the presence of inversion sites, which are known as non-natural nucleosides that can form satisfactory interactions with 2-deoxythymidine (dT) and 2-deoxycytidine (dC) in TA and CG base pairs in the target homo-purine DNA sequences, drastically restricts the formation of classically stable base triplets and even the triplex DNA. Therefore, the design of non-natural type nucleosides, which can effectively recognize CG or/and TA inversion sites with satisfactory selectivity, should be of great significance to expanding the triplex-forming sequence. Here, this review mainly provides a comprehensive review of the current development of novel non-natural nucleosides to recognize CG or/and TA inversion sites in triplex DNA formation against double-strand DNA (dsDNA).

Site-Specific Tritium Labeling at the Predefined Internal Position of the Chemically-Modified RNA.

In nucleic acid drug discovery, it is extremely important to develop a technology to understand the distribution in target organs and to trace the degradation process in the body in order to optimize the structure and improve the efficiency of the clinical trial process. Since nucleic acid drugs are essentially metabolically degraded into numerous fragments, labeling at the internal position is preferable to that at the terminus. Due to the high molar specific activity of tritium, various approaches for tritium-labeling have been studied for nucleic acid drugs. Nevertheless, a generally-applicable method for tritium labeling of the internal position of a nucleic acid has not been established. In this study, we have demonstrated a new and efficient method for site-specific tritium labeling of the cytosine base at a predefined internal position in nucleic acid drugs. This method was developed by the chemical modification of the cytosine 4-amino group with the pyridinyl vinyl keto group by the functionality-transfer reaction using the reactive oligodeoxynucleotide (ODN), followed by reduction with NaBT4. Applicability to a variety of chemical structures, such as 5-methyl cytosine, 2'-O-methyl, 2'-fluoro ribose derivatives, Locked/Bridged nucleic acid (LNA/BNA) derivatives, as well as phosphorothioate bonds, has been evidenced using nine oligoribonucleic acid (ORN) substrates. It has been clearly demonstrated that this method is an excellent method for tritium-labeling of nucleic acid with an average conversion efficiency of 74%, an average isolated labeling yield of 60%, and an average specific activity of 61 GBq/mmol. This method is expected to contribute to the preclinical absorption, distribution, metabolism, excretion (ADME) studies of nucleic acid drug candidates.

Recognition of 5-methyl-CG and CG base pairs in duplex DNA with high stability using antiparallel-type triplex-forming oligonucleotides with 2-guanidinoethyl-2'-deoxynebularine.

The formation of triplex DNA is a site-specific recognition method that directly targets duplex DNA. However, triplex DNA formation is generally formed for the GC and AT base pairs of duplex DNA, and there are no natural nucleotides that recognize the CG and TA base pairs, or even the 5-methyl-CG (5mCG) base pair. Moreover, duplex DNA, including 5mCG base pairs, epigenetically regulates gene expression in vivo, and thus targeting strategies are of biological importance. Therefore, the development of triplex-forming oligonucleotides (TFOs) with artificial nucleosides that selectively recognize these base pairs with high affinity is needed. We recently reported that 2'-deoxy-2-aminonebularine derivatives exhibited the ability to recognize 5mCG and CG base pairs in triplex formation; however, this ability was dependent on sequences. Therefore, we designed and synthesized new nucleoside derivatives based on the 2'-deoxy-nebularine (dN) skeleton to shorten the linker length connecting to the hydrogen-bonding unit in formation of the antiparallel motif triplex. We successfully demonstrated that TFOs with 2-guanidinoethyl-2'-deoxynebularine (guanidino-dN) recognized 5mCG and CG base pairs with very high affinity in all four DNA sequences with different adjacent nucleobases of guanidino-dN as well as in the promoter sequences of human genes containing 5mCG base pairs with a high DNA methylation frequency.

Application of the Functionality Transfer Oligodeoxynucleotide for the Site-Selective Modification of RNA with a Divers Molecule.

Due to the importance of the RNA chemical modifications, methods for the selective chemical modification at a predetermined site of the internal position of RNA have attracted much attention. We have developed functional artificial nucleic acids that modify a specific site of RNA in a site- and base-selective manner. In addition, the copper-catalyzed azide-alkyne cycloaddition (CuAAC) has been shown to introduce additional molecules on the alkynes attached to the pyridine ring. However, it was found that some azide compounds produced the cycloadduct in lower yields. Therefore, in this study, we synthesized the pyridinyl transfer group with the alkyne attached via a polyethylene glycol (PEG) linker with a different length and optimized its structure for both the transfer and CuAAC reaction. Three new transfer groups were synthesized by introducing an alkyne group at the end of the triethylene (11), tetraethylene (12) or pentaethylen glycol linker (13) at the 5-position of the pyridine ring of (E)-3-iodo-1-(pyridin-2-yl)prop-2-en-1-one. These transfer groups were introduced to the 6-thioguanine base in the oligodeoxynucleotide (ODN) in high yields. The transfer groups 11 and 12 more efficiently underwent the cytosine modification. For the CuAAC reaction, although 7 showed low adduct yields with the anionic azide compound, the new transfer groups, especially 12 and 13, significantly improved the yields. In conclusion, the transfer groups 12 and 13 were determined to be promising compounds for the modification of long RNAs.

Multiple-turnover single nucleotide primer extension reactions to detect 8-oxo-2'-deoxyguanosine in DNA.

The identification of the position of 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA is important to clarify the pathogenesis of many diseases. We herein developed a purine-1,3-diazaphenoxazine triphosphate (dPdapTP) and described the first example of detecting the presence of 8-oxo-dG by amplifying it several hundred times after the multiple-turnover single nucleotide primer extension reactions.

Development of a selective ligand for G-G mismatches of CGG repeat RNA inducing the RNA structural conversion from the G-quadruplex into a hairpin-like structure.

The trinucleotide CGG repeat is located in the 5'-UTR of FMR1 and its abnormal expansion and formation of a noncanonical RNA structure causes fetal genetic diseases. In this study, a small molecular dimer-type ligand consisting of dual G-clamp units for the recognition of two neighboring guanines was synthesized, and the binding properties for the r(CGG) repeats were investigated. Compound 2 was confirmed to bind to the mismatch guanines in the stem region of the r(CGG) repeat hairpin. In addition, the RNase T1 assay demonstrated that 2 induced the structural conversion of the r(CGG)8 repeat from the G-quadruplex into a hairpin-like structure.

Simple and Easy Synthesis of γ-Amido-dNTPs in Water and Their Polymerase Reaction Properties.

γ-Amido-modified 2'-deoxynucleoside triphosphates (dNTPs) and nucleoside triphosphates (NTPs) are becoming increasingly important as biological tools. We herein describe the simple and easy synthesis of γ-amido-dNTPs and -NTPs from commercially available corresponding dNTPs and NTPs in a one-pot reaction using water-soluble carbodiimide and ammonia solution. We examined the effects of synthesized γ-amido-dNTPs on the DNA polymerase reaction. The results obtained showed the incorporation of these derivatives into the DNA primer while maintaining nucleobase selectivity; however, their incorporation efficiency by DNA polymerase was lower than that of dNTP. This is the first study to demonstrate the successful synthesis of four sets of γ-amido-dNTPs and clarify their properties.

Artificial Host Molecules to Covalently Capture 8-Nitro-cGMP in Neutral Aqueous Solutions and in Cells.

New 1,3-diazaphenoxazine derivatives (nitroG-Grasp-Guanidine, NGG) have been developed to covalently capture 8-nitro-cGMP in neutral aqueous solutions, which furnish a thiol reactive group to displace the 8-nitro group and a guanidine unit for interaction with the cyclic phosphate. The thiol group was introduced to the 1,3-diazaphenoxazine skeleton through a 2-aminobenzylthiol group (NGG-H) and its 4-methyl (NGG-pMe) and 6-methyl (NGG-oMe) substituted derivatives. The covalent adducts were formed between the NGG derivatives and 8-nitro-cGMP in neutral aqueous solutions. Among the NGG derivatives, the one with the 6-methyl group (NGG-oMe) exhibited the most efficient capture reaction. Furthermore, NGG-H showed a cell permeability into HEK-293 and RAW 264.7 cells and reduced the intracellular 8-nitro-cGMP level. The NGG derivatives developed in this study would become a valuable tool to study the intracellular role of 8-nitro-cGMP.

Development of MTH1-Binding Nucleotide Analogs Based on 7,8-Dihalogenated 7-Deaza-dG Derivatives.

MTH1 is an enzyme that hydrolyzes 8-oxo-dGTP, which is an oxidatively damaged nucleobase, into 8-oxo-dGMP in nucleotide pools to prevent its mis-incorporation into genomic DNA. Selective and potent MTH1-binding molecules have potential as biological tools and drug candidates. We recently developed 8-halogenated 7-deaza-dGTP as an 8-oxo-dGTP mimic and found that it was not hydrolyzed, but inhibited enzyme activity. To further increase MTH1 binding, we herein designed and synthesized 7,8-dihalogenated 7-deaza-dG derivatives. We successfully synthesized multiple derivatives, including substituted nucleosides and nucleotides, using 7-deaza-dG as a starting material. Evaluations of the inhibition of MTH1 activity revealed the strong inhibitory effects on enzyme activity of the 7,8-dihalogenated 7-deaza-dG derivatives, particularly 7,8-dibromo 7-daza-dGTP. Based on the results obtained on kinetic parameters and from computational docking simulating studies, these nucleotide analogs interacted with the active site of MTH1 and competitively inhibited the substrate 8-oxodGTP. Therefore, novel properties of repair enzymes in cells may be elucidated using new compounds.

Design and synthesis of purine nucleoside analogues for the formation of stable anti-parallel-type triplex DNA with duplex DNA bearing the 5mCG base pair.

We herein demonstrated for the first time the direct recognition of duplex DNA bearing the 5-methyl-2'-deoxycytosine and 2'-deoxyguanosine base pair by triplex DNA formation. Triplex-forming oligonucleotides contained the novel artificial nucleoside analogues 2-amino-2'-deoxy-nebularine derivatives, and their molecular design, synthesis, and functional evaluation are described.

Synthesis of Nucleoside Derivatives of N-Acetyl-7-nitroindoline, Their Incorporation into the DNA Oligomer, and Evaluation of Their Photoreactivity in the DNA/RNA Duplex.

N-Acetyl-7-nitroindoline has a characteristic reaction in that its acetyl group is photo-activated to acetylate amines to form amides. In this study, the N-acetyl-7-nitroindoline part was connected to the 2'-deoxyribose part at the 3- or 5-position or to a glycerol unit at the 3-position through an ethylene linker (1, 2, and 3, respectively). They were incorporated into the oligodeoxynucleotides, and their photo-reactivities toward the complementary RNA were evaluated. The acetyl group of 1 was photo-activated to form the deacelylated nitroso derivative without affecting the RNA strand. The photoreaction with 2 suggested acetylation of the RNA strand. In contrast, compound 3 formed the photo-cross-linked adduct with the RNA. These results have shown the potential application of N-acetyl-7-nitroindoline unit in aqueous solutions.

Protection of all cleavable sites of DNA with the multiple CGCG or continuous CGG sites from the restriction enzyme, indicative of simultaneous binding of small ligands.

The anthracenone ligands (1-12) with a keto-phenol and a hydroxamic acid unit were synthesized and evaluated by a restriction enzyme inhibition assay. DNA substrates composed of multiple CGCG or CGG sites are fully hydrolyzed by a restriction enzyme that is selective for each sequence. Under such conditions, the full-length DNA substrate remains only when the ligand binds to all binding sites and protects it from hydrolysis by the restriction enzymes. In the assay using AccII and the 50-mer DNA substrates containing a different number of CGCG sites at different non-binding AT base pair intervals, the more the CGCG sites, the more the full-length DNA increased. Namely, simultaneous binding of the ligand (5) to the CGCG sites increased in the order of (CGCG)5>(CGCG)2>(CGCG)1. Furthermore, the length of the spacer of the hydroxamic acid to the anthracenone skeleton played an important role in the preference for the number of the d(A/T) base pairs between the CGCG sites. The long spacer-ligand (5) showed a preference to the CGCG sites with five AT pairs, and the short spacer-ligand (10) to that with two AT pairs. The ligand (12) with the shortest spacer showed a preference in simultaneous binding to the 54-mer DNA composed of 16 continuous CGG sites in the assay using the restriction enzyme Fnu4HI that hydrolyzes the d(GCGGC)/d(CGCCG) site. Application of these ligands to biological systems including the repeat DNA sequence should be of significant interest.

Synthesis of C-nucleoside analogues based on the pyrimidine skeleton for the formation of anti-parallel-type triplex DNA with a CG mismatch site.

The triplex DNA forming method is an attractive tool as a gene-targeting agent. Using artificial nucleoside analogues based on C-nucleoside, stable and selective triplex DNA can be formed in a specific region of duplex DNA, and its biotechnology applications will greatly expand. In this study, we designed and synthesized novel C-nucleoside analogues based on the pyrimidine skeleton, 3MeAP-d(Y-Cl) and 3MeAP-d(Y-H), capable of recognizing a CG mismatch site that is not recognized by natural nucleosides. After incorporating them into the oligonucleotides, their triplex forming abilities were evaluated by gel-shift assay. Although it was only one sequence, the 3'-GZG-5' sequence, the stability of the CG mismatch site recognition was greatly improved compared with previous nucleoside analogues.

Enhancements in the utilization of antigene oligonucleotides in the nucleus by booster oligonucleotides.

We recently found the translocation of double-stranded DNA into the nucleus. We herein describe the concept of novel booster oligodeoxynucleotides including 2'-deoxy uridine, which release antigene oligonucleotides in the nucleus by enzymatic digestion. This system exhibited stronger hTERT mRNA expression inhibitory activity than single-stranded antigene oligonucleotides.

Development of novel C-nucleoside analogues for the formation of antiparallel-type triplex DNA with duplex DNA that includes TA and dUA base pairs.

Expansion of the triplex DNA forming sequence is required in the genomic targeting fields. Basically, triplex DNA is formed by the interaction between the triplex-forming oligonucleotides and homo-purine region with the target duplex DNA. The presence of the base pair conversion sites hampers stable triplex formation. To overcome this limitation, it is necessary to develop an artificial nucleic acid to recognize the base conversion sites, and the CG and TA base pairs. We describe the synthesis of C-nucleoside analogues and an evaluation of the ability of triplex formation. Consequently, the combined use of the novel C-nucleoside analogues, AY - AY-d(Y-NH2), AY-d(Y-Cl) and IAP-d(Y-Cl), is capable of recognizing duplex DNA including the TA or dUA base pair.

Oxidative-stress-driven mutagenesis in the small intestine of the gpt delta mouse induced by oral administration of potassium bromate.

Tumorigenesis induced by oxidative stress is thought to be initiated by mutagenesis, but via an indirect mechanism. The dose-response curves for agents that act by this route usually show a threshold, for unknown reasons. To gain insight into these phenomena, we have analyzed the dose response for mutagenesis induced by the oral administration of potassium bromate, a typical oxidative-stress-generating agent, to gpt delta mice. The agent was given orally for 90 d to either Nrf2+ or Nrf2-knockout (KO) mice and mutants induced in the small intestine were analyzed. In Nrf2+mice, the mutant frequency was significantly greater than in the vehicle controls at a dose of 0.6 g/L but not at 0.2 g/L, indicating that a practical threshold for mutagenesis lies between these doses. At 0.6 g/L, the frequencies of G-to-T transversions (landmark mutations for oxidative stress) and G-to-A transitions were significantly elevated. In Nrf2-KO mice, too, the total mutant frequency was increased only at 0.6 g/L. G-to-T transversions are likely to have driven tumorigenesis in the small intestine. A site-specific G-to-T transversion at guanine (nucleotide 406) in a 5'-TGAA-3' sequence in gpt, and our primer extension reaction showed that formation of the oxidative DNA base modification 8-oxo-deoxyguanosine (8-oxo-dG) at nucleotide 406 was significantly increased at doses of 0.6 and 2 g/L in the gpt delta mice. In the Apc oncogene, guanine residues in the same or similar sequences (TGAA or AGAA) are highly substituted by thymine (G-to-T transversions) in potassium bromate-induced tumors. We propose that formation of 8-oxo-dG in the T(A)GAA sequence is an initiating event in tumor formation in the small intestine in response to oxidative stress.

The adduct formation between the thioguanine-polyamine ligands and DNA with the AP site under UVA irradiated and non-irradiated conditions.

The AP sites are representative of DNA damage and known as an intermediate in the base excision repair (BER) pathway which is involved in the repair of damaged nucleobases by reactive oxygen species, UVA irradiation, and DNA alkylating agents. Therefore, it is expected that the inhibition or modulation of the AP site repair pathway may be a new type of anticancer drug. In this study, we investigated the effects of the thioguanine-polyamine ligands (SG-ligands) on the affinity and the reactivity for the AP site under UVA irradiated and non-irradiated conditions. The SG-ligands have a photo-reactivity with the A-F-C sequence where F represents a tetrahydrofuran AP site analogue. Interestingly, the SG-ligands promoted the ß-elimination of the AP site followed by the formation of a covalent bond with the ß-eliminated fragment without UVA irradiation.