Design and synthesis of an environment-sensitive 3-methyleneisoindolin-1-one fluorophore for labeling DNA-interacting proteins.

We designed 6-dimethylamino 3-methyleneisoindolin-1-one as an environment-sensitive fluorophore, examining its applications for protein labeling. Synthesized 3-methyleneisoindolin-1-one exhibits solvatochromic fluorescence (λemmax; 472 nm in 2-PrOH, 512 nm in H2O). A positive linear dependence between λemmax and solvent dielectric constant (DC), as well as between Stokes shift and DC, and a negative correlation between fluorescence quantum yield and DC are observed in protic solvents. These properties are similar to those of the oxygen isosteric fluorophore, 4-dimethylaminophthalimide, a slovatochromic fluorophore utilized for labeling oligodeoxynucleotides (ODNs) and peptides. Notably, fluorescence intensity of 3-methyleneisoindolin-1-one is higher than the phthalimide in protic solvents used in this study. The 3-methyleneisoindolin-1-one demonstrated the higher stability in pH 8 solution than in pH 6 solution in contrast to the stability profile of the phthalimide, which was stable at pH 6 but was hydrolyzed at pH 8. We also synthesized an o-keto benzaldehyde derivative that converts a primary amine to 6-dimethylamino 3-methyleneisoindolin-1-one under biocompatible conditions and introduced it into ODNs for turn-on fluorescent protein labeling. The synthesized ODN with a protein-binding sequence of Escherichia coli DnaA was employed to modify the DNA-binding domain of DnaA, and the fluorescent properties of the modified protein were investigated.

The selective chemical modification of the 6-amino group of adenosine of the premature termination codon induces readthrough to produce full-length peptide in the reconstituted E. Coli translation system.

Nonsense mutations in the coding region turn amino acid codons into termination codons, resulting in premature termination codons (PTCs). In the case of the in-frame PTC, if translation does not stop at the PTC but continues to the natural termination codon (NTC) with the insertion of an amino acid, known as readthrough, the full-length peptide is formed, albeit with a single amino acid mutation. We have previously developed the functionality-transfer oligonucleotide (FT-Probe), which forms a hybrid complex with RNA of a complementary sequence to transfer the functional group, resulting in modification of the 4-amino group of cytosine or the 6-amino group of adenine. In this study, the FT-Probe was used to chemically modify the adenosines of the PTC (UAA, UAG, and UGA) of mRNA, which were assayed for the readthrough in a reconstituted Escherichia coli translation system. The third adenosine-modified UAA produced three readthrough peptides incorporating tyrosine, glutamine and lysine at the UAA site. It should be noted that the additional modification with a cyclodextrin only induced glutamine incorporation. The adenosine modified UGA induced readthrough very efficiently with selective tryptophan incorporation. Readthrough of the modified UGA is caused by inhibition of the RF2 function. This study has demonstrated that the chemical modification of the adenosine 6-amino group of the PTC is a strategy for effective readthrough in a prokaryotic translation system.

Novel strategy for activating gene expression through triplex DNA formation targeting epigenetically suppressed genes.

Triplex DNA formation is a useful genomic targeting tool that is expected to have a wide range of applications, including the antigene method; however, there are fundamental limitations in its forming sequence. We recently extended the triplex DNA-forming sequence to methylated DNA sequences containing 5mCG base pairs by developing guanidino-dN, which is capable of recognizing a 5mCG base pair with high affinity. We herein investigated the effect of triplex DNA formation using TFOs with guanidino-dN on methylated DNA sequences at the promoter of the RASSF1A gene, whose expression is epigenetically suppressed by DNA methylation in MCF-7 cells, on gene expression. Interestingly, triplex DNA formation increased the expression of the RASSF1A gene at the transcript and protein levels. Furthermore, RASSF1A-activated MCF-7 cells exhibited cell growth suppressing activity. Changes in the expression of various genes associated with the promotion of apoptosis and breast cancer survival accompanied the activation of RASSF1A in cells exhibited antiproliferative activity. These results suggest the potential of increases in gene expression through triplex DNA formation as a new genomic targeting tool.

Trigonometric Bundling Disulfide Unit Starship Synergizes More Effectively to Promote Cellular Uptake.

A small molecule disulfide unit technology platform based on dynamic thiol exchange chemistry at the cell membrane has the potential for drug delivery. However, the alteration of the CSSC dihedral angle of the disulfide unit caused by diverse substituents directly affects the effectiveness of this technology platform as well as its own chemical stability. The highly stable open-loop relaxed type disulfide unit plays a limited role in drug delivery due to its low dihedral angle. Here, we have built a novel disulfide unit starship based on the 3,4,5-trihydroxyphenyl skeleton through trigonometric bundling. The intracellular delivery results showed that the trigonometric bundling of the disulfide unit starship effectively promoted cellular uptake without any toxicity, which is far more than 100 times more active than that of equipment with a single disulfide unit in particular. Then, the significant reduction in cell uptake capacity (73-93%) using thiol erasers proves that the trigonometric bundling of the disulfide starship is an endocytosis-independent internalization mechanism via a dynamic covalent disulfide exchange mediated by thiols on the cell surface. Furthermore, analysis of the molecular dynamics simulations demonstrated that trigonometric bundling of the disulfide starship can significantly change the membrane curvature while pushing lipid molecules in multiple directions, resulting in a significant distortion in the membrane structure and excellent membrane permeation performance. In conclusion, the starship system we built fully compensates for the inefficiency deficiencies induced by poor dihedral angles.

Recognition of 8-Oxo-2'-deoxyguanosine in DNA Using the Triphosphate of 2'-Deoxycytidine Connecting the 1,3-Diazaphenoxazine Unit, dCdapTP.

DNA is constantly damaged by various external and internal factors. In particular, oxidative damage occurs in a steady state, and 8-oxo-2'-deoxyguanosine (oxodG) is known as the main oxidative damage. OxodG is a strong genotoxic nucleoside and is thought to be involved in the pathogenesis of cancer and neurological diseases. However, a breakthrough method to detect the position of oxodG in DNA has not yet been developed. Therefore, we attempted to develop a novel method to detect oxodG in DNA using artificial nucleosides. Recently, we have succeeded in the recognition of oxodG in DNA by a single nucleotide elongation reaction using nucleoside derivatives based on a purine skeleton with a 1,3-diazaphenoxazine unit. In this study, we developed a new nucleoside derivative with a pyrimidine skeleton in order to further improve the recognition ability and enzymatic reaction efficiency. We, therefore, designed and synthesized 2'-deoxycytidine-1,3-diazaphenoxazine (Cdap) and its triphosphate derivatives. The results showed that it was incorporated into the primer strand relative to the dG template because of its cytidine skeleton, but it was more effective at the complementary position of the oxodG template. These results indicate that the new nucleoside derivative can be considered as one of the new candidates for the detection of oxodG in DNA.

Precise immunofluorescence canceling for highly multiplexed imaging to capture specific cell states.

Cell states are regulated by the response of signaling pathways to receptor ligand-binding and intercellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving a comprehensive acquisition of spatial protein information from cells. However, the specificity of antibodies is still compromised when visualizing activated signals. Here, we develop Precise Emission Canceling Antibodies (PECAbs) that have cleavable fluorescent labeling. PECAbs enable high-specificity sequential imaging using hundreds of antibodies, allowing for reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes.

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.

The effect of balloon-expandable stent and self-expanding stent on changes in mitral annular motion after aortic valve replacement in patients with aortic stenosis.

OBJECTIVES: The purpose of this study was to evaluate the effect of decalcification and existence of stent at the aortic annulus on mitral annular motion after surgery. METHODS: Patients receiving Inspiris (Edwards, CA, USA, n = 117), Intuity (Edwards, n = 36), Perceval (Corcym, London, UK, n = 36), Evolut (Medtronics, MN, USA, n = 81) and Sapien 3 (Edwards, n = 250) were included in the study. Mitral annular motion was evaluated by E', using tissue doppler imaging. RESULTS: After surgery, a significant increase in E' was observed in patients receiving Inspiris (Before: 4.2 ± 1.21 cm/s vs. Discharge: 5.0 ± 1.23 cm/s, p < 0.001). Mid-term echocardiogram performed at 11.8 ± 2.2 months after the surgery, showed a significant increase in E' in patients receiving Inspiris (Before: 4.2 ± 1.21 cm/s vs. Mid-term: 5.2 ± 1.20 cm/s, p < 0.001) and Perceval (Before: 3.9 ± 1.34 cm/s vs. Mid-term: 4.5 ± 1.24 cm/s, p = 0.008). Univariable analysis showed a higher increase in E' in patients with decalcified annulus compared to those without decalcified annulus (Decalcification: 0.15 ± 1.321 cm/s vs. No Decalcification: 0.66 ± 1.420 cm/s, p < 0.001). Multivariable analysis showed that balloon-expandable stent (ß = - 0.6960, p < 0.001) and self-expanding stent (r = - 0.3592, p = 0.042) were independent limiting factors for an increase in E' at discharge. However, balloon-expandable stent (ß = - 0.8382, p < 0.001), and not self-expanding stent (ß = - 0.3682, p = 0.089), was a remaining independent factor associated with E' at mid-term follow-up. CONCLUSIONS: Decalcification was associated with improvement in E' after surgery. Balloon-expandable stent was an independent limiting factor for improvement in E' up to 1 year after the surgery, while self-expanding stent was not a significant factor after 1 year.

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.

Selective Unnatural Base Pairing and Recognition of 2-Hydroxy-2'-deoxyadenosine in DNA Using Pseudo-dC Derivatives.

The formation of unnatural base pairs within duplex DNA would facilitate DNA nanotechnology and biotechnology. Iso-2'-deoxyguanosine (iso-dG) forms base pairs with iso-2'-deoxycytidine, and its use as an unnatural base pair was investigated. Iso-dG is one of the tautomers of 2-hydroxy-2'-deoxyadenosine (2-OH-dA), known as an oxidatively damaged nucleobase, and its selective recognition in DNA plays an important role in the diagnosis and pathogenesis of disease. Therefore, we focused on pseudo-dC (ψdC) as a suitable molecule that recognizes 2-OH-dA in DNA. Since 2-OH-dA shows tautomeric structures in DNA, we designed and used ψdC, which also has a tautomeric structure. We successfully synthesized a ψdC phosphoramidite compound for the synthesis of oligonucleotides (ODNs) as well as its triphosphate derivative (ψdCTP). Tm measurements revealed that ODNs including ψdC showed stable base pair formation with ODNs having 2-OH-dA. In contrast, low Tm values were observed for other bases (dG, dA, dC, and T). The results obtained for the single-nucleotide primer extension reaction revealed that ψdCTP was incorporated into the complementary position of 2-OH-dA in template DNA with high selectivity. In addition, the primer elongation reaction was confirmed to proceed in the presence of dNTPs. The present study reports an artificial nucleic acid that selectively and stably forms unnatural base pairs with 2-OH-dA in DNA.

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.

Implications of N7-hydrogen and C8-keto on the base pairing, mutagenic potential and repair of 8-oxo-2'-deoxy-adenosine: Investigation by nucleotide analogues.

Oxidative lesions, such as 8-oxo-dG and 8-oxo-dA, are continuously generated from exposure to reactive oxygen species. While 8-oxo-dG has been extensively studied, 8-oxo-dA has not received as much attention until recently. Herein, we report the synthesis of duplex DNAs incorporating dA, 8-oxo-dA, 7-deaza-dA, 8-Br-dA, and 8-Br-7-deaza-dA, which have different substitutions at 7- and 8-position, for the investigation into the implications of N7-hydrogen and C8-keto on the base pairing preference, mutagenic potential and repair of 8-oxo-dA. Base pairing study suggested that the polar N7-hydrogen and C8-keto of 8-oxo-dA, rather than the syn-preference, might be essential for 8-oxo-dA to form a stable base pair with dG. Insertion and extension studies using KF-exo- and human DNA polymerase ß indicated that the efficient dGTP insertion opposite 8-oxo-dA and extension past 8-oxo-dA:dG are contingent upon not only the stable base pair with dG, but also the flexibility of the active site in polymerase. The N7-hydrogen in 8-oxo-dA or C7-hydrogen in 7-deaza-dA and 8-Br-7-deaza-dA was suggested to be important for the recognition by hOGG1, although the excision efficiencies of 7-deaza-dA and 8-Br-7-deaza-dA were much lower than 8-oxo-dA. This study provides an insight into the structure-function relationship of 8-oxo-dA by nucleotide analogues.

The effect of stent and decalcification on mitral annular motion after aortic valve replacement.

OBJECTIVE: The purpose of this study was to evaluate the changes in mitral annular motion after surgery in patients with aortic stenosis. METHODS: Patients receiving Edwards (Edwards) valves were included in the study. Echocardiographic findings were compared among the three treatments postoperatively, at discharge, and at 1 year after the surgery. Mitral annular motion was evaluated by e prime, using tissue doppler imaging. RESULTS: There were 111 patients receiving Inspiris, 30 patients receiving Intuity and 241 patients receiving Sapien 3. The patients receiving Sapien 3 were significantly older, (Inspiris: 71 ± 6.7 years vs. Intuity: 75 ± 5.2 years vs. Sapien 3: 84 ± 5.1 years, p < .001), and prevalence of hemodialysis were significantly higher in patients receiving Intuity (Inspiris: 11.7% vs. Intuity: 46.7% vs. Sapien 3: 0.0%, p < .001). There was a significant improvement in mean pressure gradient in all groups (Inspiris: 55 ± 21.2-13 ± 5.2 mmHg, p < .001; Intuity: 48 ± 17.6-12 ± 4.9 mmHg, p < .001, Sapien 3: 55 ± 16.6-14 ± 5.2 mmHg, p < .001). Decalcification was associated with increase in e prime after surgery (no decalcification: 0.10 ± 1.280 cm/s vs. decalcification: 0.68 ± 1.405 cm/s, p < .001) Further, existence of stent was associated with less increase in e prime after surgery (no stent: 0.83 ± 1.210 cm/s vs. stent: 0.10 ± 1.356; p < .001). Multivariate analysis showed that existence of stent but not decalcification of the aortic valve was independently associated with changes in e prime after surgery (ß: -.4679, 95% confidence interval: -0.93389 to -0.00200, p = .049). CONCLUSIONS: Although improvement in pressure gradient was achieved in all treatments, existence of stent inhibited mitral annular motion after surgery.

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.

Detection and structural analysis of pyrimidine-derived radicals generated on DNA using a profluorescent nitroxide probe.

The oxidative damage of DNA is associated with aging and the development of various diseases. Although nucleoside-derived radicals play an important role in DNA oxidation, their analysis methods are limited. Herein, we propose a fluorometric detection and structural analysis of radicals on the surface of oxidatively damaged DNA using a profluorescent nitroxide probe combined with liquid chromatography-fluorometry and high-resolution tandem mass spectrometry.

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.