Predictive value of local control by 4'-[methyl-11C]-thiotymidine PET volume parameters in p16-negative oropharyngeal, hypopharyngeal, and supraglottic squamous cell carcinoma.

PURPOSE: We investigated the potential of baseline 4'-[methyl- 11 C]-thiothymidine ([ 11 C]4DST) PET for predicting loco-regional control of head and neck squamous cell carcinoma (HNSCC). METHODS: A retrospective analysis was performed using volumetric parameters, such as SUVmax, proliferative tumor volume (PTV), and total lesion proliferation (TLP), of pretreatment [ 11 C]4DST PET for 91 patients with HNSCC with primary lesions in the oral cavity, hypopharynx, supraglottis, and oropharynx, which included p16-negative patients. PTV and TLP were calculated for primary lesions and metastatic lymph nodes combined. We examined the association among the parameters and relapse-free survival and whether case selection focused on biological characteristics improved the accuracy of prognosis prediction. RESULTS: The area under the curves (AUCs) using PTV and TLP were high for the oropharyngeal/hypopharyngeal/supraglottis groups (0.91 and 0.87, respectively), whereas that of SUVmax was 0.66 ( P  < 0.01). On the other hand, the oral group had lower AUCs for PTV and TLP (0.72 and 0.77, respectively). When all cases were examined, the AUCs using PTV and TLP were 0.84 and 0.83, respectively. CONCLUSION: Baseline [ 11 C]4DST PET/CT volume-based parameters can provide important prognostic information with p16-negative oropharyngeal, hypopharyngeal, and supraglottic cancer patients.

Caging of a Strongly Pairing Fluorescent Thymidine Analog with Soft Nucleophiles.

Controlling the pairing strength of nucleobases in DNA through reactions with compounds found inside the cell is a formidable challenge. Here we report how a thiazolyl substituent turns a strongly pairing ethynylpyridone C-nucleoside into a reactive residue in oligonucleotides. The thiazolyl-bearing pyridone reacts with soft nucleophiles, such as glutathione, but not with hard nucleophiles like hydroxide or carbonate. The addition products pair much more weakly with adenine in a complementary strand than the starting material, and also change their fluorescence. This makes oligonucleotides containing the new deoxynucleoside interesting for controlled release. Due to its reactivity toward N, P, S, and Se-nucleophiles, and the visual signal accompanying chemical conversion, the fluorescent nucleotide reported here may also have applications in chemical biology, sensing and diagnostics.

Synthesis, Duplex-Forming Ability, and Nuclease Resistance of Oligonucleotides Containing a Thymidine Derivative with a 1-Oxaspiro[4.5]decane Skeleton.

Chemically modified nucleic acids are essential for the therapeutic application of oligonucleotides. In this study, 6'-C-spiro-thymidine exhibiting a fixed torsion angle γ was designed, synthesized, and incorporated into oligonucleotides. The conformational analysis of the 6'-C-spiro-thymidine monomer revealed that its torsion angle γ was in the +synclinal range (approx. 60°), which is similar to that in a natural RNA duplex, as expected. On the other hand, the sugar conformation of the RNA duplex is known to be predominantly an N-type, whereas that of the synthesized monomer was an S-type. The results of the UV melting analysis demonstrated that the duplex-forming ability of 6'-C-spiro-thymidine was inferior to that of natural DNA. Contrarily, 6'-C-spiro-thymidine could enhance the stability of oligonucleotides toward nucleases. Particularly, the incorporation of 6'-C-spiro-thymidine on the 3'-ends of the oligonucleotides significantly increased the nuclease resistance of the oligonucleotides.

Novel 3'-[4-fluoroaryl-(1,2,3-triazol-1-yl)]-3'-deoxythymidine analogues: Design, synthesis, characterization and their potential as anticancer agents.

Novel 3'-[4-fluoroaryl-(1,2,3-triazol-1-yl)]-3'-deoxythymidine analogues (7a-l) were developed by the Cu alkyne-azide cycloaddition (CuAAC) reaction. The obtained lead compounds were confirmed by using 1H NMR, 13C NMR, 2 D NMR, HRMS and their anticancer activities were screened against Huh-7 liver cancer cells and U87MG human glioblastoma cells. Among the synthesized fluorinated 1,2,3-triazolyl nucleosides, three compounds (7i, 7a-b) demonstrated promising anti-proliferative against Huh-7 and U87MG cell lines. Significantly, compound 7i has displayed remarkable promising anticancer activity with IC50 value in the micromole range (22.41-24.92 µM) and (18.12-21.36 µM) against Huh-7 cancer cells and U87MG glioblastoma cells, respectively.

Metal-free hydroxy and aminocyanation of furanos-3-uloses.

TSAO-T and ATSAO-T analogues are molecules of interest that are able to inhibit the reverse transcriptase (RT) of HIV-1 and HCV. We also recently highlighted their antiproliferative properties. In all cases, the spiro cycle was a required group for biological activities, which led chemists to produce many derivatives, especially on this ring. These structures can be accessed through the formation of glycoaminonitriles and glycocyanhydrins using methodologies not always adapted to the synthesis of large quantities. Moreover, these latter are poorly versatile (substrate-dependent), need expensive cyanogenic agents and implies the use of a metal in non-catalytic amounts. For this reason, we report here a new metal-free methodology for the synthesis of glycoaminonitriles and glycocyanhydrins using molecular iodine (I2).

Comparative Study of the γH2AX Foci Forming in Human Lung Fibroblasts Incubated in Media Containing Tritium-Labeled Thymidine or Amino Acids.

We compared the formation of γH2AX foci (marker of DNA double-strand breaks) in human lung fibroblasts (MRC-5 line) during their 24-h incubation in a medium containing 3H-labeled thymidine or amino acids (glycine, alanine, and proline) with specific radioactivity from 100 to 400 MBq/liter. A linear dependence of changes in the number of γH2AX foci on the specific radioactivity of the medium was revealed. The quantitative yield of DNA double-strand breaks under the influence of 3H-thymidine was more than 2-fold higher than under the influence of 3H-labeled amino acids. Comparative analysis of the yields of DNA double-strand breaks during cell incubation with 3H-labeled amino acids showed that 3H-alanine produced more pronounced effect that 3H-proline, which is consistent with the data on the content of their non-radioactive analogs in chromatin proteins.

Uric Acid as a Photosensitizer in the Reaction of Deoxyribonucleosides with UV Light of Wavelength Longer than 300 nm: Identification of Products from 2'-Deoxycytidine.

DNA reacts directly with UV light with a wavelength shorter than 300 nm. Although ground surface sunlight includes little of this short-wavelength UV light due to its almost complete absorption by the atmosphere, sunlight is the primary cause of skin cancer. Photosensitization by endogenous substances must therefore be involved in skin cancer development mechanisms. Uric acid is the final metabolic product of purines in humans, and is present at relatively high concentrations in cells and fluids. When a neutral mixed solution of 2'-deoxycytidine, 2'-deoxyguanosine, thymidine, and 2'-deoxyadenosine was irradiated with UV light with a wavelength longer than 300 nm in the presence of uric acid, all the nucleosides were consumed in a uric acid dose-dependent manner. These reactions were inhibited by the addition of radical scavengers, ethanol and sodium azide. Two products from 2'-deoxycytidine were isolated and identified as N4-hydroxy-2'-deoxycytidine and N4,5-cyclic amide-2'-deoxycytidine, formed by cycloaddition of an amide group from uric acid. A 15N-labeled uric acid, uric acid-1,3-15N2, having two 14N and two 15N atoms per molecule, produced N4,5-cyclic amide-2'-deoxycytidine containing both 14N and 15N atoms from uric acid-1,3-15N2. Singlet oxygen, hydroxyl radical, peroxynitrous acid, hypochlorous acid, and hypobromous acid generated neither N4-hydroxy-2'-deoxycytidine nor N4,5-cyclic amide-2'-deoxycytidine in the presence of uric acid. These results indicate that uric acid is a photosensitizer for the reaction of nucleosides by UV light with a wavelength longer than 300 nm, and that an unidentified radical derived from uric acid with a delocalized unpaired electron may be generated.

Structural insights into RNA polymerase III-mediated transcription termination through trapping poly-deoxythymidine.

Termination of the RNA polymerase III (Pol III)-mediated transcription requires the conversion of an elongation complex (EC) to a pre-termination complex (PTC) on poly-deoxythymidine (dT)-containing non-template strand, a mechanism distinct from Pol I and Pol II. Here, our in vitro transcription elongation assay showed that 5-7 dT-containing DNA template led to transcription termination of Pol III, but not Pol I or Pol II. We assembled human Pol III PTC on a 7 dT-containing DNA template and determined the structure at 3.6 Å resolution. The structure reveals that poly-dT are trapped in a narrow exit tunnel formed by RPC2. A hydrophobic gate of the exit tunnel separates the bases of two connected deoxythymidines and may prevent translocation of the non-template strand. The fork loop 2 stabilizes both template and non-template strands around the transcription fork, and may further prevent strand translocation. Our study shows that the Pol III-specific exit tunnel and FL2 allow for efficient translocation of non-poly-dT sequence during transcription elongation but trap poly-dT to promote DNA retention of Pol III, revealing molecular mechanism of poly-dT-dependent transcription termination of Pol III.

Recent advances in nucleotide analogue-based techniques for tracking dividing stem cells: An overview.

Detection of thymidine analogues after their incorporation into replicating DNA represents a powerful tool for the study of cellular DNA synthesis, progression through the cell cycle, cell proliferation kinetics, chronology of cell division, and cell fate determination. Recent advances in the concurrent detection of multiple such analogues offer new avenues for the investigation of unknown features of these vital cellular processes. Combined with quantitative analysis, temporal discrimination of multiple labels enables elucidation of various aspects of stem cell life cycle in situ, such as division modes, differentiation, maintenance, and elimination. Data obtained from such experiments are critically important for creating descriptive models of tissue histogenesis and renewal in embryonic development and adult life. Despite the wide use of thymidine analogues in stem cell research, there are a number of caveats to consider for obtaining valid and reliable labeling results when marking replicating DNA with nucleotide analogues. Therefore, in this review, we describe critical points regarding dosage, delivery, and detection of nucleotide analogues in the context of single and multiple labeling, outline labeling schemes based on pulse-chase, cumulative and multilabel marking of replicating DNA for revealing stem cell proliferative behaviors, and determining cell cycle parameters, and discuss preconditions and pitfalls in conducting such experiments. The information presented in our review is important for rational design of experiments on tracking dividing stem cells by marking replicating DNA with thymidine analogues.

Molecular basis for DarT ADP-ribosylation of a DNA base.

ADP-ribosyltransferases use NAD+ to catalyse substrate ADP-ribosylation1, and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria2-4. Reversible ADP-ribosylation has traditionally been considered a protein-specific modification5, but recent in vitro studies have suggested nucleic acids as targets6-9. Here we present evidence that specific, reversible ADP-ribosylation of DNA on thymidine bases occurs in cellulo through the DarT-DarG toxin-antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis, enteropathogenic Escherichia coli and Pseudomonas aeruginosa)10. We report the structure of DarT, which identifies this protein as a diverged member of the PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP-HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class of ADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details of this PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thymine-linked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT-DarG regulates growth by ADP-ribosylation of DNA at the origin of chromosome replication.

A new family of globally distributed lytic roseophages with unusual deoxythymidine to deoxyuridine substitution.

Marine bacterial viruses (bacteriophages) are abundant biological entities that are vital for shaping microbial diversity, impacting marine ecosystem function, and driving host evolution.1-3 The marine roseobacter clade (MRC) is a ubiquitous group of heterotrophic bacteria4,5 that are important in the elemental cycling of various nitrogen, sulfur, carbon, and phosphorus compounds.6-10 Bacteriophages infecting MRC (roseophages) have thus attracted much attention and more than 30 roseophages have been isolated,11-13 the majority of which belong to the N4-like group (Podoviridae family) or the Chi-like group (Siphoviridae family), although ssDNA-containing roseophages are also known.14 In our attempts to isolate lytic roseophages, we obtained two new phages (DSS3_VP1 and DSS3_PM1) infecting the model MRC strain Ruegeria pomeroyi DSS-3. Here, we show that not only do these phages have unusual substitution of deoxythymidine with deoxyuridine (dU) in their DNA, but they are also phylogenetically distinct from any currently known double-stranded DNA bacteriophages, supporting the establishment of a novel family ("Naomiviridae"). These dU-containing phages possess DNA that is resistant to the commonly used library preparation method for metagenome sequencing, which may have caused significant underestimation of their presence in the environment. Nevertheless, our analysis of Tara Ocean metagenome datasets suggests that these unusual bacteriophages are of global importance and more diverse than other well-known bacteriophages, e.g., the Podoviridae in the oceans, pointing to an overlooked role for these novel phages in the environment.

On the population of triplet states of 2-seleno-thymine.

The population and depopulation mechanisms leading to the lowest-lying triplet states of 2-Se-Thymine were studied at the MS-CASPT2/cc-pVDZ level of theory. Several critical points on different potential energy hypersurfaces were optimized, including minima, conical intersections, and singlet-triplet crossings. The accessibility of all relevant regions on the potential energy hypersurfaces was investigated by means of minimum energy paths and linear interpolation in internal coordinates techniques. Our analysis indicates that, after the population of the bright S2 state in the Franck-Condon region, the first photochemical event is a barrierless evolution towards one of its two minima. After that, three viable photophysical deactivation paths can take place. In one of them, the population in the S2 state is transferred to the T2 state via intersystem crossing and subsequently to the T1 state by internal conversion. Alternatively, the S1 state could be accessed by internal conversion through two distinct conical intersections with S2 state followed by singlet-triplet crossing with the T2 state. The absence of a second minimum on the T1 state and a small energy barrier on pathway along the potential energy surface towards the ground state from the lowest triplet state are attributed as potential reasons to explain why the lifetime of the triplet state of 2-Se-Thymine might be reduced in comparison with its thio-analogue.

Optimized Biocatalytic Synthesis of 2-Selenopyrimidine Nucleosides by Transglycosylation*.

Selenium-modified nucleosides are powerful tools to study the structure and function of nucleic acids and their protein interactions. The widespread application of 2-selenopyrimidine nucleosides is currently limited by low yields in established synthetic routes. Herein, we describe the optimization of the synthesis of 2-Se-uridine and 2-Se-thymidine derivatives by thermostable nucleoside phosphorylases in transglycosylation reactions using natural uridine or thymidine as sugar donors. Reactions were performed at 60 or 80 °C and at pH 9 under hypoxic conditions to improve the solubility and stability of the 2-Se-nucleobases in aqueous media. To optimize the conversion, the reaction equilibria in analytical transglycosylation reactions were studied. The equilibrium constants of phosphorolysis of the 2-Se-pyrimidines were between 5 and 10, and therefore differ by an order of magnitude from the equilibrium constants of any other known case. Hence, the thermodynamic properties of the target nucleosides are inherently unfavorable, and this complicates their synthesis significantly. A tenfold excess of sugar donor was needed to achieve 40-48 % conversion to the target nucleoside. Scale-up of the optimized conditions provided four Se-containing nucleosides in 6-40 % isolated yield, which compares favorably to established chemical routes.

Thermally reversible and irreversible interstrand photocrosslinking of 5-chloro-2'-deoxy-4-thiouridine modified DNA oligonucleotides.

We describe highly efficient interstrand photocrosslinking of a DNA duplex containing 5-chloro-2'-deoxy-4-thiouridine (ClSdU) in one strand, proceeding via a two-step photochemical cascade, involving the formation of a thermally reversible crosslink between ClSdU and thymidine in the target strand and its subsequent conversion to a thermally stable fluorescent crosslink. These results show that ClSdU has great potential to be a valuable DNA photo-crosslinking reagent for chemical biology applications.

Determination of substituent effects on the proton affinities of natural nucleosides by the kinetic method.

The kinetics of the unimolecular dissociations of proton-bound dimers produced by fast-atom bombardment from nucleosides and reference amines enables the evaluation of the proton affinities (PAs) of ribonucleosides. The PAs of cytosine, guanosine, adenosine, uridine, and deoxyuridine have been thus determined. These values and those already available for the corresponding DNA homologues allow the evaluation of the effect of the hydroxyl group in position 2' of the sugar moiety, which lowers the PAs of RNA nucleosides by 0.6-1 kcal/mol, and of the methyl group in position 5 of the thymine ring, which enhances the basicity of deoxythymidine over deoxyuridine by 0.6 kcal/mol.

Clinical value of PET/CT with carbon-11 4DST in the evaluation of malignant and benign lung tumors.

OBJECTIVES: The aim of this study was to assess the clinical value of [11C]4DST uptake in patients with lung nodules, including benign and malignant tumors, and to assess the correlation between [11C]4DST uptake and proliferative activity of tumors in comparison with [18F]FDG uptake. METHODS: Twenty-six patients (22 males and 4 females, mean age of 65.5-year-old) were analyzed in this prospective study. Patients underwent [11C]4DST and [18F]FDG PET/CT imaging on the same day. Diagnosis of each lung nodule was confirmed by histopathological examination of tissue specimens at surgery, or during clinical follow-up after the PET/CT studies. To assess the utility of the semi-quantitative evaluation method, the SUVmax was calculated of [11C]4DST and [18F]FDG uptake by the lesion. Proliferative activities of each tumor as indicated by the immunohistochemical Ki-67 index was also estimated using surgical specimens of patients. Then the relationship between the SUVmax of both PET/CT and the Ki-67 index was examined. Furthermore, the relationship between the uptake of [11C]4DST or [18F]FDG and the histopathological findings, the clinical stage, and the clinical outcome of patients were also assessed. RESULTS: There was a positive linear relationship between the SUVmax of [11C]4DST images and the Ki-67 index (Correlation coefficients = 0.68). The SUVmax of [11C]4DST in the 26 lung nodules were 1.65 ± 0.40 for benign lesions, 3.09 ± 0.83 for adenocarcinomas (P < 0.001 between benign and adenocarcinoma), and 2.92 ± 0.58 for SqCCs (P < 0.001 between benign and SqCC). Whereas, the SUVmax of [18F]FDG were 2.38 ± 2.27 for benign lesions, 6.63 ± 4.24 for adenocarcinomas (n.s.), and 7.52 ± 2.84 for SqCCs (n.s.). The relationship between TNM tumor stage and the SUVmax of [11C]4DST were 2.54 ± 0.37 for T1, 3.48 ± 0.57 for T2, and 4.17 ± 0.72 for T3 (P < 0.005 between T1 and T2, and P < 0.001 between T1 and T3). In comparison with the TNM pathological stage, SUVmax of [11C]4DST were 2.63 ± 0.49 for stage I, 3.36 ± 0.23 for stage II, 3.40 ± 1.12 for stage III, and 4.65 for stage IV (P < 0.05 between stages I and II). In comparison of the clinical outcome, the SUVmax of [11C]4DST were 2.72 ± 0.56 for the no recurrence (No Rec.) group, 3.10 ± 0.33 for the recurrence-free with adjuvant chemotherapy after the surgery (the No Rec. Adjv. CTx. group) and 4.66 ± 0.02 for the recurrence group (Rec. group) (P < 0.001 between the No Rec and Rec. groups, and P < 0.005 between the No Rec. Adjv. CTx. and Rec. groups). CONCLUSIONS: PET/CT with [11C]4DST is as feasible for imaging of lung tumors as [18F]FDG PET/CT. For diagnosing lung tumors, [11C]4DST PET is useful in distinguishing benign nodules from malignancies. [11C]4DST uptake in lung carcinomas is correlated with the proliferative activity of tumors, indicating a promising noninvasive PET imaging of DNA synthesis in malignant lung tumors.

Synthesis and properties of oligonucleotides bearing thymidine derivatives with 1,6-dioxaspiro[4.5]decane skeleton.

Thymidine derivatives bearing spiroacetal moieties on the C4'-position (5'R-spiro-thymidine and 5'S-spiro-thymidine) were synthesized and incorporated into oligonucleotides. The duplex- and triplex-forming abilities of both the oligonucleotides were evaluated from UV melting experiments. Oligonucleotides with the 5'S-spiro modifications could form thermally stable duplexes with complementary RNA and DNA; however, the 5'R-spiro modification significantly decreased the thermal stabilities of the duplexes and triplexes. Oligonucleotides with these spiro-thymidines showed significantly high resistance towards enzymatic degradation.

Novel Dioxane and Morpholino Nucleotide Analogues: Syntheses and RNA-Hybridization Properties.

A novel class of nucleotide analogues with a dioxane ring as central scaffold has been developed. Synthetic routes in two diastereomeric series were realized, and the final thymidine analogues were synthesized with common functionalities for the automated oligonucleotide synthesis. The chemical space of the initially derived nucleotides was expanded by changing the central dioxane to analogous morpholine derivatives. This opens up the possibility for further derivatization by attaching different substituents at the morpholine nitrogen. The novel nucleotide building blocks were incorporated into double-stranded RNA sequences, and their hybridization properties investigated by melting-temperature analysis. Both scaffolds, dioxanes and morpholines, had an equal impact on double-strand stability, but Tm values differed depending on the chirality in the six-membered ring.

Mitochondrial Transcription Factor A Binds to and Promotes Mutagenic Transcriptional Bypass of O4-Alkylthymidine Lesions.

O2- and O4-alkylated thymidine lesions are known to be poorly repaired and persist in mammalian tissues. To understand how mammalian cells sense the presence and regulate the repair of these lesions, we employed a quantitative proteomic method to discover regioisomeric O2- and O4-n-butylthymidine (O2- and O4-nBudT)-binding proteins. We were able to identify 21 and 74 candidate DNA damage recognition proteins for O2-nBudT- and O4-nBudT-bearing DNA probes, respectively. Among these proteins, DDB1 and DDB2 selectively bind to O2-nBudT-containing DNA, whereas three high-mobility group (HMG) proteins (i.e., HMGB1, HMGB2, and mitochondrial transcription factor A (TFAM)) exhibit preferential binding to O4-nBudT-bearing DNA. We further demonstrated that TFAM binds directly and selectively with O4-alkyldT-harboring DNA, and the binding capacity depends mainly on the HMG box-A domain of TFAM. We also found that TFAM promotes transcriptional mutagenesis of O4-nBudT and O4-pyridyloxobutylthymidine, which is a DNA adduct induced by tobacco-specific N-nitrosamines, in vitro and in human cells. Together, we explored, for the first time, the interaction proteomes of O-alkyldT lesions, and our study expanded the functions of TFAM by revealing its capability in the recognition of O4-alkyldT-bearing DNA and uncovering its modulation of transcriptional mutagenesis of these lesions in human cells.

Quantitative LC-MS/MS analysis of 5-hydroxymethyl-2'-deoxyuridine to monitor the biological activity of J-binding protein.

Base J replaces 1% of thymine in most kinetoplastid flagellates, and is implicated in transcription regulation. Base J is synthesized in two steps: first, a thymine base in DNA is converted to 5-hydroxymethyluracil by J-binding proteins (JBP1, JBP2); secondly, a glucosyl transferase glycosylates the 5-hydroxymethyluracil to form base J. Here, we present a highly sensitive and selective LC-MS/MS method to quantify the in vitro JBP1 activity on synthetic oligonucleotide substrates. The method demonstrated successful to support biochemical studies of JBPs and can be used as a template for additional JBP activity studies or for inhibitor screening in the future.