Thymine Groups Research Articles

Straightforward Preparation of Telechelic H-bonding Polymers From Difunctional Trithiocarbonates and Supramolecular Block Copolymers Thereof

We report an original strategy toward the straightforward preparation of precisely defined telechelic H-bonding polymers and the generation of supramolecular block copolymers thereof. Making use of an α,ω-functionalized symmetrical trithiocarbonate bearing thymine groups at both chain ends, a series of heterocomplementary H-bonding polymers were effortlessly generated by RAFT polymerization in a one step process. The resulting telechelic macromolecules selectively interacted with heterocomplementary α-DAP-functionalized chains to afford supramolecular block copolymers in solution and in bulk. These self-assemblies were evidenced by 1H NMR and rheological measurements. As a consequence of these associations, whereas non functional homopolymer blends tended to microphase separate as observed by AFM analysis, H-bonding homopolymer blends exhibited homogeneous microstructures in accord with the formation of supramolecular block copolymers promoting the stabilization of the interface between the polymers.

Molecular Basis for Recognition of Nucleoside Triphosphate by Gene 4 Helicase of Bacteriophage T7

The translocation of DNA helicases on single-stranded DNA and the unwinding of double-stranded DNA are fueled by the hydrolysis of nucleoside triphosphates (NTP). Although most helicases use ATP in these processes, the DNA helicase encoded by gene 4 of bacteriophage T7 uses dTTP most efficiently. To identify the structural requirements of the NTP, we determined the efficiency of DNA unwinding by T7 helicase using a variety of NTPs and their analogs. The 5-methyl group of thymine was critical for the efficient unwinding of DNA, although the presence of a 3'-ribosyl hydroxyl group partially overcame this requirement. The NTP-binding pocket of the protein was examined by randomly substituting amino acids for several amino acid residues (Thr-320, Arg-504, Tyr-535, and Leu-542) that the crystal structure suggests interact with the nucleotide. Although positions 320 and 542 required aliphatic residues of the appropriate size, an aromatic side chain was necessary at position 535 to stabilize NTP for efficient unwinding. A basic side chain of residue 504 was essential to interact with the 4-carbonyl of the thymine base of dTTP. Replacement of this residue with a small aliphatic residue allowed the accommodation of other NTPs, resulting in the preferential use of dATP and the use of dCTP, a nucleotide not normally used. Results from this study suggest that the NTP must be stabilized by specific interactions within the NTP-binding site of the protein to achieve efficient hydrolysis. These interactions dictate NTP specificity.

Precision synthesis and characterization of thymine-functionalized polyisobutylene

A new two-step synthesis of polyisobutylene (PIB) with precisely one thymine functionality per chain (PIB-T) is reported. The primary hydroxyl-functionalized PIB (PIB-OH) precursor was prepared by direct functionalization via living carbocationic polymerization of isobutylene initiated by the α-methylstyrene epoxide/TiCl4 system. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) of a low molecular weight PIB-OH precursor demonstrated the effectiveness of direct functionalization by this method. A PIB-acrylate precursor (PIB-Ac) was obtained from such a PIB-OH, and the PIB-T was subsequently prepared by Michael addition of thymine across the acrylate double bond. MALDI-ToF MS of the products verified that all polymer chains carried precisely one thymine group. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 3501–3506, 2010

The 5-Methyl Group in Thymine Dynamically Influences the Structure of A-Tracts in DNA at the Local and Global Level

DNA sequences containing a stretch of several A:T basepairs without a 5'-TA-3' step are known as A-tracts and have been the subject of extensive investigation because of their unique structural features such as a narrow minor groove and their crucial role in several biological processes. One of the aspects under investigation has been the influence of the 5-methyl group of thymine on the properties of A-tracts. Detailed molecular dynamics simulation studies of the sequences d(CGCAAAUUUGCG) and d(CGCAAATTTGCG) indicate that the presence of the 5-methyl group in thymine increases the frequency of a narrow minor groove conformation, which could facilitate its specific recognition by proteins, and reduce its susceptibility to cleavage by DNase I. The bias toward a wider minor groove in the absence of the thymine 5-methyl group is a static structural feature. Our results also indicate that the presence of the thymine 5-methyl group is necessary for calibrating the backbone conformation and the basepair and dinucleotide step geometry of the core A-tract as well as the flanking CA/TG and the neighboring GC/GC steps, as observed in free and protein-bound DNA. As a consequence, it also fine-tunes the curvature of the longer DNA fragment in which the A-tract is embedded.

LNA 5′-phosphoramidites for 5′→3′-oligonucleotide synthesis

Hereby we report an efficient synthesis of LNA thymine and LNA 5-methylcytosine 5'-phosphoramidites, allowing incorporation of LNA thymine and LNA 5-methylcytosine into oligonucleotides synthesized in the 5'→3' direction. Key steps include regioselective enzymatic benzoylation of the 5'-hydroxy group of unprotected LNA thymine, and subsequent 4,4'-dimethoxytritylation of the 3'-hydroxy group of the O5'-benzoylated LNA thymine nucleoside.

UV degradation of deoxyribonucleic acid

The effects of UV synchrotron radiation on deoxyribonucleic acid (DNA) cast films have been systematically investigated by vacuum ultraviolet and infrared spectrophotometry as a function of irradiation time. Cast DNA films exposed at 140 nm (8.85 eV) for different irradiation times, revealed consistent changes in their VUV spectra which indicate a decrease of thymine groups and an increase of π → π* transition spectral signature associated with the C O group of the open sugar chain. This result was corroborated by a decrease in C O stretching vibration at 1061 cm −1 observed in the infrared spectra. Both these results are consistent with the creation of single strand breaks in the deoxyribose component of DNA molecule and a decrease in the phosphate groups. It was also shown that UV radiation is effective in damaging the thymine groups involved in Hoogsteen base pairing with adenine. The analysis of the infrared data suggests that the usual spectroscopic fingerprints of DNA denaturation are not necessarily a reliable measure of DNA damage.

Thymine-Protected Gold Nanoparticles: Assembly and Disassembly of Gold Nanoparticles in the Presence of Hg<SUP>2+</SUP>

In this paper, we describe thymine-protected gold nanoparticles. Assembly of thymine-protected gold nanoparticles into interconnected network was observed by gradual addition of Hg2+. Moreover, the plasmon absorption intensity varied almost linearly with the concentration of Hg2+, indicating that thymine-protected gold nanoparticles are potentially useful for signaling Hg2+. It is interesting to note that disassembly of gold nanoparticles occurred when the concentration of Hg2+ in the solution was high. It is proposed that the assembly of gold nanoparticles is due to the coordination of Hg2+ with thymine groups of adjacent nanoparticles, while the disassembly of gold nanoparticles is attributed to the electrostatic repulsion among the nanoparticles with positive charges which are generated through the coordination of Hg2+ with thymine groups absorbed on the surface of each nanoparticle.

Syntheses of 5‐Chlorouracils/Thymines with 1‐[Phosphono(Methyl/Difluoromethyl)]‐1,2‐Unsaturated‐Moiety‐Substituted Methyl Groups at N(1) and Human Thymidine Phosphorylase Inhibitory Activity

Abstractmagnified imageBy attaching (methyl)‐ or (difluoromethyl)‐phosphonate groups to the 1‐positions of ethene, cyclopentene or benzene, and attaching 1‐(methyl)‐5‐chlorouracil or 1‐(methyl)thymine groups to the corresponding 2‐positions, compounds 1–5 were prepared as potential inhibitors of recombinant human thymidine phosphorylase (TP). The products were designed to mimic the interatomic distance (ca. 3.41 Å) between the incoming phosphate and leaving pyrimidine groups at the transition state for the putative SN2 mechanism of TP. Free rotation around the (unsaturated‐CH2)–pyrimidine bonds in 1–5 enabled a span of ca. 2.40–4.40 Å between the CH2 or CF2 C‐atoms in the phosphonates and N(1) of the pyrimidines to be covered. The products were found to be ineffective inhibitors, and some reasons for this are given.

Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.

DNA cytosine methylation is crucial for retrotransposon silencing and mammalian development. In a computational search for enzymes that could modify 5-methylcytosine (5mC), we identified TET proteins as mammalian homologs of the trypanosome proteins JBP1 and JBP2, which have been proposed to oxidize the 5-methyl group of thymine. We show here that TET1, a fusion partner of the MLL gene in acute myeloid leukemia, is a 2-oxoglutarate (2OG)- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro. hmC is present in the genome of mouse embryonic stem cells, and hmC levels decrease upon RNA interference-mediated depletion of TET1. Thus, TET proteins have potential roles in epigenetic regulation through modification of 5mC to hmC.

Cloning and Characterization of Rhodotorula glutinis Thymine Hydroxylase

Thymine hydroxylase (TH) is a member of the alpha-ketoglutarate-dependent nonheme iron dioxygenase family that includes a series of DNA repair proteins including alkB. Substantial interest in this family of enzymes derives from their capacity to modify DNA bases and precursors by oxidation. Previously, a sequence has been published for cloned Rhodotorula glutinis TH. However, the minimal reported activity of this enzyme, coupled with inconsistencies with previously published mass spectrometry data, compelled us to reexamine TH. The sequence reported here differs from the previously reported sequence at two amino acid positions and is consistent with previously reported mass spectrometry data. The cloned enzyme characterized in this report displayed substantial activity, indicating that the sequence differences are critical for activity. The substrate selectivity of TH against a series of pyrimidine analogues is consistent with that reported for the wild-type enzyme and, in part, explains the mode of selection of uracil analogues. A preliminary model of the active site has been constructed for the purposes of comparing TH with other members of this family. TH and alkB share in common the capacity to oxidize N-methyl groups. However, TH has the added capacity to oxidize the 5-methyl group of thymine, a property that is potentially important for enzymes that could act on DNA and modify DNA-protein interactions.

An Analysis of the Different Behavior of DNA and RNA through the Study of the Mutual Relationship between Stacking and Hydrogen Bonding

The mutual relationship between stacking and hydrogen bonding and the possible influence of stacking in the different behavior of DNA and RNA base pairs have been studied through complete DFT optimization of different structures of A-U and A-T dimers (i.e., A-U/U-A and A-T/T-A), using some functionals developed by the group of Truhlar. The results obtained in this work clearly show that stacking and hydrogen bonding are deeply connected. The different behavior of DNA and RNA when replacing uracil by thymine can be interpreted through the formation of a stabilizing CH/pi interaction between the methyl group of thymine and the five-member ring of adenine.

A “plug and play” polymer through biocomplementary hydrogen bonding

AbstractThis article describes a DNA‐like polymer that exhibits the ability to self‐assemble through hydrogen bonding. We synthesized poly[1‐(4‐vinylbenzyl)thymine] (PVBT) and 9‐hexadecyladenine (A‐C16) through an atom transfer radical polymerization (ATRP) and alkylation, respectively. Biocomplementary PVBT/A‐C16 hierarchical supramolecular complexes formed in dilute DMSO solution through nucleobase recognition, that is, hydrogen bonding interactions between the thymine (T) groups of PVBT and the adenine (A) group of A‐C16; evidence for this molecular recognition was also gained from dynamic light scattering studies. 1H NMR titration studies in CDCl3 showed that T–A complexes formed rapidly on the NMR time scale with high association constants (up to 534 M−1). Moreover, FTIR spectroscopic, differential scanning calorimetry, wide‐angle X‐ray diffraction, and small‐angle X‐ray scattering analyses provided further details into the nature of the self‐assembly of these systems. In the bulk state, these complexes self‐assemble into well‐ordered lamellar structures; the changing d‐spacing distance (ranging from 4.98 to 2.32 nm) at different A‐C16 loadings reveals that the molecular structures of the PVBT/A‐C16 complexes are readily tailored. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 6416–6424, 2008

Photo-sensible (thymine containing) azo-polysiloxanes: synthesis and light induced effects

.The paper presents the possibility to obtain azo-polysiloxanes modified with thymine and their light induced processing with potential interest in opto-electronics or biomolecules nanomanipulation. The presence of the thymine group in the polymeric structure can confer to material biological properties, in the same time the capacity of tymine to generate H-bonds being useful to the relief geometry stabilization in time. The investigated polymers were obtained in a two step reaction, starting from a polysiloxane containing chlorobenzyl groups in the side chain. The azopolysiloxanes' photochromic behaviour was investigated in solid state, using thin films etalated on the surface of a quartz slide. The effect of surface relief structuration process under the action of UV (355 nm) laser radiation was studied. Laser induced effects on the material surface depends on the incident laser fluence and number of pulses.

阴离子作用下的小牛胸腺DNA的表面增强拉曼光谱研究

Dependence of surface-enhanced Raman scattering (SERS) from Calf thymus DNA on anions is investigated. With the silver colloid, the bands at 732, 960 and 1333 cm-1 for adenine (A), 1466 cm-1 for deoxyribose, and 1652 cm-1 for the C=O group of thymine (T) are observably enhanced. With the presence of the Cl- or SO^(2-)_(4) anions, the bands at 732 and 1326/1329 cm-1 for the symmetric stretching and skeletal vibrational modes of adenine (A) are dramatically enhanced, and the enhancement effect with the SO^(2-)_(4) ion is more than that with the Cl_ ion. The experimental results show that the DNA molecule can be adsorbed on the silver colloid particles through the C6N and N7 of adenine (A), the C=O of thymine (T) and deoxyribose. Moreover, the formed hydrogen bonding of the Cl_ or SO^(2-)_(4) ions to the C6NH2 group of adenine (A) can induce larger C6N electronegativity, which is favor for the C6N/N7 cooperative adsorption on the (Ag)^(+)_(n) colloid particles.

Hydrogen-bonding tectons for the construction of bimolecular framework materials

Three hydrogen-bonding tectons functionalised with pyrimidine-dione hydrogen-bonding moieties, either uracil or thymine groups, have been synthesised and their solid-state structures studied by single crystal X-ray diffraction. The tectons have different linkers between the pyrimidine-dione hydrogen-bonding groups. Whereas tectons 1 and 2 have either naphthalenetetracarboxylic diimide or p-xylyl spacers, respectively, linking two hydrogen-bonding appendages, tecton 3 comprises three thymine moieties linked via a 2,4,6-tris(methyl)mesitylene spacer. All three tectons have been structurally characterised and in the case of 2 and 3 exhibit the anticipated R22(8) double hydrogen-bonded arrangement between the imide moieties. In contrast, the single crystal structure of the DMSO solvate of 1 does not exhibit inter-tecton hydrogen-bonding but adopts N–H⋯O interactions with guest DMSO molecules. However, bimolecular hydrogen-bonded adducts have successfully been prepared exploiting the complementary triple hydrogen-bonding interactions between 1, or 2, and melamine to give two-dimensional hydrogen-bonded sheets.

Functional Characterization of the Escherichia coli Fis–DNA Binding Sequence

The Escherichia coli protein Fis is remarkable for its ability to interact specifically with DNA sites of highly variable sequences. The mechanism of this sequence-flexible DNA recognition is not well understood. In a previous study, we examined the contributions of Fis residues to high-affinity binding at different DNA sequences using alanine-scanning mutagenesis and identified several key residues for Fis–DNA recognition. In this work, we investigated the contributions of the 15-bp core Fis binding sequence and its flanking regions to Fis–DNA interactions. Systematic base-pair replacements made in both half sites of a palindromic Fis binding sequence were examined for their effects on the relative Fis binding affinity. Missing contact assays were also used to examine the effects of base removal within the core binding site and its flanking regions on the Fis–DNA binding affinity. The results revealed that: (1) the − 7G and + 3Y bases in both DNA strands (relative to the central position of the core binding site) are major determinants for high-affinity binding; (2) the C 5 methyl group of thymine, when present at the + 4 position, strongly hinders Fis binding; and (3) AT-rich sequences in the central and flanking DNA regions facilitate Fis–DNA interactions by altering the DNA structure and by increasing the local DNA flexibility. We infer that the degeneracy of specific Fis binding sites results from the numerous base-pair combinations that are possible at noncritical DNA positions (from − 6 to − 4, from − 2 to + 2, and from + 4 to + 6), with only moderate penalties on the binding affinity, the roughly similar contributions of − 3A or G and + 3T or C to the binding affinity, and the minimal requirement of three of the four critical base pairs to achieve considerably high binding affinities.

Hydrogen-Abstracted Adenine−Thymine Radicals with Interesting Transferable Properties

The formation of radicals on DNA bases through various pathways can lead to harmful structural alterations. Such processes are of interest for preventing alteration of healthy DNA and, conversely, to develop more refined methods for inhibiting the replication of unwanted mutagenic DNA. In the present work, we explore theoretically the energetic and structural properties of the nine possible neutral radicals formed via hydrogen abstraction from the adenine-thymine base pair. The lowest energy radical is formed by loss of a hydrogen atom from the methyl group of thymine. The next lowest energy radicals, lying 8 and 9 kcal mol-1 higher than the global minimum, are those in which hydrogens are removed from the two nitrogens that would join the base pair to 2-deoxyribose in double-stranded DNA. The other six radicals lie between 16 and 32 kcal mol-1 higher in energy. Unlike the guanine-cytosine base pair, adenine-thymine (A-T) exhibits only minor structural changes upon hydrogen abstraction, with all A-T derived radicals maintaining planarity. Moreover, the energetic ordering for the radicals of the two isolated bases (adenine and thymine) is preserved upon formation of the base pair, though with a wider spread of energies. Even more significantly, the energetic interleaving of the (A-H)*-T and A-(T-H)* radicals is correctly predicted from the X-H bond dissociation energies of the isolated adenine and thymine. This suggests that the addition of the hydrogen-bonded complement base only marginally affects the bond energies.

Synthesis of “bioinspired” copolymers: experimental and theoretical investigation on poly(vinyl benzyl thymine-co-triethyl ammonium chloride)

“Bioinspired” copolymers based on vinylbenzyl thymine (VBT) and an ionically-charged monomer, such as vinylbenzyl triethylammonium chloride (VBA), were synthesized and theoretically investigated. These water-soluble copolymers are polystyrene- (PS) based, and their structure mimics DNA. In the presence of short-wavelength UV light, the thymine groups dimerize into non-toxic, environmentally benign, and biodegradable photo-resistant materials. Copolymerizations with different comonomer ratios were carried out at 65°C. Samples were taken along the reactions to determine monomer conversion, chemical composition, and molecular weight distribution. While average molecular weights fall along the reaction, the average composition remains almost constant and coincident with the initial comonomer ratios, thus indicating a similar reactivity of all the comonomer radicals. A mathematical model was developed that simulates the synthesis of the base biopolymer, in the sense of predicting the evolution of the global reaction variables and molecular structure of the polymer. The termination and propagation kinetic constants were adjusted to the experimental data. The resulting values are quite different to those of a normal styrene homopolymerization, thus suggesting a noticeable effect of the solvent and the comonomer pending groups.

Recombinant Schizosaccharomyces pombe Nth1 Protein Exhibits DNA Glycosylase Activities for 8-oxo-7,8-dihydroguanine and Thymine Residues Oxidized in the Methyl Group

Bacteria and eukaryotes possess redundant enzymes that recognize and remove oxidatively damaged bases from DNA through base excision repair. DNA glycosylases remove damaged bases to initiate the base excision repair. The exocyclic methyl group of thymine does not escape oxidative damage to produce 5-formyluracil (5-foU) and 5-hydroxymethyluracil (5-hmU). 5-foU is a potentially mutagenic lesion. A homolog of E. coli endonuclease III (SpNth1) had been identified and characterized in Schizosaccharomyces pombe. In this study, we found that SpNth1 recognizes and removes 5-foU and 5-hmU from DNA with similar efficiency. The specific activities for the removal of 5-foU and 5-hmU were comparable with that for thymine glycol. The expression of SpNth1 reduced the hydrogen peroxide toxicity and the frequency of spontaneous mutations in E. coli nth nei mutant. It was also revealed that SpNth1 had DNA glycosylase activity for removing 8-oxo-7,8-dihydroguanine (8-oxoG) from 8-oxoG/G and 8-oxoG/A mispairs. These results indicated that SpNth1 has a broad substrate specificity and is involved in the base excision repair of 8-oxoG and thymine residues oxidized in the methyl group in S. pombe.

Association of Star-Shaped Poly(d,l-lactide)s Containing Nucleobase Multiple Hydrogen Bonding

A new family of associating polymers based on four-arm, star-shaped poly(D,L-lactide) (PDLLA) containing peripheral complementary hydrogen-bonding sites is described. Hydroxy-terminated, four-arm, star-shaped PDLLAs of controlled molar mass were functionalized with complementary DNA base pairs, adenine (A) and thymine (T), to obtain PDLLA-A and PDLLA-T, respectively. 1H NMR spectroscopy confirmed quantitative functionalization and the subsequent formation of PDLLA-A and PDLLA-T hydrogen-bonded complexes. Job's analysis revealed a 1:1 optimal stoichiometry for the hydrogen-bonded complexes, and the association constant (Ka) that was determined using the 1H NMR-based Benesi-Hildebrand treatment was 84 M(-1) for the low molar mass complementary polymers. Furthermore, the PDLLA-based hydrogen-bonded complexes exhibited higher solution viscosities compared to the corresponding non-hydrogen-bonded precursors, which further confirmed strong complementary multiple-hydrogen-bonding associations between the star-shaped polymers with terminal adenine and thymine groups. Moreover, variable-temperature 1H NMR studies demonstrated the thermoreversibility of the hydrogen-bonded PDLLA-based complexes in solution.