Deoxyribose Moiety Research Articles

A nontargeted screening method for covalent DNA adducts and DNA modification selectivity using liquid chromatography-tandem mass spectrometry

A method for nontargeted screening for covalent DNA adducts was developed using combination of neutral loss scan and product ion scan in a hybrid linear-ion-trap - triple quadrupole mass spectrometer system. DNA 2′-deoxynucleosides and adducts eluted from liquid chromatography were first analyzed in neutral loss mode to screen for the neutral loss of the deoxyribose moiety ([M+H-116]+) from the protonated molecular ion ([M+H]+). The product ion scan was subsequently used to elucidate the structures for the molecular ions observed from the peaks in the neutral loss scan chromatogram. The synthesized DNA adducts were used to evaluate the developed method by reaction of 20-mer DNA oligonucleotide with two direct agents respectively, specifically phenyl glycidyl ether and styrene-7,8-oxide. The modification selectivity of two compounds to the four nitrogenous bases on DNA sequence was also investigated in this study. The results showed that the two compounds had different modification selectivity to the four bases. Both compounds could modify all four nitrogenous bases (i.e. adenine, guanine, thymine, and cytosine) on DNA sequences to form various covalent DNA adducts. While phenyl glycidyl ether modified almost all of thymidine on DNA sequence, styrene-7,8-oxide, on the other hand, modified only a small portion of thymidine. The developed method proved possibly a potential tool for screening of unknown DNA adducts as exposure biomarkers of contaminants to human in the environment.

Long-Range Vibrational Dynamics Are Directed by Watson-Crick Base Pairing in Duplex DNA.

Ultrafast two-dimensional infrared (2D-IR) spectroscopy of a 15-mer A-T DNA duplex in solution has revealed structure-dependent vibrational coupling and energy transfer processes linking bases with the sugar-phosphate backbone. Duplex melting induces significant changes in the positions of off-diagonal peaks linking carbonyl and ring-stretching vibrational modes of the adenine and thymine bases with vibrations of the phosphate group and phosphodiester linkage. These indicate that Watson-Crick hydrogen bonding and helix formation lead to a unique vibrational coupling arrangement of base vibrational modes with those of the phosphate unit. On the basis of observations from time-resolved 2D-IR data, we conclude that rapid energy transfer processes occur between base and backbone, mediated by additional modes located on the deoxyribose moiety within the same nucleotide. These relaxation dynamics are insensitive to duplex melting, showing that efficient intramolecular energy relaxation to the solvent via the phosphate groups is the key to excess energy dissipation in both single- and double-stranded DNA.

Screening for DNA Alkylation Mono and Cross-Linked Adducts with a Comprehensive LC-MS(3) Adductomic Approach.

A high-resolution/accurate-mass DNA adductomic approach was developed to investigate anticipated and unknown DNA adducts induced by DNA alkylating agents in biological samples. Two new features were added to a previously developed approach to significantly broaden its scope, versatility, and selectivity. First, the neutral loss of a base (guanine, adenine, thymine, or cytosine) was added to the original methodology's neutral loss of the 2'-deoxyribose moiety to allow for the detection of all DNA base adducts. Second, targeted detection of anticipated DNA adducts based on the reactivity of the DNA alkylating agent was demonstrated by inclusion of an ion mass list for data dependent triggering of MS(2) fragmentation events and subsequent MS(3) fragmentation. Additionally, untargeted screening of the samples, based on triggering of an MS(2) fragmentation event for the most intense ions of the full scan, was included for detecting unknown DNA adducts. The approach was tested by screening for DNA mono and cross-linked adducts in purified DNA and in DNA extracted from cells treated with PR104A, an experimental DNA alkylating nitrogen mustard prodrug currently under investigation for the treatment of leukemia. The results revealed the ability of this new DNA adductomic approach to detect anticipated and unknown PR104A-induced mono and cross-linked DNA adducts in biological samples. This methodology is expected to be a powerful tool for screening for DNA adducts induced by endogenous or exogenous exposures.

ChemInform Abstract: Selenium‐Mediated Dehalogenation of Halogenated Nucleosides and Its Relevance to the DNA Repair Pathway.

Halogenated nucleosides can be incorporated into the newly synthesized DNA of replicating cells and therefore are commonly used in the detection of proliferating cells in living tissues. Dehalogenation of these modified nucleosides is one of the key pathways involved in DNA repair mediated by the uracil-DNA glycosylase. Herein, we report the first example of a selenium-mediated dehalogenation of halogenated nucleosides. We also show that the mechanism for the debromination is remarkably different from that of deiodination and that the presence of a ribose or deoxyribose moiety in the nucleosides facilitates the deiodination. The results described herein should help in understanding the metabolism of halogenated nucleosides in DNA and RNA.

Selenium‐Mediated Dehalogenation of Halogenated Nucleosides and its Relevance to the DNA Repair Pathway

AbstractHalogenated nucleosides can be incorporated into the newly synthesized DNA of replicating cells and therefore are commonly used in the detection of proliferating cells in living tissues. Dehalogenation of these modified nucleosides is one of the key pathways involved in DNA repair mediated by the uracil‐DNA glycosylase. Herein, we report the first example of a selenium‐mediated dehalogenation of halogenated nucleosides. We also show that the mechanism for the debromination is remarkably different from that of deiodination and that the presence of a ribose or deoxyribose moiety in the nucleosides facilitates the deiodination. The results described herein should help in understanding the metabolism of halogenated nucleosides in DNA and RNA.

Selenium-Mediated Dehalogenation of Halogenated Nucleosides and its Relevance to the DNA Repair Pathway.

Halogenated nucleosides can be incorporated into the newly synthesized DNA of replicating cells and therefore are commonly used in the detection of proliferating cells in living tissues. Dehalogenation of these modified nucleosides is one of the key pathways involved in DNA repair mediated by the uracil-DNA glycosylase. Herein, we report the first example of a selenium-mediated dehalogenation of halogenated nucleosides. We also show that the mechanism for the debromination is remarkably different from that of deiodination and that the presence of a ribose or deoxyribose moiety in the nucleosides facilitates the deiodination. The results described herein should help in understanding the metabolism of halogenated nucleosides in DNA and RNA.

High-resolution crystal structure of Z-DNA in complex with Cr(3+) cations.

This work is part of our project aimed at characterizing metal-binding properties of left-handed Z-DNA helices. The three Cr3+ cations found in the asymmetric unit of the d(CGCGCG)2–Cr3+ crystal structure do not form direct coordination bonds with atoms of the Z-DNA molecule. Instead, the hydrated Cr3+ ions are engaged in outer-sphere interactions with phosphate groups and O6 and N7 guanine atoms of the DNA. The Cr3+(1) and Cr3+(2) ions have disordered coordination spheres occupied by six water molecules each. These partial-occupancy chromium cations are 2.354(15) Å apart and are bridged by three water molecules from their hydration spheres. The Cr3+(3) cation has distorted square pyramidal geometry. In addition to the high degree of disorder of the DNA backbone, alternate conformations are also observed for the deoxyribose and base moieties of the G2 nucleotide. Our work illuminates the question of conformational flexibility of Z-DNA and its interaction mode with transition-metal cations.Electronic supplementary materialThe online version of this article (doi:10.1007/s00775-015-1247-5) contains supplementary material, which is available to authorized users.

Remarkable effects of solvent and substitution on the photo-dynamics of cytosine: a femtosecond broadband time-resolved fluorescence and transient absorption study.

Cytosine (Cyt) among all the nucleic acid bases features the most complex and least understood nonradiative deactivation, a process that is crucially important for its photostability. Herein, the excited state dynamics of Cyt and a series of its N1- and C5-derivatives, including the full set of Cyt nucleosides and nucleotides in DNA and RNA and the nucleosides of 5-methyl cytosine, 5-methylcytidine and 2'-deoxy-5-methylcytidine, have been investigated in water and in methanol employing femtosecond broadband time-resolved fluorescence coupled with fs transient absorption spectroscopy. The results reveal remarkable state-specific effects of the substitution and solvent in tuning distinctively the timescales and pathways of the nonradiative decays. For Cyt and the N1-derivatives, the nonradiative deactivations occur in a common two-state process through three channels, two from the light-absorbing ππ* state with respectively the sub-picosecond (∼0.2 ps) and the picosecond (∼1.5 ps) time constant, and the third is due to an optically dark nπ* state with the lifetime ranging from several to hundreds of picoseconds depending on solvents and substitutions. Compared to Cyt, the presence of the ribose or deoxyribose moiety at the N1 position of N1-derivatives facilitates the formation of the nπ* at the sub-picosecond timescale and at the same time increases its lifetime by ∼4-6 times in both water and methanol. In sharp contrast, the existence of the methyl group at the C5 position of the C5-derivatives eliminates completely the sub-picosecond ππ* channel and the channel due to the nπ*, but on the other hand slows down the decay of the ππ* state which after relaxation exhibits a single time constant of ∼4.1 to ∼7.6 ps depending on solvents. Varying the solvent from water to methanol accelerates only slightly the decay of the ππ* state in all the compounds; while for Cyt and its N1-derivatives, this change of solvent also retards strongly the nπ* channel, prolongs its lifetime from such as ∼7.7 ps in water to ∼52 ps in methanol for Cyt and from ∼30 ps in water to ∼186 ps in methanol for deoxycytidine. The spectral signatures we obtained for the ππ* and the nπ* states allow unambiguous evidence for clarifying uncertainties in the excited states of Cyt and the derivatives. The results provide a unifying experimental characterization at an improved level of detail about the photophysics of Cyt and its analogues under biologically relevant conditions and may help in understanding the photostability as well as photo-damages of the bases and related DNAs.

Kinetic Measurement of Leukemia-Cell Proliferation Rate By Deuterium Labeling Predicts Time to Initial Treatment of Patients with Chronic Lymphocytic Leukemia

Background: Chronic lymphocytic leukemia (CLL) patients exhibit variable times from diagnosis to the need to initiate therapy. Previous studies have demonstrated that the rates of CLL cell proliferation (birth) and elimination (death plus recirculation to solid tissues) could be estimated using the incorporation of deuterium (2H), taken in the form of heavy water (2H2O), into the DNA of dividing cells. In a cross-sectional study of patients with early and late stage CLL, birth rates above 0.35% per day were associated with more aggressive disease. The NIH-sponsored CLL Research Consortium (CRC) conducted a prospective study on patients with early stage disease to assess the relationship between CLL-cell birth rate and the length of time to initiation of initial therapy.Methods: Eligible patients had untreated early stage (Rai 0, 1 or 2) disease, and were no more than 3 years from diagnosis. Additional clinical parameters included age at diagnosis, gender, mutational status of the immunoglobulin heavy chain variable region (IGHV), 70-kD zeta-associated protein (ZAP-70) (binary split at 20%) and CD38 (binary split at 30%) expression, and FISH cytogenetics by the Dohner hierarchy. Heavy water was administered daily for 6 weeks, and peripheral blood was obtained for kinetic measurements at the end of weeks 3, 6, 9, 12, and 16. CLL birth rates for blood samples were measured at the same facility (KineMed, Inc, Emeryville, CA) by quantifying deuterium incorporation into the deoxyribose moiety of DNA of tumor cells, using isotope ratio mass spectrometry (IRMS). Subjects were followed for time to administration of therapy; subjects still without treatment at time of analysis were censored, as were subjects who exited the study to receive treatment outside of the established criteria. To assess the impact of birth rate and other factors on time to first therapy, Kaplan-Meier curves were compared using the logrank test for univariable analyses; stepwise Cox proportional hazards regression models were used to build multivariable models.Results: Between July 2005 and February 2009, 119 subjects were enrolled through the CRC. 107 subjects were eligible. For 10 eligible patients, exit from the study prior to completion of protocol-mandated heavy water consumption (n=5) or technical issues (n=5) made it impossible to obtain estimates of birth rate. The 97 eligible subjects for whom birth rates were available form the basis of this report. 60% of subjects were male; median age 57 years (range 40-85). Median birth rate was 0.32%/day (range 0.07-1.31%/day). 43 subjects (44%) had birth rates higher than the previously reported threshold of 0.35%/day. With a median follow up of 4 years, 33 eligible subjects (34%) required treatment. IGHV status (31 subjects unmutated), elevated ZAP-70 (31 subjects), elevated CD38 (21 subjects), and hierarchical categorization of FISH anomalies (4 del(17p), 5 del(11q), 8 tri(12), 50 del(13q)) were examined for association with differential time to first therapy in univariable analyses. All were associated with earlier need for therapy (p <0.001) except for genomic aberrations defined by FISH (p = 0.96). Birth rate > 0.35%/day was also significantly associated with shorter time to first therapy (p < 0.001). In multivariable modeling, only birth rate and IGHV status met the criteria for inclusion (Wald p-value < 0.05). Hazard ratios were 3.51 (p = 0.002) for birth rate above 0.35%/day and 5.0 for unmutated IGHV status (p < 0.001). The association between elevated birth rate and shorter time to first treatment was observed within subjects with both unmutated and mutated IGHV (see Figure 1).Conclusion: An increased birth rate of CLL cells early in the disease course is a strong predictor of an earlier need for treatment in univariable analysis, as are unmutated IGHV, elevated ZAP-70 expression and elevated CD38 expression. However, only unmutated IGHV and an increased birth rate contribute significantly to the multivariable model. This adds a direct measure of B-cell kinetics to the array of biomarkers available to subjects with CLL. [Display omitted] DisclosuresMurphy:KineMed, Inc: Equity Ownership. Emson:KineMed, Inc.: Employment, Equity Ownership. Li:KineMed, Inc.: Employment, Equity Ownership. McConnel:KineMed, Inc.: Equity Ownership. Turner:KineMed, Inc.: Employment, Equity Ownership. Busch:KineMed, Inc.: Equity Ownership. Hellerstein:KineMed Inc: Consultancy, Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Chiorazzi:KineMed, Inc.: Stock options Other.

RADAMOL tool: Role of radiation quality and charge transfer in damage distribution along DNA oligomer

Theoretical modeling is a powerful tool to predict radiation damage of biomolecules such as DNA, proteins and more complex biological targets. In this paper, we present damage distributions along a DNA oligomer irradiated by 10 keV electrons, 1, 2, 5, 10 and 20 MeV protons and alpha particles predicted by the simulation tool RADAMOL. The scavengeable damage of base and deoxyribose moieties due to radical attack is more important in the studied system than unscavengeable ionizations of the DNA target and surrounding bound water layer. Radiation quality does not modify distribution of primary damages along the DNA, but changes overall damage yields. For the first time, electron and hole migration along the DNA macromolecule has been taken into account in the simulation procedure. The effect of these processes on distribution of DNA damages is demonstrated.

Cell-permeable dual inhibitors of protein kinases CK2 and PIM-1: structural features and pharmacological potential.

It has been proposed that dual inhibitors of protein kinases CK2 and PIM-1 are tools particularly valuable to induce apoptosis of cancer cells, a property, however, implying cell permeability, which is lacking in the case of selective CK2/PIM-1 inhibitors developed so far. To fill this gap, we have derivatized the scaffold of the promiscuous CK2 inhibitor TBI with a deoxyribose moiety, generating TDB, a selective, cell-permeable inhibitor of CK2 and PIM-1. Here, we shed light on the structural features underlying the potency and narrow selectivity of TDB by exploiting a number of TDB analogs and by solving the 3D structure of the TDB/CK2 complex at 1.25 Å resolution, one of the highest reported so far for this kinase. We also show that the cytotoxic efficacy of TDB is almost entirely due to apoptosis, is accompanied by parallel inhibition of cellular CK2 and PIM-1, and is superior to both those observed combining individual inhibitors of CK2 and PIM-1 and by treating cells with the CK2 inhibitor CX4945. These data, in conjunction with the observations that cancer cells are more susceptible than non-cancer cells to TDB and that such a sensitivity is maintained in a multi-drug resistance background, highlight the pharmacological potential of this compound.

Amentoflavone Protects against Hydroxyl Radical-induced DNA Damage via Antioxidant Mechanism

Objective: Oxidative DNA damage is involved in mutation, cell death, carcinogenesis, and aging. Since biflavonoid amentoflavone exhibited beneficial effects on DNA, we therefore investigated its protection against •OH-induced DNA oxidative damage then discussed the mechanism. It will enhance the understanding of interaction between biflavonoid vs DNA mediated by free radicals. Methods: The protective effect of amentoflavone against •OH-induced DNA damage was measured using our method. To explore the mechanism, it was further determined by •OH-induced bases damage, deoxyribose damage, and various antioxidant assays. Results: Amentoflavone increased dose-dependently its protective percentages against •OHinduced damage on DNA, bases, and deoxyribose; The IC50 were 31.85±4.75, 198.75±33.53, 147.14±20.95, 75.15±10.52, 93.75±16.36, 167.69±13.90, and 137.95±19.86 μM, respectively for DNA, cytosine, uracil, adenine, thymine, guanine, and deoxyribose damages. Radical-scavenging assays suggested that amentoflavone could effectively scavenge •O2 -, DPPH•, ABTS•+ radicals (IC50 values were respectively 8.98±0.23, 432.25±84.05, 7.25±0.35 μM). Conclusions: Based on the mechanistic analysis, it is concluded that amentoflavone can effectively protect against •OH-induced oxidative damage DNA (including base & deoxyribose moieties), via deoxynucleotide radical repairing, and reactive oxygen species (ROS) scavenging approaches which may be mediated by donating hydrogen atom (H•) and electron (e). Further analysis indicated that both scavenging and repairing approaches can be primarily attributed to its antioxidant mechanism which may ultimately arise from to the stability of its oxidized product semi-quinone form. Its protection against DNA damage may be generally responsible for the radioprotective and anti-inflammation effects.

Uncoupling of the apyrimidinic/apurinic endonucleolytic and 3′→5′ exonucleolytic activities of the Nfo protein of Mycoplasma pneumoniae through mutation of specific amino acid residues

The DNA recombination and repair machineries of Mycoplasma pneumoniae and Mycoplasma genitalium were predicted to consist of a set of ~11 proteins. The function of one of these proteins was inferred from its homology with proteins belonging to the Endo IV enzyme family. The members of this family function in the repair of apyrimidinic/apurinic (AP) sites in DNA. As such activity may be crucial in the mycoplasmal life cycle, we set out to study the Endo IV-like proteins encoded by M. pneumoniae and M. genitalium. Both proteins, termed NfoMpn and NfoMge, respectively, were assessed for their ability to interact with damaged and undamaged DNA. In the absence of divalent cations, both proteins exhibited specific cleavage of AP sites. Surprisingly, the proteins also recognized and cleaved cholesteryl-bound deoxyribose moieties in DNA, showing that these Nfo proteins may also function in repair of large DNA adducts. In the presence of Mg(2+), NfoMpn and NfoMge also showed 3'→5' exonucleolytic activity. By introduction of 13 single point mutations at highly conserved positions within NfoMpn, two major types of mutants could be distinguished: (i) mutants that showed no, or limited, AP cleavage activity in the presence of EDTA, but displayed significant levels of AP cleavage activity in the presence of Mg(2+); these mutants displayed no, or very low, exonucleolytic activity; and (ii) mutants that only demonstrated marginal levels of AP site cleavage activity in the presence of Mg(2+) and did not show exonucleolytic activity. Together, these results indicated that the AP endonucleolytic activity of the NfoMpn protein can be uncoupled from its 3'→5' exonucleolytic activity.

Identification of a 5'-Deoxyadenosine Deaminase in Methanocaldococcus jannaschii and Its Possible Role in Recycling the Radical S-Adenosylmethionine Enzyme Reaction Product 5'-Deoxyadenosine

We characterize here the MJ1541 gene product from Methanocaldococcus jannaschii, an enzyme that was annotated as a 5'-methylthioadenosine/S-adenosylhomocysteine deaminase (EC 3.5.4.31/3.5.4.28). The MJ1541 gene product catalyzes the conversion of 5'-deoxyadenosine to 5'-deoxyinosine as its major product but will also deaminate 5'-methylthioadenosine, S-adenosylhomocysteine, and adenosine to a small extent. On the basis of these findings, we are naming this new enzyme 5'-deoxyadenosine deaminase (DadD). The Km for 5'-deoxyadenosine was found to be 14.0 ± 1.2 μM with a kcat/Km of 9.1 × 10(9) M(-1) s(-1). Radical S-adenosylmethionine (SAM) enzymes account for nearly 2% of the M. jannaschii genome, where the major SAM derived products is 5'-deoxyadenosine. Since 5'-dA has been demonstrated to be an inhibitor of radical SAM enzymes; a pathway for removing this product must be present. We propose here that DadD is involved in the recycling of 5'-deoxyadenosine, whereupon the 5'-deoxyribose moiety of 5'-deoxyinosine is further metabolized to deoxyhexoses used for the biosynthesis of aromatic amino acids in methanogens.

Model of biological quantum logic in DNA.

The DNA molecule has properties that allow it to act as a quantum logic processor. It has been demonstrated that there is coherent conduction of electrons longitudinally along the DNA molecule through pi stacking interactions of the aromatic nucleotide bases, and it has also been demonstrated that electrons moving longitudinally along the DNA molecule are subject to a very efficient electron spin filtering effect as the helicity of the DNA molecule interacts with the spin of the electron. This means that, in DNA, electrons are coherently conducted along a very efficient spin filter. Coherent electron spin is held in a logically and thermodynamically reversible chiral symmetry between the C2-endo and C3-endo enantiomers of the deoxyribose moiety in each nucleotide, which enables each nucleotide to function as a quantum gate. The symmetry break that provides for quantum decision in the system is determined by the spin direction of an electron that has an orbital angular momentum that is sufficient to overcome the energy barrier of the double well potential separating the C2-endo and C3-endo enantiomers, and that enantiomeric energy barrier is appropriate to the Landauer limit of the energy necessary to randomize one bit of information.

Electron-Induced Degradation of 8-Bromo-2′-deoxyadenosine 3′,5′-Diphosphate, a DNA Radiosensitizing Nucleotide

The phosphodiester bond cleavage in 8-bromo-2'-deoxyadenosine 3',5'-diphosphate (BrdADP), as a model of electron induced single strand break (SSB) in labeled DNA, was investigated at the B3LYP/6-31++G(d,p) level of theory both in the gas phase and in water solution. Barrier-free and highly exergonic, especially in water solution (-2.83 eV), release of the bromide anion due to electron attachment confirms radiosensitizing properties of 8-bromoadenine. Thermodynamically (-19 kcal/mol) and kinetically (barrier of 10-13 kcal/mol) feasible hydrogen atom transfer from the C3' or C5' sites of the deoxyribose moiety to the C8 center of adenine radical is followed by a relatively low (14-18 kcal/mol) activation barrier O-P bond cleavage at either the 3'- or the 5'-site. The C5' radical may also stabilize via the formation of 5',8-cycloadenosine. The latter process has favorable thermodynamic and kinetic characteristics, which makes the O-P bond breakage at the 5'-site highly unlikely. Thus, the O-P cleavage reaction, being an equivalent of SSB in DNA labeled with 8-bromoadenine, should lead to the formation of cyclic ketone, which if identified in a radiolytic experiment, would confirm the mechanism proposed in the current study.

A New Method of the Visualization of the Double-Stranded Mitochondrial and Nuclear DNA

The study describes the method of a sensitive detection of double-stranded DNA molecules in situ. It is based on the oxidative attack on the deoxyribose moiety by copper(I) in the presence of oxygen. We have shown previously that the oxidative attack leads to the formation of frequent gaps in DNA. Here we have demonstrated that the gaps can be utilized as the origins for an efficient synthesis of complementary labeled strands by DNA polymerase I and that such enzymatic detection of the double-stranded DNA is a sensitive approach enabling in-situ detection of both the nuclear and mitochondrial genomes in formaldehyde-fixed human cells.

Fragmentation of protonated oligonucleotides by energetic photons and Cq+ions

The ionization and fragmentation of trapped protonated dGCAT oligonucleotides upon interaction with energetic photons ($h\ensuremath{\nu}=$ 10--570 eV) and keV C${}^{q+}$ ions was investigated by means of time-of-flight mass spectrometry. The observed fragmentation patterns are dominated by protonated and nonprotonated nucleobase ions and fragments of the deoxyribose moiety. Fragments exceeding the size of nucleosides are almost completely absent. Absorption of VUV photons as well as interaction with keV ions predominantly involves ionization or excitation of molecular valence electrons and accordingly the observed fragmentation patterns exhibit qualitatively similar features. Soft-x-ray-induced ionization of core level electrons accompanied by subsequent emission of an Auger electron shifts the fragment distributions towards smaller masses. This systematic study allows for insights into differences and similarities between ion- and photon-induced excitation and fragmentation mechanisms. In particular, the crucial role of the deoxyribose moiety for radiation-induced DNA damage that was predicted on the basis of gas-phase experiments using isolated deoxyribose molecules is confirmed.

Exploring the limits of nucleobase expansion: computational design of naphthohomologated (xx-) purines and comparison to the natural and xDNA purines

The properties of doubly-expanded (xx-) purine analogues are compared to the natural and singly-expanded (x-) purines using quantum chemical (B3LYP, MP2) methods. Purine expansion upon incorporation of a benzene or naphthalene spacer affects the preferred orientation of the base about the glycosidic bond in the corresponding nucleoside to a similar extent. Although the natural purines preferentially adopt the anti orientation with respect to the 2'-deoxyribose moiety, the syn and anti conformations are almost isoenergetic in the case of the expanded analogues. However, the anti/syn rotational barrier either remains similar to or is slightly elevated upon purine expansion, and therefore the expanded analogues will likely display similar rigidity with respect to rotation about the glycosidic bond as the natural purines. The A:T Watson-Crick hydrogen-bond strength is slightly enhanced, while the G:C interaction energy decreases slightly, upon incorporation of either expanded purine. As expected, the largest effect of base expansion occurs on the stacking energies. Specifically, the maximum (most negative) stacking energies in isolated purine dimers formed by aligning the nucleobase centers of mass can be increased by up to 34% by including a single x-purine and by up to 51% by including an xx-purine. Although increases in the purinepurine intrastrand stacking interactions are found even when a simplified duplex model composed of two stacked (hydrogen-bonded) base pairs is considered, the purinepyrimidine stacking energies decrease upon purine expansion. However, the total stability (sum of all hydrogen-bonding and stacking interactions) of natural duplex models is up to 10% and 22% greater for duplexes containing expanded x- and xx-purines respectively, which is mainly due to enhanced inter and intrastrand stacking interactions. Thus, unidirectional expansion in the size of the nucleobase spacer can continuously enhance the stability of expanded duplexes.

Atomic Scissors: A New Method of Tracking the 5-Bromo-2′-Deoxyuridine-Labeled DNA In Situ

A new method of the light microscopy detection of BrdU-labeled DNA in situ is described. It is based on the oxidative attack at the deoxyribose moiety by copper(I) in the presence of oxygen, which leads to the abstraction of hydrogen atom from deoxyribose culminating in the elimination of the nucleobase, scission of the nucleic-acid strand and formation of frequent gaps. The gaps allow the reaction of the antibodies with the commonly used markers of replication (e.g. 5-bromo-2′-deoxyuridine), which are otherwise masked. The method developed makes it possible to detect nuclear and mitochondrial DNA replication efficiently. In most cases, it does not inhibit effective protein detections and in addition enables simultaneous localization of newly-synthesized RNA. The alternative presently-used methods result in protein denaturation and/or extensive DNA cleavage followed by the DNA-bound proteins peeling off.