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The reaction of HO• radical with DNA is intensively studied both mechanistically and analytically for lesions formation. Several aspects related to the reaction paths of purine moieties with the formation of 5',8-cyclopurines (cPu), 8-oxopurines (8-oxo-Pu), and their relationship are not well understood. In this study, we investigated the reaction of HO• radical with a 21-mer double-stranded oligodeoxynucleotide (ds-ODNs) in γ-irradiated aqueous solutions under various oxygen concentrations and accurately quantified the six purine lesions (i.e., four cPu and two 8-oxo-Pu) by LC-MS/MS analysis using isotopomeric internal standards. In the absence of oxygen, 8-oxo-Pu lesions are only ∼4 times more than cPu lesions. By increasing oxygen concentration, the 8-oxo-Pu and the cPu gradually increase and decrease, respectively, reaching a gap of ∼130 times at 2.01 × 10-4 M of O2. Kinetic treatment of the data allows to estimate the C5' radical competition between cyclization and oxygen trapping in ds-ODNs, and lastly the rate constants of the four cyclization steps. Tailored computational studies by means of dispersion-corrected DFT calculations were performed on the CGC and TAT in their double-strand models for each cPu diastereoisomer along with the complete reaction pathways of the cyclization steps. Our findings reveal unheralded reaction mechanisms that resolve the long-standing issues with C5' radical cyclization in purine moieties of DNA sequences.

Cockayne Syndrome (CS) is an autosomal recessive neurodegenerative premature aging disorder associated with defects in nucleotide excision repair (NER). Cells from CS patients, with mutations in

Despite the widespread use of silver nanoparticles (AgNPs) in different fields and the amount of investigations available, to date, there are many contradictory results on their potential toxicity. In the present study, extensively characterized 20-nm AgNPs were investigated using optimized protocols and standardized methods to test several toxicological endpoints in different cell lines. The agglomeration/aggregation state of AgNPs in culture media was measured by dynamic light scattering (DLS). DNA and chromosomal damage on BEAS-2B and RAW 264.7 cells were evaluated by comet and micronucleus assays, while oxidative DNA damage by modified comet assay and 8-oxodG/8-oxodA detection. We also investigated immunotoxicity and immunomodulation by cytokine release and NO production in RAW 264.7 and MH-S cells, with or without lipopolysaccharide (LPS) stimulus. Transmission electron microscope (TEM) analysis was used to analyze cellular uptake of AgNPs. Our results indicate different values of AgNPs hydrodynamic diameter depending on the medium, some genotoxic effect just on BEAS-2B and no or slight effects on function of RAW 264.7 and MH-S in absence or presence of LPS stimulus. This study highlights the relevance of using optimized protocols and multiple endpoints to analyze the potential toxicity of AgNPs and to obtain reliable and comparable results.

Xeroderma Pigmentosum (XP) is a DNA repair disease characterized by nucleotide excision repair (NER) malfunction, leading to photosensitivity and increased incidence of skin malignancies. The role of XP-A in NER pathways has been well studied while discrepancies associated with ROS levels and the role of radical species between normal and deficient XPA cell lines have been observed. Using liquid chromatography tandem mass spectrometry we have determined the four 5&rsquo, 8-cyclopurines (cPu) lesions (i.e., 5&prime, R-cdG, 5&prime, S-cdG, 5&prime, R-cdA and 5&prime, S-cdA), 8-oxo-dA and 8-oxo-dG in wt (EUE-pBD650) and XPA-deficient (EUE-siXPA) human embryonic epithelial cell lines, under different oxygen tension (hyperoxic 21%, physioxic 5% and hypoxic 1%). The levels of Fe and Cu were also measured. The main findings of our study were: (i) the total amount of cPu (1.82&ndash, 2.52 lesions/106 nucleotides) is the same order of magnitude as 8-oxo-Pu (3.10&ndash, 4.11 lesions/106 nucleotides) in both cell types, (ii) the four cPu levels are similar in hyperoxic and physioxic conditions for both wt and deficient cell lines, whereas 8-oxo-Pu increases in all cases, (iii) both wt and deficient cell lines accumulated high levels of cPu under hypoxic compared to physioxic conditions, whereas the 8-oxo-Pu levels show an opposite trend, (iv) the diastereoisomeric ratios 5&prime, R/5&prime, S are independent of oxygen concentration being 0.29 for cdG and 2.69 for cdA for EUE-pBD650 (wt) and 0.32 for cdG and 2.94 for cdA for EUE-siXPA (deficient), (v) in deficient cell lines Fe levels were significantly higher. The data show for the first time the connection of oxygen concentration in cells with different DNA repair ability and the levels of different DNA lesions highlighting the significance of cPu. Membrane lipidomic data at 21% O2 indicated differences in the fatty acid contents between wild type and deficient cells, envisaging functional effects on membranes associated with the different repair capabilities, to be further investigated.

The reaction of hydroxyl radical (HO&bull, ) with DNA produces many primary reactive species and many lesions as final products. In this study, we have examined the optical spectra of intermediate species derived from the reaction of HO&bull, with a variety of single- and double-stranded oligodeoxynucleotides and ct-DNA in the range of 1 &mu, s to 1 ms by pulse radiolysis using an Intensified Charged Coupled Device (ICCD) camera. Moreover, we applied our published analytical protocol based on an LC-MS/MS system with isotopomeric internal standards to enable accurate and precise measurements of purine lesion formation. In particular, the simultaneous measurement of the four purine 5&prime, 8-cyclo-2&prime, deoxynucleosides (cPu) and two 8-oxo-7,8-dihydro-2&prime, deoxypurine (8-oxo-Pu) was obtained upon reaction of genetic material with HO&bull, radicals generated either by &gamma, radiolysis or Fenton-type reactions. Our results contributed to the debate in the literature regarding absolute level of lesions, method of HO&bull, radical generation, 5&prime, R/5&prime, S diastereomeric ratio in cPu, and relative abundance between cPu and 8-oxo-Pu.

Purine 5′,8-cyclo-2′-deoxynucleosides (cPu) are tandem-type lesions observed among the DNA purine modifications and identified in mammalian cellular DNA in vivo. These lesions can be present in two diasteroisomeric forms, 5′R and 5′S, for each 2′-deoxyadenosine and 2′-deoxyguanosine moiety. They are generated exclusively by hydroxyl radical attack to 2′-deoxyribose units generating C5′ radicals, followed by cyclization with the C8 position of the purine base. This review describes the main recent achievements in the preparation of the cPu molecular library for analytical and DNA synthesis applications for the studies of the enzymatic recognition and repair mechanisms, their impact on transcription and genetic instability, quantitative determination of the levels of lesions in various types of cells and animal model systems, and relationships between the levels of lesions and human health, disease, and aging, as well as the defining of the detection limits and quantification protocols.

5&rsquo, 8-Cyclo-2&rsquo, deoxyadenosine (cdA), in the 5&rsquo, R and 5&rsquo, Sdiastereomeric forms, are typical non strand-break oxidative DNA lesions, induced by hydroxyl radicals, with emerging importance as a molecular marker. These lesions are exclusively repaired by the nucleotide excision repair (NER) mechanism with a low efficiency, thus readily accumulating in the genome. Poly(ADP-ribose) polymerase1 (PARP1) acts as an early responder to DNA damage and plays a key role as a nick sensor in the maintenance of the integrity of the genome by recognizing nicked DNA. So far, it was unknown whether the two diastereomeric cdA lesions could induce specific PARP1 binding. Here, we provide the first evidence of PARP1 to selectively recognize the diastereomeric lesions of 5&rsquo, S-cdA and 5&rsquo, R-cdA in vitro as compared to deoxyadenosine in model DNA substrates (23-mers) by using circular dichroism, fluorescence spectroscopy, immunoblotting analysis, and gel mobility shift assay. Several features of the recognition of the damaged and undamaged oligonucleotides by PARP1 were characterized. Remarkably, PARP1 exhibits different affinities in binding to a double strand (ds) oligonucleotide, which incorporates cdA lesions in R and S diastereomeric form. In particular, PARP1 proved to bind oligonucleotides, including a 5&rsquo, S-cdA, with a higher affinity constant for the 5&rsquo, S lesion in a model of ds DNA than 5&rsquo, R-cdA, showing different recognition patterns, also compared with undamaged dA. This new finding highlights the ability of PARP1 to recognize and differentiate the distorted DNA backbone in a biomimetic system caused by different diastereomeric forms of a cdA lesion.

The non-bulky 5’,8-cyclopurine DNA lesions (cP) and the bulky, benzo[a]pyrene diol epoxide-derived stereoisomeric cis and trans-N(2)-guanine adducts (BPDE-dG) are good substrates of the human nucleotide excision repair (NER) mechanism. These DNA lesions were embedded at the In or Out rotational settings near the dyad axis in nucleosome core particles reconstituted either with native histones extracted from HeLa cells (HeLa-NCP), or with recombinant histones (Rec-NCP). The cP lesions are completely resistant to NER in human HeLa cell extracts. The BPDE-dG adducts are also NER-resistant in Rec-NCPs, but are good substrates of NER in HeLa NCPs. The four BPDE-dG adduct samples are excised with different efficiencies in free DNA, but in HeLa-NCPs the efficiencies are reduced by a common factor of 2.2 ± 0.2 relative to the NER efficiencies in free DNA. The NER response of the BPDE-dG adducts in HeLa-NCPs is not directly correlated with the observed differences in the thermodynamic destabilization of HeLa NCPs, the Förster Resonance Energy Transfer (FRET) values, or hydroxyl radical footprint patterns, and are weakly dependent on the rotational settings. These and other observations suggest that NER is initiated by the binding of the DNA damage-sensing NER factor XPC-RAD23B to a transiently opened BPDE-modified DNA sequence that corresponds to the known footprint of XPC-DNA-RAD23B complexes ( ≥ 30 base pairs). These observations are consistent with the hypothesis that post-translation modifications and the dimensions and properties of the DNA lesions are the major factors that have an impact on the dynamics and initiation of NER in nucleosomes.

The use of copper complexes for redox and oxidative-based mechanisms in therapeutic strategies is an important field of multidisciplinary research. Here, a novel Cu(II) complex [Cu(TPMA)(Phen)](ClO4)2 (Cu-TPMA-Phen, where TPMA = tris-(2-pyridylmethyl)amine and Phen = 1,10-phenanthroline) was studied using both the free and encapsulated forms. A hollow pH-sensitive drug-delivery system was synthesized, characterized, and used to encapsulate and release the copper complex, thus allowing for the comparison with the free drug. The human neuroblastoma-derived cell line NB100 was treated with 5 ?M Cu-PMA-Phen for 24 h, pointing to the consequences on mono- and polyunsaturated fatty acids (MUFA and PUFA) present in the membrane lipidome, coupled with cell viability and death pathways (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium viability assay, flow cytometry, microscopy, caspase activation). In parallel, the Cu-TPMA-Phen reactivity with the fatty acid moieties of phospholipids was studied using the liposome model to work in a biomimetic environment. The main results concerned: (i) the membrane lipidome in treated cells, involving remodeling with a specific increase of saturated fatty acids (SFAs) and a decrease of MUFA, but not PUFA; (ii) cytotoxic events and lipidome changes did not occur for the encapsulated Cu-TPMA-Phen, showing the influence of such nanocarriers on drug activity; and (iii) the liposome behavior confirmed that MUFA and PUFA fatty acid moieties in membranes are not affected by oxidative and isomerization reactions, proving the different reactivities of thiyl radicals generated from amphiphilic and hydrophilic thiols and Cu-TPMA-Phen. This study gives preliminary but important elements of copper(II) complex reactivity in cellular and biomimetic models, pointing mainly to the effects on membrane reactivity and remodeling based on the balance between SFA and MUFA in cell membranes that are subjects of strong interest for chemotherapeutic activities as well as connected to nutritional strategies.

L' Alternanza Scuola Lavoro è diventata obbligatoria con l'approvazione della legge n. 107/2017 nota come "La Buona Scuola". Gli Istituti di Ricerca Pubblici possono essere dei partner educativi della scuola nella realizzazione di progetti/percorsi di ASL. L'offerta di moduli formativi per ASL da parte degli Istituti di Ricerca si motiva con l'obiettivo di accrescere l'interesse dei giovani allo studio delle discipline scientifiche, mentre acquisiscono nuove competenze lavorative. D'altra parte queste attività formative/lavorative possono essere viste come attività di divulgazione delle nuove scoperte scientifiche verso la cittadinanza da parte degli Istituti di Ricerca. Nella presentazione saranno relazionati i progetti/percorsi educativi svolti nell'anno scolastico 2016-2017, presso L'Istituto di Cristallografia (sede di Monterotondo), dagli studenti degli Istituti Scolastici delle provincie di Roma e Rieti. A dimostrazione delle nuove competenze acquisite dagli studenti, che hanno frequentato questi percorsi, saranno mostrati alcuni dei prodotti didattici da loro realizzati. Tra questi prodotti didattici la mostra multimediale "Il Lato Rosa della Scienza" presentata alla Notte della Scienza 2017 presso l'Area della Ricerca di Roma 2 (Tor Vergata). Gli studenti delle classi III e IV del Liceo Scientifico G.Peano durante il periodo di ASL presso IC hanno realizzato la mostra con gli strumenti di scrittura collaborativa accademica. Per lo svolgimento dei percorsi formativi si sono utilizzate le nuove tecnologie di internet, note con il nome web 2.0, sulla piattaforma informatica per la formazione dell'Istituto di Cristallografia. Il software utilizzato è quello adottato da molte università, Moodle, ma la configurazione hardware dei server è stata realizzata e modificata dal gruppo Smart ELab dell'IC per ottenere alte prestazioni e alta affidabilità.

Epigenetic modifications, reversibly controlling gene expression, are crucial for interpreting the genome under the influence of physiological factors. The basic processes (DNA methylation and histone modification, DNA accessibility, and chromatin structure) which characterize epigenetics are regulated by an orchestrated series of events. The spectrum of these events is becoming wider and wider, increasing the complexity of the interplay between the basic processes and the multiple side mechanisms responsive to environmental stimuli. The "histone code hypothesis" suggests that combinations of different histone modifications may regulate chromatin structure and transcriptional activity. Among these modifications, a crucial role is played by poly(ADP-ribosyl)ation, the reaction catalyzed by poly(ADP-ribose)polymerase-1 widely recognized as a "genome guardian" for driving the repair of damaged DNA. Increasing evidence indicates also that alterations in membrane phospholipid composition and lipid metabolism may play a role in epigenetics. Moreover, a "lipid code" has been proposed since in the nucleus a lipid fraction is present that seems tightly bound to DNA. This review will analyze these topics and their possible interplay in epigenetic regulation and discuss the relative role of nutritional and environmental challenges

Fatty acids, as structural components of membranes and inflammation/anti-inflammatory mediators, have well-known protective and regulatory effects. They are studied as biomarkers of pathological conditions, as well as saturated and unsaturated hydrophobic moieties in membrane phospholipids that contribute to homeostasis and physiological functions. Lifestyle, nutrition, metabolism and stress—with an excess of radical and oxidative processes—cause fatty acid changes that are examined in the human body using blood lipids. Fatty acid-based membrane lipidomics represents a powerful diagnostic tool for assessing the quantity and quality of fatty acid constituents and also for the follow-up of the membrane fatty acid remodeling that is associated with different physiological and pathological conditions. This review focuses on fatty acid biomarkers with two examples of recent lipidomic research and health applications: (i) monounsaturated fatty acids and the analytical challenge offered by hexadecenoic fatty acids (C16:1); and (ii) the cohort of 10 fatty acids in phospholipids of red blood cell membranes and its connections to metabolic and nutritional status in healthy and diseased subjects.

Epigenetic modifications, reversibly controlling gene expression, are crucial for interpreting the genome under the influence of physiological factors. The basic processes (DNA methylation and histone modification, DNA accessibility and chromatin structure) which characterize epigenetics, are regulated by an orchestrated series of events. The spectrum of these events is becoming wider and wider, increasing the complexity of the interplay between the basic processes and the multiple side mechanisms responsive to environmental stimuli. The ‘Histone Code Hypothesis’ suggests that combinations of different histone modifications may regulate chromatin structure and transcriptional activity. Among these modifications a crucial role is played by Poly(ADP-ribosyl)ation, the reaction catalyzed by Poly(ADP-ribose)polymerase-1 widely recognized as a “genome guardian” for driving the repair of damaged DNA. Increasing evidence indicates also that alterations in membrane phospholipid composition and lipid metabolism may play a role in epigenetics. Moreover, a ‘lipid code’ has been proposed since in the nucleus a lipid fraction is present that seems tightly bound to DNA. This review will analyze these topics, their possible interplay in epigenetic regulation, and discuss the relative role of nutritional and environmental challenges.

The hydroxyl radical is a powerful oxidant that generates DNA lesions including the stereoisomeric R and S 5?,8-cyclo-2?-deoxyadenosine (cdA) and 5?,8-cyclo-2?-deoxyguanosine (cdG) pairs that have been detected in cellular DNA. Unlike some other oxidatively generated DNA lesions, cdG and cdA are repaired by the human nucleotide excision repair (NER) apparatus. The relative NER efficiencies of all four cyclopurines were measured and compared in identical human HeLa cell extracts for the first time under identical conditions, using identical sequence contexts. The cdA and cdG lesions were excised with similar efficiencies, but the efficiencies for both 5?R cyclopurines were greater by a factor of ~2 than for the 5?S lesions. Molecular modeling and dynamics simulations have revealed structural and energetic origins of this difference in NER-incision efficiencies. These lesions cause greater DNA backbone distortions and dynamics relative to unmodified DNA in 5?R than in 5?S stereoisomers, producing greater impairment in van der Waals stacking interaction energies in the 5?R cases. The locally impaired stacking interaction energies correlate with relative NER incision efficiencies, and explain these results on a structural basis in terms of differences in dynamic perturbations of the DNA backbone imposed by the R and S covalent 5?,8 bonds. © 2014 © The Author(s) 2014. Published by Oxford University Press.

We report the synthesis and fibrillogenesis inhibiting activity of the new peptide derivatives 1–4, related to the pentapeptide Ac-LPFFD-NH2 (iAβ5p), proposed by Soto and co-workers and widely recognized as one of the most active β-sheet breaker agents. The Aβ25–35 fragment of the parent full-length Aβ1–42 was used as fibrillogenesis model. The activity of peptide derivatives 1–4 was tested in vitro by thioflavin T binding assay, far UV CD spectroscopy, and scanning electron microscopy. Their ability to hinder the toxic effect of Aβ25–35in vivo was studied by monitoring the viability of human SH-SY5Y neuroblastoma cells and the prevention of superoxide anion radical release from BV2 microglial cells. The results point to a favourable role in the fibrillogenesis inhibitory activity of the sulphonamide junction for compounds 1 and 2, containing an N,N-dimethyltaurine and a taurine amino-terminal moiety, respectively. Furthermore, compounds 1 and 2 show a significant protective effect on cell viability, rescuing the cells from the toxicity exerted by Aβ25–35 treatment.

Combining various techniques in solution we proved that Doxorubicin, also called Adriamycin, and Sabarubicin, also known as MEN 10755, bind to the human telomeric sequence, 5'-d[GGG(TTAGGG)(3)]-3' (21-mer), assuming a G-quadruplex structure in the presence of K(+). Complexes of drugs with the 21-mer in 1 : 1 and 2 : 1 stoichiometry coexist in solution. Association constants were obtained from titration experiments and confirmed by isothermal titration calorimetry. The fluorescence of the drugs was quenched upon complexation. UV circular dichroism (CD) spectra of the complexes were characterized by the G-quadruplex signal and indicated that drug binding influences the equilibrium between quadruplex conformations. The visible CD spectra were exclusively due to the drug and show differences in the complexation modes of the two drugs. Spectroscopic and thermodynamic parameters of the 1 : 1 complexes point to drug stacking with the G-quadruplex top or bottom tetrad. Thermodynamic data suggests that the binding of the second drug molecule in the 2 : 1 complex may occur in a groove. Complexation caused a small increase in the thermal stability of the G-quadruplex main conformation, shifting T(m) from 62 to 67 °C.

NH 4 [NiGa 1.84 In 0.16 (PO 4 ) 3 (H 2 O) 2 ] orthophosphate was prepared through hydrothermal synthesis and analyzed by means of single crystal X-ray diffractometry. It was solved within C2/c monoclinic space group; a = 13.2483(8) A, b = 10.2468(8) A, c = 8.9033(7) A, β = 108.637(1)°, V = 1145.27(15) A 3 . Phosphorous atoms display tetrahedral arrangement, occupying two distinct sites; Ni atoms form NiO 4 (OH 2 ) 2 distorted octahedra, while Ga and In atoms share the same site forming (Ga,In)O 5 units with trigonal bipyramidal geometry. (Ga,In)O 5 , NiO 4 (OH 2 ) 2 and P0 4 units are linked together through edge- and vertex-sharing arrangements, making up the three-dimensional framework, completed by (NH 4 ) + counter-ions. Strong O—H ... O and N―H...O hydrogen bonds contribute to stabilize the array: O―H...O interactions were also investigated by means of Libowitzky formula (Libowitzky, 1999) application, owing to the results of IR spectroscopy.

5',8-Cyclopurine-2'-deoxynucleosides including 5',8-cyclo-dA (cdA) and 5',8-cyclo-dG (cdG) are induced by hydroxyl radicals resulting from oxidative stress such as ionizing radiation. 5',8-cyclopurine-2'-deoxynucleoside lesions are repaired by nucleotide excision repair with low efficiency, thereby leading to their accumulation in the human genome and lesion bypass by DNA polymerases during DNA replication and base excision repair (BER). In this study, for the first time, we discovered that DNA polymerase β (pol β) efficiently bypassed a 5'R-cdA, but inefficiently bypassed a 5'S-cdA during DNA replication and BER. We found that cell extracts from pol β wild-type mouse embryonic fibroblasts exhibited significant DNA synthesis activity in bypassing a cdA lesion located in replication and BER intermediates. However, pol β knock-out cell extracts exhibited little DNA synthesis to bypass the lesion. This indicates that pol β plays an important role in bypassing a cdA lesion during DNA replication and BER. Furthermore, we demonstrated that pol β inserted both a correct and incorrect nucleotide to bypass a cdA at a low concentration. Nucleotide misinsertion was significantly stimulated by a high concentration of pol β, indicating a mutagenic effect induced by pol β lesion bypass synthesis of a 5',8-cyclopurine-2'-deoxynucleoside. Moreover, we found that bypass of a 5'S-cdA by pol β generated an intermediate that failed to be extended by pol β, resulting in accumulation of single-strand DNA breaks. Our study provides the first evidence that pol β plays an important role in bypassing a 5',8-cyclo-dA during DNA replication and repair, as well as new insight into mutagenic effects and genome instability resulting from pol β bypassing of a cdA lesion.

Diastereomeric 5',8-cyclo-2'-deoxypurines: brief overview of synthetic strategies, modelling and in vitro biological activity. 5?,8-cyclo-2?-deoxypurines (cdPus) are typical DNA lesions resulting from endogenous and environmental free radical stress. The interest in these lesions is connected with the mechanism of their formation due to the HOo attack at the H5? atom of the 2-deoxyribose moiety, followed by intramolecular cyclization between C5?-C8 bond and subsequent oxidation of the resulting N7-radical.[1,2] Two diastereomeric cdPus are formed in the 5?R and 5?S forms (Fig.1). The two diastereomeric forms are repaired by nucleotide excision repair (NER) with different efficiency, the 5?R isomer being 2 times more efficiently repaired than the 5?S isomer. Molecular dynamics simulation elucidated that 5?R diastereoisomeric forms cause greater DNA backbone distortions than the 5?S diastereomers, thus theoretically supporting a different efficiency of NER[3] mechanism. We recently discovered that DNA polymerase ? (pol ?) has different behavior with 5?R-cdA lesion (efficiently bypassed) than 5?S-cdA (inefficiently bypassed) during DNA replication and base excision repair (BER),[4,5] highlighting that the nature of the DNA lesion can play a crucial role in biological processes. The diastereoisomeric 5?S- and 5?R-cdPus lesions are discussed in terms of differences in: i.Synthetic strategy and automated synthesis efficiency. ii.Physical-chemical properties (MD simulations, NMR, Melting Temperature) iii.Biological Activity in vitro References [1] Chatgilialoglu, C.;Ferreri, C.; Terzidis, M.A. Chem.Soc.Rev. 2011, 40, 1153. [2]Boussicault, F.; Kaloudis, P.; Caminal, C.; Mulazzani, Q. G.; Chatgilialoglu C. J. Am Chem. Soc. 2008, 130, 8377. [3]Kropachev, K.; Ding, S.; Terzidis, M.A.; Masi, A.; Liu, Z.; Cai, Y.; Kolbanovskiy, M.; Chatgilialoglu, C.; Broyde, S.; Nicholas E. Geacintov, N.E.; Shafirovich, V. Nucleic Acids Research, 2014, 42, 5020. [4]Xu, M.; Lai, Y.; Jiang, Z.; Terzidis, M.A.; Masi, A.; Chatgilialoglu, C.; Liu, Y. Nucleic Acids Research, 2014. 42,13749 [5]Jiang, Z.; Xu, M.; Lai, Y.; Laverde, E.E.; Terzidis, M.A.; Masi, A.; Chatgilialoglu, C.; Liu, Y. DNA Repair, 2015, 33, 24.

Differential pulse voltammetries of guanine-rich single- and double-stranded oligonucleotides containing up to six consecutive guanines are reported. The observed progressive lowering of the first voltammetric peak potential as the number of adjacent guanines increases unambiguously points toward the establishment of delocalized hole domains; the hole stabilization energy is ca. 0.1 eV per GG step, significantly lower than that observed for AA steps.

The association of 5′,8-cyclo-2′-deoxyguanosine (cdG), a DNA tandem lesion, with its complementary base cytosine has been studied by voltammetry and NMR in chloroform, using properly silylated derivatives of the two nucleobases for increasing their solubilities. Both voltammetric data and NMR titrations indicated that the Watson-Crick complex of cytidine with cdG is weaker than that with guanosine, the difference being approximately of one order of magnitude between the two association constants.

5',8-cyclo-2'-deoxypurines (cdPus) are common forms of oxidized DNA lesions resulting from endogenous and environmental oxidative stress such as ionizing radiation. The lesions can only be repaired by nucleotide excision repair with a low efficiency. This results in their accumulation in the genome that leads to stalling of the replication DNA polymerases and poor lesion bypass by translesion DNA polymerases. Trinucleotide repeats (TNRs) consist of tandem repeats of Gs and As and therefore are hotspots of cdPus. In this study, we provided the first evidence that both (5'R)- and (5'S)-5',8-cyclo-2'-deoxyadenosine (cdA) in a CAG repeat tract caused CTG repeat deletion exclusively during DNA lagging strand maturation and base excision repair. We found that a cdA induced the formation of a CAG loop in the template strand, which was skipped over by DNA polymerase beta (pol beta) lesion bypass synthesis. This subsequently resulted in the formation of a long flap that was efficiently cleaved by flap endonuclease 1, thereby leading to repeat deletion. Our study indicates that accumulation of cdPus in the human genome can lead to TNR instability via a unique lesion bypass by pol beta.

A short, efficient and high-yielding approach for the diastereoselective syntheses of the four (5′S)- and (5′R)-5′,8-cyclopurine lesions and their phosphoramidites has been developed. The intermediates prepared by these synthetic pathways provide an easy access to those modified nucleosides that constitute an important molecular library for recognition of DNA damage. With respect to previous synthesis, the key methodologies for cyclization steps and phosphoramidite synthesis were ameliorated.

We investigated with spectroscopic techniques the noncovalent interaction of a bimetallic water-soluble (Zn(II)/Pt(II)) porphyrazine hexacation, [(PtCl(2))(CH(3))(6)LZn](6+), and its octacationic analogue [(CH(3))(8)LZn](8+), lacking the cis-platin-like functionality, with a 21-mer double strand (ds) 5'-d[GGG(TTAGGG)(3)]-3'/3'-d[CCC(AATCCC)(3)]-5', as model for B-DNA. Both hexacation and octacation tend to aggregate in water. The structure as well as the ground and excited-state electronic properties of the Zn(II)/Pt(II) hexacation [(PtCl(2))(CH(3))(6)LZn](6+) in water solution were investigated using density functional theory (DFT) and time-dependent DFT (TDDFT) methods. TDDFT calculations of the lowest excited states of [(PtCl(2))(CH(3))(6)LZn](6+) in water provided an accurate description of the Q-band spectral region. In particular, the calculated optical spectra were in agreement with the experimental ones, obtained in the presence of micelles favoring complete disruption of the aggregates. The model for dsDNA binding that emerges from the analysis of UV-vis absorption and time-resolved fluorescence data shows the presence of complexes of 1 dsDNA molecule with 1, 2, and 4 macrocycles. Comparing the results for the hexacation [(PtCl(2))(CH(3))(6)LZn](6+) with those for the [(CH(3))(8)LZn](8+)octacation, we observed a higher degree of monomerization for the [(PtCl(2))(CH(3))(6)LZn](6+) derivative.

The biological consequences of free radical production is the central subject of a very lively scientific debate, focusing on the estimation of the type and extent of damage, as well as the efficiency of the protective and repair systems. When studying free radical based chemical mechanisms, it is very important to establish biomimetic models, which allow the experiments to be performed in a simplified environment, but suitably designed to be in strict connection with cellular conditions. The biomimetic modeling approach has been coupled with physical organic chemistry methodologies and knowledge of free radical reactivity. Molecular basis of important processes have been identified, building up molecular libraries of products concerning unsaturated lipids, sulfur-containing proteins and nucleic acids, to be developed as biomarkers. Ongoing projects in our group deal with lipidomics, genomics and proteomics of free radical stress and some examples will be described.

A water soluble Zn(II) porphyrazine drives the conformational equilibrium of the G-quadruplex of a human telomeric sequence exclusively towards a parallel conformation upon complexation.

The opioid agonists endomorphins (Tyr-Pro-Trp-Phe-NH(2); EM1 and Tyr-Pro-Phe-Phe-NH(2); EM2) and morphiceptin (Tyr-Pro-Phe-Pro-NH(2)) exhibit an extremely high selectivity for mu-opioid receptor. Here a series of novel EM2 and morphiceptin analogues containing in place of the proline at position 2 the S and R residues of beta-homologues of proline (HPro), of 2-pyrrolidinemethanesulphonic acid (HPrs) and of 3-pyrrolidinesulphonic acid (betaPrs) have been synthesized and their binding affinity and functional activity have been investigated. The highest micro-receptor affinity is shown by [(S)betaPrs(2)]EM2 analogue (6e) which represents the first example of a beta-sulphonamido analogue in the field of opioid peptides.

Peptide derivatives 1 – 5 , incorporating synthetic non-proteinogenic amino acids, related to the β-amyloid 17–21 fragment of the amyloidogenic Aβ 1–40 , and the N -protected decapeptide 6 , corresponding to a dimeric sequence of the same fragment, have been synthesized. These compounds were designed by using Soto's pentapeptide Ac–LPFFD–NH 2 (iAβ5p) as lead compound. Their activity as inhibitors of fibrillogenesis and stability against enzymatic degradation have been determined. Compounds 1 , 5 and 6 are potent inhibitors in comparison to the lead compound. Exposure to chymotrypsin of peptide derivatives 1 – 5 , all containing unnatural amino acids, shows increased stability as compared with iAβ5p and 6 . Conformational properties of the new compounds have been determined by CD and FT-IR spectroscopies.

For-Met-betaAlapsi[CSNH]-Phe-OMe (3), For-Met-betaAlapsi[CH2NH]-Phe-OMe (5), For-Met-NH-pC6H4-SO(2-Phe-OMe 8a), For-Met-NH-mCH4-SO2-Phe-OMe (8b) and the corresponding N-Boc precursors (2, 4, 7a, b) have been synthesized and their activity towards human neutrophils has been evaluated in comparison with that shown by the reference tripeptide For-Met-Leu-Phe-OMe (fMLF-OMe). Chemotaxis, lysozyme release and superoxide anion production have been measured. (1)H NMR titration experiments and IR spectra have been discussed in order to ascertain the preferred solution conformation adopted by the tripeptide 3 with particular reference to the presence of a folded conformation centred at the centrally positioned thionated beta-residue.

In order to gain information on the activity shown by alpha-peptide/beta-sulfonamidopeptide hybrid analogs of the potent chemotactic agent fMLF-OMe, a structure-activity study is reported on N-Boc- and N-formyl tripeptide models containing an aminoalkanesulfonic acid as central residue. Directed migration (chemotaxis), superoxide anion production, and lysozyme release have been measured. The biochemical functions and the conformational properties of the new compounds are discussed and related to previously studied models containing beta-residues.