Final Report Summary - CYCLOGUO (Formation and roles of the 5',8-cyclo-2'-deoxyguanosine lesion)
DNA is the repository of genetic information in each living cell, and its integrity and stability are essential to life. DNA can react under oxidative and free radical conditions which are generated during aerobic metabolism or under different environmental conditions (UV light, smoke, etc…), and chemical studies of DNA reactivity is of fundamental importance since it has been estimated that an individual cell can suffer up to one million DNA changes per day, endangering the above mentioned integrity and stability. The hydroxyl radical OH● represents one of the most dangerous species that alter biomolecules, involved in several biological damages and ageing, and in the last decades was thoroughly studied in its reactivity with DNA. This reactivity can cause changes of the four fundamental nucleotide units (adenosine, guanosine, thymine, citosine) and chemical studies have contributed to the understanding of the DNA damages, as well as of the array of enzymatic systems involved in DNA repair.
The research topic of DNA damage and repair is of outmost importance in medicine, since the alteration of DNA and the impairment of the repair systems will lead to mutation and possibly diseases.
The CycloGuo project addressed specifically one of the DNA lesions known as tandem damage of the purine nucleobases, 2’-deoxyadenosine and 2’-deoxyguanosine, and in Figure 1 the lesions of purine 5',8-cyclonucleosides (5',8-cPus) are shown as couple of diastereoisomeric compounds.
The CycloGuo project intended to give a chemical contribution for the investigation of the cyclopurine lesions, solving the problems in the synthesis of the four diastereomeric structures (see in Figure 1). This is an important issue since the availability of the four lesions is necessary in order to conduct subsequent studies on their characterization, in terms of structural, functional and biological properties. Moreover, the incorporation of these modified structures as phosphoroamidite analogues in oligonucleotide sequences can simulate the lesions occurring on the DNA strands, and this biomimetic study is preliminary for the understanding of the potential consequences in vivo.
The CYCLOGUO project was dealing with the synthesis of the 5',8-cPus and their phosphoramidites in particular deriving from 2'-deoxyguanosine. In 2013, we published a new synthetic protocol for the 5',8-cyclo-dGuo lesions (…lit….) (Figure 1) and particularly giving for the first time an access for the (5'S)- diastereoisomer. The new methodology was completed with the synthesis of 5',8-cyclo-dGuo phosphoramidites (Figure 2).
The two adenine 5',8-cPu phosphoramidites were also synthesised and incorporated into oligonucleotides strands allowing the comparison of the final overall yields taking into account also the DNA solid phase synthesis steps for each lesion. Together with improvements in the overall yield of 5'R 5',8-cyclo-dGuo phosphoramidite the latter results have just been submitted for publication (…lit….). The biological consequences given by the cyclopurine lesions have been the subject of several investigations in an interdisciplinary context, and will be reported in scientific papers that are in the writing process. The article published in Nucleic Acids Research journal, fruit of collaboration with Prof. N. Geancintov’s and Prof. S. Broyde’s groups at the New York University, showed the enzymatic repair by NER for the four 5',8-cPus were measured in comparison with identical human HeLa cell extracts using identical sequence contexts. The purine base, adenine or guanine, was found not to have influence on the efficiency of the repair. However, the geometry of the phosphate group linked to the C5' carbon showed high influence, with the 5'R lesions being ~2 times more efficiently repaired than the 5'S. In silico studies have revealed structural and energetic origins of this difference in NER-incision efficiencies. The backbone distortion and dynamics in modified DNA are caused by the existence of the C5'−C8 bond in the lesions and greater impairment in van der Waals stacking interaction energies is produced in the 5'R cases. This work contains part of the data we have collected on the NER efficiency and the other part is expected to be published very soon.
The 5’,8-cPus compounds have also been developed as DNA oxidative stress biomarkers in comparison with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) which is the most used biomarker, although suffers from oxidative artefacts during the assays. As part of the CYCLOGUO project, we have synthesised the isotopic enriched- 15N equivalents of the four 5’,8-cPus, used for development and validation of an analytical protocol for the evaluation of DNA samples based on the stable isotope dilution tandem mass spectrometry technique. The synthetic methodologies for the preparation of the standards are described in the Journal of Visualised Experiments. Crucial work has been carried out for the enzymatic digestion conditions, that could liberate quantitatively the lesions at nucleoside level, and this protocol is now the best available in terms of efficiency and cost per analysis. The examination of biological samples and the significance of these analyses will be soon published.
The structural characteristics if the 5’,8-cPus compounds have been studied for applications as advanced materials, in particular with the 5'S diastereoisomer of 5’,8-cdG in G-quadruplex assembly, using NMR experiments and computational modelling to support the observed properties, and these results have been submitted for publication.
The research topic of DNA damage and repair is of outmost importance in medicine, since the alteration of DNA and the impairment of the repair systems will lead to mutation and possibly diseases.
The CycloGuo project addressed specifically one of the DNA lesions known as tandem damage of the purine nucleobases, 2’-deoxyadenosine and 2’-deoxyguanosine, and in Figure 1 the lesions of purine 5',8-cyclonucleosides (5',8-cPus) are shown as couple of diastereoisomeric compounds.
The CycloGuo project intended to give a chemical contribution for the investigation of the cyclopurine lesions, solving the problems in the synthesis of the four diastereomeric structures (see in Figure 1). This is an important issue since the availability of the four lesions is necessary in order to conduct subsequent studies on their characterization, in terms of structural, functional and biological properties. Moreover, the incorporation of these modified structures as phosphoroamidite analogues in oligonucleotide sequences can simulate the lesions occurring on the DNA strands, and this biomimetic study is preliminary for the understanding of the potential consequences in vivo.
The CYCLOGUO project was dealing with the synthesis of the 5',8-cPus and their phosphoramidites in particular deriving from 2'-deoxyguanosine. In 2013, we published a new synthetic protocol for the 5',8-cyclo-dGuo lesions (…lit….) (Figure 1) and particularly giving for the first time an access for the (5'S)- diastereoisomer. The new methodology was completed with the synthesis of 5',8-cyclo-dGuo phosphoramidites (Figure 2).
The two adenine 5',8-cPu phosphoramidites were also synthesised and incorporated into oligonucleotides strands allowing the comparison of the final overall yields taking into account also the DNA solid phase synthesis steps for each lesion. Together with improvements in the overall yield of 5'R 5',8-cyclo-dGuo phosphoramidite the latter results have just been submitted for publication (…lit….). The biological consequences given by the cyclopurine lesions have been the subject of several investigations in an interdisciplinary context, and will be reported in scientific papers that are in the writing process. The article published in Nucleic Acids Research journal, fruit of collaboration with Prof. N. Geancintov’s and Prof. S. Broyde’s groups at the New York University, showed the enzymatic repair by NER for the four 5',8-cPus were measured in comparison with identical human HeLa cell extracts using identical sequence contexts. The purine base, adenine or guanine, was found not to have influence on the efficiency of the repair. However, the geometry of the phosphate group linked to the C5' carbon showed high influence, with the 5'R lesions being ~2 times more efficiently repaired than the 5'S. In silico studies have revealed structural and energetic origins of this difference in NER-incision efficiencies. The backbone distortion and dynamics in modified DNA are caused by the existence of the C5'−C8 bond in the lesions and greater impairment in van der Waals stacking interaction energies is produced in the 5'R cases. This work contains part of the data we have collected on the NER efficiency and the other part is expected to be published very soon.
The 5’,8-cPus compounds have also been developed as DNA oxidative stress biomarkers in comparison with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) which is the most used biomarker, although suffers from oxidative artefacts during the assays. As part of the CYCLOGUO project, we have synthesised the isotopic enriched- 15N equivalents of the four 5’,8-cPus, used for development and validation of an analytical protocol for the evaluation of DNA samples based on the stable isotope dilution tandem mass spectrometry technique. The synthetic methodologies for the preparation of the standards are described in the Journal of Visualised Experiments. Crucial work has been carried out for the enzymatic digestion conditions, that could liberate quantitatively the lesions at nucleoside level, and this protocol is now the best available in terms of efficiency and cost per analysis. The examination of biological samples and the significance of these analyses will be soon published.
The structural characteristics if the 5’,8-cPus compounds have been studied for applications as advanced materials, in particular with the 5'S diastereoisomer of 5’,8-cdG in G-quadruplex assembly, using NMR experiments and computational modelling to support the observed properties, and these results have been submitted for publication.