A general scheme for the synthesis of C8-arylpurine phosphoramidites has been developed. C8-Arylation of C8-bromo-2′-deoxyguanosine is the key step and has been achieved through the use of a Suzuki coupling. Since the coupling reaction is conducted under aqueous conditions, it is unnecessary to protect and then deprotect the hydroxyl groups, thus saving several steps and improving overall yields. Once the C8-arylgroup is introduced, the glycosidic bond becomes very sensitive to acid catalyzed cleavage. Protection of the amino groups as the corresponding N,N-dimethylformamidine derivative improves stability of the derivatives. Synthetic C8-arylpurines were successfully used to prepare synthetic oligonucleotides.
Z-DNA is the only DNA conformation that has a left-handed helical twist. Although Z-DNA has been implicated in both carcinogenesis and mutagenesis, its specific biological role remains uncertain. We have demonstrated that the formation of C8-arylguanine DNA adducts, derived from arylhydrazines, shifts the B/Z-DNA equilibrium toward the Z-DNA conformation in d(CG)5 sequences. However, our previous work examined the effect of two adducts in the duplex, and it was unclear whether the two base modifications were working together to cause the equilibrium shift toward the Z-DNA conformation. Here we report the synthesis and characterization of a hairpin oligonucleotide sequence (d(CG)5T4(CG)5) containing only one C8-arylguanine modified base. The unmodified hairpin and the previously studied unmodified double-stranded oligonucleotide were conformationally similar, and each required ∼3 M NaCl to yield a B-/Z-DNA ratio of 1:1. The introduction of a single C8-arylguanine modification significantly reduced the NaCl concentration needed to produce a 1:1 B-/Z-DNA ratio in the hairpin. Further, the addition of MgCl2 and spermine to the C8-arylguanine-modified hairpin shifts the B/Z-DNA equilibrium such that the Z form predominated under physiological conditions. NMR and molecular modeling indicated the conformational effects produced by the C8-arylguanine modification occurred locally at the site of modification while CD data demonstrated that the C8-arylguanine-modified base destabilized the B form. Additionally, our data show that adopting the Z-DNA conformation is preferred over denaturation to the single-stranded form. Finally, the conformational effects of the C8-arylguanine modifications were not additive and the introduction of any such modifications drive Z-DNA formation under physiological conditions, which may provide a novel carcinogenesis mechanism where DNA adducts confer their carcinogenicity through a Z-DNA-mediated mechanism.
Base modifications are known to affect the structure and function of DNA. C8-guanine adducts from various carcinogenic compounds have been shown to be potent Z-DNA inducers. Hence, it has been hypothesized that Z-DNA plays a role in cancer and other genetic diseases. In this comprehensive review, Z-DNA and the effect of prevalent C8-guanine adducts on the B-Z transition are addressed. The discoveries of Z-DNA binding proteins including ADAR1, E3L, DLM1, and PKZ have suggested the relevance of Z-DNA in living systems. In addition, increasing evidence on the Z-DNA connection to gene transcription and inhibition reveals potential biological functions of the left-handed DNA. Finally, C8-guanine adducts that promote Z-DNA formation can be used as a tool to explore the Z-DNA function and its role in carcinogenesis.
C8-Aryl purines, their nucleosides, and phosphoramidites has been synthetic targets for more than 60 years. Interest in these compounds stems from their utility as fluorescent markers, they have therapeutic uses, are biomarkers, biomolecular probes, supramolecular building blocks, and for conformational studies. Until recently, the selective arylation of the C8-position of purines has been a challenging task. Several approaches have been explored including building them up from a pyrimidine or selective C8-modification of an unsubstituted purine. Neither of these approaches has proven to have broad scope. The discovery that C8-aryl purine nucleosides can be made via the Suzuki cross-coupling reaction has allowed a diverse array of analogues to be prepared and, in turn, the corresponding phosphoramidites. The latter is particularly significant as C8-aryl purine adducts are a major mutation observed from aromatic carcinogens and ready access to C8-aryl phosphoramidites will facilitate the synthesis and study of C8-aryl purine biomarkers and modified oligonucleotides.
Methylation of cytosine has been known to play a significant role in epigenetic regulation. 5-Methylcytosine was among the first base modification that was discovered for the capability to facilitate B/Z-DNA transition as observed in CG repeated tracks. A study on gene repression by Z-DNA prone sequence as in ADAM-12 has ignited our research interest for the Z-DNA role in epigenetics. Ten eleven translocation family proteins are responsible to catalyze 5-methylcytosine to produce oxidative products including 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine, which each may have unique function rather than the sole purpose of 5-methylcytosine clearance. Although the Z-DNA-promoting effect of 5-methylcytosine was well established, the effect of its oxidative products on Z-DNA remain unknown. In this study, the Z-DNA-promoting effect of 5-hydroxymethylcytosine, 5-formylcytosine, and 5carboxycytosine on the CG decamer model were investigated along with known Z-DNA stabilizers, 5-methylcytosine and 8oxoguanine. Experimental results from circular dichroism (CD) and NMR indicates that all oxidative products of 5-methylcytosine hinder B/Z-DNA transition as high salt concentration suitable to stabilize and convert unmodified CG decamer to Z-DNA conformation is insufficient to facilitate the B/Z-DNA transition of CG decamer containing 5-hydroxymethylcytosine, 5formylcytosine, or 5-carboxycytosine. Molecular dynamic simulation and free energy calculation by MM-PBSA are in agreement with the experimental finding that 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine destabilize Z-DNA conformation of CG decamer, in contrast to its precursor. Investigation of Z-DNA switch-on/switch-off regulated by 5-methylcytosine and its oxidative products is a further step to elucidate the potential of epigenetic regulated via Z-DNA.
Attempting to elucidate biological significance of the left-handed Z-DNA is a research challenge due to Z-DNA potential role in many diseases. Discovery of Z-DNA binding proteins has ignited the interest in search for Z-DNA functions. Biosensor with Z-DNA forming probe can be useful to study the interaction between Z-DNA conformation and Z-DNA binding proteins. In this study, 5-methylcytosine (C) containing CG decamers were characterized for their suitability to form Z-DNA and to be used in Z-DNA forming probe. The 5'-thiol oligonucleotide embedded with 5'-CGCGCGCG CG-3' was designed and developed as a potential Z-DNA forming probe for Z-DNA binding protein screening.
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