C8-Arylguanine Modified Hairpin Oligonucleotides: Synthesis, Conformational Analysis, and Tools for the Investigation of Z-DNA Formation

Z-DNA is the only DNA conformation that has a left-handed helical twist. Z-DNA has been implicated in carcinogenesis from its role in gene expression. Z-DNA formation has been linked to mutagenesis since it may play a role in genetic instability and can induce gene deletions. However, the specific biological role for Z-DNA in vivo remains uncertain. Arylhydrazines are carcinogenic compounds that are known to form DNA adducts, although it is unknown how DNA adduct formation is related to their carcinogenicity. Previous work in our lab has 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 sought to determine the conformational effects of a hairpin oligonucleotide sequence (d(CG) 5T4(CG)5) containing only one C8-arylguanine modified base to compare with our previous results.;Synthesis of C8-arylguanine modified hairpin oligonucleotides began at the nucleoside level and then the C8-aryl-2'-deoxyguanosine phosphoramidite was incorporated into a hairpin sequence (d(5'CGCGCG*CGCGTTTTCGCGCGCGCG3') where G*= C8-arylguanine). The conformational effects of a single C8-arylguanine modification on the hairpin oligonucleotide were determined and quantitated by a combination of NMR, CD and molecular modeling. 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, the 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.

Name thesis:degree_name

PhD

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Pharmaceutical Sciences

Year dc:date.available

2014

Author dc:creator

• Train, Brian C.

Contributors dc:contributor

• Peter M. Gannett
• Patrick S. Callery
• Robert K. Griffith
• Yongyut Rojanasakul
• X. Michael Shi

• Pharmaceutical sciences

Identifier

https://researchrepository.wvu.edu/etd/592

OAI identifier oai:identifier

oai:researchrepository.wvu.edu:etd-1595