Synthesis and characterization of oligodeoxyribonucleotides containing a site-specifically incorporated N6-carboxymethyl-2'-deoxyadenosine or N4-carboxymethyl-2'-deoxycytidine

Nucleic Acids Research
Jianshuang Wang, Yinsheng Wang

Abstract

Humans are exposed to both endogenous and exogenous N-nitroso compounds (NOCs), and many NOCs can be metabolically activated to generate a highly reactive species, diazoacetate, which is capable of inducing carboxymethylation of nucleobases in DNA. Here we report, for the first time, the chemical syntheses of authentic N(6)-carboxymethyl-2'-deoxyadenosine (N(6)-CMdA) and N(4)-carboxymethyl-2'-deoxycytidine (N(4)-CMdC), liquid chromatography-ESI tandem MS confirmation of their formation in calf thymus DNA upon diazoacetate exposure, and the preparation of oligodeoxyribonucleotides containing a site-specifically incorporated N(6)-CMdA or N(4)-CMdC. Additionally, thermodynamic studies showed that the substitutions of a dA with N(6)-CMdA and dC with N(4)-CMdC in a 12-mer duplex increased Gibbs free energy for duplex formation at 25°C by 5.3 and 6.8 kcal/mol, respectively. Moreover, primer extension assay revealed that N(4)-CMdC was a stronger blockade to Klenow fragment-mediated primer extension than N(6)-CMdA. The polymerase displayed substantial frequency of misincorporation of dAMP opposite N(6)-CMdA and, to a lesser extent, misinsertion of dAMP and dTMP opposite N(4)-CMdC. The formation and the mutagenic potential of N(6)-CMdA ...Continue Reading

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Citations

Jul 26, 2013·Langmuir : the ACS Journal of Surfaces and Colloids·Ioannis A TrantakisShana J Sturla
Jun 22, 2012·PloS One·Adyasha BharatiSurendra Chandra Sabat
May 23, 2017·Nucleic Acids Research·Jun WuYinsheng Wang
Jan 19, 2019·The Journal of Biological Chemistry·Pengcheng WangYinsheng Wang

Related Concepts

Potassium diazoacetate
Calf thymus DNA
N6-carboxymethyl-2'-deoxyadenosine
N4-carboxymethyl-2'-deoxycytidine
Azo Compounds
Deoxyadenosines
Deoxycytidine
DNA, Double-Stranded
DNA Replication
Glycine, Sodium Hydrogen Carbonate

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