Rates of Chemical Cleavage of DNA and RNA Oligomers Containing Guanine Oxidation Products

Abstract

The nucleobase guanine in DNA (dG) and RNA (rG) has the lowest standard reduction potential of the bases, rendering it a major site of oxidative damage in these polymers. Mapping the sites at which oxidation occurs in an oligomer via chemical reagents utilizes hot piperidine for cleaving oxidized DNA and aniline (pH 4.5) for cleaving oxidized RNA. In the present studies, a series of time-dependent cleavages of DNA and RNA strands containing various guanine lesions were examined to determine the strand scission rate constants. The guanine base lesions 8-oxo-7,8-dihydroguanine (OG), spiroiminodihydantoin (Sp), 5-guanidinohydantoin (Gh), 2,2,4-triamino-2H-oxazol-5-one (Z), and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) were evaluated in piperidine-treated DNA and aniline-treated RNA. These data identified wide variability in the chemical lability of the lesions studied in both DNA and RNA. Further, the rate constants for cleaving lesions in RNA were generally found to be significantly smaller than for lesions in DNA. The OG nucleotides were poorly cleaved in DNA and RNA; Sp nucleotides were slowly cleaved in DNA and did not cleave significantly in RNA; Gh and Z nucleotides cleaved in both DNA and RNA at intermediate rates; and 2Ih oligonucleotides cleaved relatively quickly in both DNA and RNA. The data are compared and contrasted with respect to future experimental design.

pH-Dependent Equilibrium between 5-Guanidinohydantoin and Iminoallantoin Affects Nucleotide Insertion Opposite the DNA Lesion

“…NMR studies were not conducted at pH values >10.5 due to concern about instability of the glycosidic bond leading to free bases in solution that would complicate the results. 48 …”

Biophysical properties, thermal stability and functional impact of 8-oxo-7,8-dihydroguanine on oligonucleotides of RNA—a study of duplex, hairpins and the aptamer for preQ1as models

“…The lesion rendered a higher stability and resistance compared to its DNA analogs (using piperidine), however, it remains to be seen if 8-oxoGua may survive for longer periods of time in cellular environments (32). …”

8-Oxo-7,8-dihydro-2′-deoxyguanosine and abasic site tandem lesions are oxidation prone yielding hydantoin products that strongly destabilize duplex DNA

“…The dsODN with the two dOGs was 5′- 32 P labeled and then oxidized followed by piperidine treatment under conditions that yield nearly quantitative cleavage of dSp and dGh and negligible cleavage at dOG. 54 The piperidine cleavage yields at the two sites were determined by denaturing polyacrylamide gel electrophoresis (PAGE) followed by quantification via storage-phosphor autoradiography. Further, the reactions were conducted under single-hit reaction conditions (<30% yield) to ensure only one reaction per strand occurred (ESI Fig.…”

“…The piperidine cleavage reactions were previously demonstrated to yield quantitative cleavage at dGh and the dSp isomers while not cleaving dOG. 54 Briefly, the dialyzed samples were lyophilized to dryness and resuspended in fresh 1 M piperidine in ddH 2 O with 200 mM β-mercaptoethanol. The solutions were heated at 90 °C for 2 h, after which the samples were lyophilized to remove the piperidine prior to PAGE analysis.…”