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Biochemistry. 1993 Aug 17;32(32):8276-83.

Synthesis and damage specificity of a novel probe for the detection of abasic sites in DNA.

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  • 1Department of Polymer Science and Engineering, Kyoto Institute of Technology, Japan.

Abstract

The abasic site (apurinic/apyrimidinic site) is the most common lesion in DNA and is suggested to be an important intermediate in mutagenesis and carcinogenesis. We have recently reported a novel assay for the detection and quantitation of abasic sites in DNA [Kubo, K., Ide, H., Wallace, S. S., & Kow, Y. W. (1992) Biochemistry 31, 3703-3708]. In this assay, the aldehyde group in an abasic site is first modified by a probe bearing a biotin residue, called the Aldehyde Reactive Probe (ARP) and then the tagged biotin is quantified by an ELISA-like assay. However, in the previous study, ARP was prepared only in a crude form, and no solid chemical data concerning the structure and specificity of ARP were reported. In this study, an improved method for the preparative synthesis of ARP has been established, and its structure has been unambiguously characterized using spectroscopic means. In order to elucidate the specificity of ARP to DNA damages, ARP was incubated with a variety of damaged bases or nucleosides and the reaction mixtures were analyzed by HPLC. Of the 14 compounds tested for their reactivity to ARP, 2-deoxyribose (a model compound for an abasic site) and 5-formyluracil reacted with ARP. Interestingly, compounds bearing a formamide group such as formamidopyrimidine and deoxyribosylformamide did not react with ARP, indicating that ARP is specific to damages having an alkyl or allyl aldehyde group. Furthermore, the ability of ARP synthesized by the defined chemical route to detect abasic sites has been substantiated using natural DNA containing abasic sites. Potential applications and limitations of the ARP assay are discussed.

PMID:
8347625
[PubMed - indexed for MEDLINE]
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