Effective molarity in a nucleic acid-controlled reaction

Abstract

Positioning of reactive functional groups within a DNA duplex can enable chemical reactions that otherwise would not occur to an appreciable extent. However, few studies have quantitatively defined the extent to which the enforced proximity of reaction partners in duplex DNA can favor chemical processes. Here, we measured substantial effective molarities (as high as 25 M) afforded by duplex DNA to a reaction involving interstrand cross-link formation between 2′-deoxyadenosine and a 2-deoxyribose abasic (Ap) site.

A New Cross-Link for an Old Cross-Linking Drug: The Nitrogen Mustard Anticancer Agent Mechlorethamine Generates Cross-Links Derived from Abasic Sites in Addition to the Expected Drug-Bridged Cross-Links

“…We also observed a “late-forming” cross-link band that became visible after 12 h and predominant after 48 h [4% yield (Figure )]. It seemed unlikely that this could be a drug-linked cross-link because the chloroethyl groups of HN2 in both DNA monoadducts [ 1 (Scheme )] and free HN2 hydrolyze rapidly ( t 1/2 = 1–30 min). ,− On the other hand, in light of our recent studies, − it was reasonable to consider that a buildup of Ap sites resulting from depurination of HN2 adducts ( 1–3 ) might permit the generation of Ap-derived cross-links. In the following sections, we provide evidence that the late-forming cross-link band seen in Figure A is a dA-Ap cross-link resulting from alkylation-induced generation of an Ap site at position 2 of duplex A (Figure ).…”

“…Toward this end, we conducted a series of experiments in which an authentic Ap site was introduced at position 1, 2, 6, or 8 in duplex A . The 5′- 32 P-labeled Ap-containing duplexes B–E were prepared as described in our previous work − by the action of uracil DNA glycosylase (UDG) on the corresponding 2′-deoxyuridine-containing oligodeoxynucleotide duplexes. , The formation of slowly migrating, Ap-derived cross-link bands was easily detected in each of the Ap-containing duplexes B–E (Figure ). The equilibrium yields of the cross-link generated in these duplexes varied substantially, with values of 5, 30, 14, and 2%, for duplexes B–E , respectively (Figure S1).…”

“…Previously characterized Ap-derived cross-links have been located at sequences in which an adenine residue directly opposes the Ap site. − In biological systems, Ap sites with opposing adenine residues arise via the enzymatic removal of misincorporated 2′-deoxyuridine by the base excision repair enzyme UDG . This may be a major source of Ap sites in eukaryotic cells .…”

“… ,,, We recently showed that Ap sites can forge DNA–DNA interstrand cross-links in some sequences via reaction of the Ap aldehyde residue with the exocyclic amino groups of nucleobases such as adenine and guanine on the opposing strand of the DNA duplex (Scheme ). − The cross-linking reactions considered here, involving “true” Ap sites, are distinct from those involving oxidized abasic sites. , Here, we show that Ap sites generated by the nitrogen mustard mechlorethamine (HN2) give rise to interstrand cross-links of a type not previously associated with this drug. We present gel electrophoretic data that are consistent with an early evolution of the expected drug-bridged cross-links 3 followed by the appearance of Ap-derived cross-links.…”

Interstrand cross-links arising from strand breaks at true abasic sites in duplex DNA

“…Catalano et al . previously proposed a product of this type to explain a band observed in the denaturing gel analysis of Ap-containing DNA duplexes ( 80 ) but information related to the structure, origin and properties of this cross-link have been lacking until now. In addition, cross-links of this type were proposed to explain high molecular weight impurities generated in the manufacture of therapeutic oligonucleotides, but again the structure, origin and properties of these cross-linked species were not characterized ( 81 ).…”