A Simple, High‐Yield Synthesis of DNA Duplexes Containing a Covalent, Thermally Cleavable Interstrand Cross‐Link at a Defined Location

Abstract

Interstrand DNA-DNA cross-links are highly toxic to cells because these lesions block the extraction of information from the genetic material. The pathways by which cells repair cross-links are important, but not well understood. The preparation of chemically well-defined cross-linked DNA substrates represents a significant challenge in the study of cross-link repair. Here we report a simple method that employs “post-synthetic” modifications of commercially available 2’-deoxyoligonucleotides to install a single cross-link in high yield at a specified location within a DNA duplex. The cross-linking process exploits hydrazone formation between a non-natural N4-amino-2’-deoxycytidine nucleobase and the aldehyde residue of an abasic site in duplex DNA. The resulting cross-link is stable under physiological conditions, but can be readily dissociated and re-formed through heating-cooling cycles.

A role for the base excision repair enzyme NEIL3 in replication-dependent repair of interstrand DNA cross-links derived from psoralen and abasic sites

“…In addition, chemical biologists can help address the roles of other BER glycosylases and ALKB family enzymes in the replication-independent unhooking of interstrand cross-links. For instance, methods are in place for the preparation of duplex DNA substrates housing a site-specific and structurally defined interstrand cross-link [17, 110, 146, 162–174]. This will enable shuttle vector-based methods, in conjunction with genetic manipulation, for interrogating the roles of BER glycosylases and ALKB family proteins in the removal of interstrand cross-link lesions in cells [164, 175].…”

Effective molarity in a nucleic acid-controlled reaction

“…41 The Ap-containing, 5′- 32 P-labeled duplexes A and B were generated as described previously 40–44 by the action of the enzyme uracil DNA glycosylase on the corresponding 2′-deoxyuridine-containing duplexes. 45–47 Subsequent time-dependent conversion of the resulting Ap-containing duplexes to cross-linked DNA was monitored by the appearance of a characteristic 17,40–44 slow-moving band on denaturing polyacrylamide gels (Figs. 1 and S1).…”

Simple, High‐Yield Syntheses of DNA Duplexes Containing Interstrand DNA‐DNA Cross‐Links Between an N4‐Aminocytidine Residue and an Abasic Site

“…dU residues near the end of oligodeoxynucleotides are less effective substrates for UDG (Varshney and van de Sande, 1991). The Ap-dC* cross-link in duplex DNA is thermally reversible (Gamboa Varela and Gates, 2015). That is, the cross-link can be broken by heating the duplex to its melting point, but the cross-link is then spontaneously regenerated upon cooling and rehybridization.…”

“…This protocol describes the preparation of an oligodeoxynucleotide 2 (Figure 4) containing a single N 4 -aminocytidine ( dC* ) residue (Gamboa Varela and Gates, 2015; Gao and Orgel, 1999; Negishi et al, 1987). The procedure involves “post-synthetic modification” of a commercially available oligodeoxynucleotide 1 containing a single dC residue with bisulfite and hydrazine (Figure 1).…”

“…It is important that the incubation time does not exceed 5 h because unreacted Ap-site undergoes cleavage and a low molecular weight cross-link begins to form (see Supporting Information of (Gamboa Varela and Gates, 2015)). The cross-linked DNA duplex 5 can be stored in solution at −20 °C for 1 d. The cross-linked DNA also can be ethanol precipitated and stored dry at −20 °C for up to 3 d. Radiolytic degradation of the DNA can occur if the 5’- 32 P-labeled material is stored for long periods of time.…”