Synthesis of DNA Interstrand Cross‐Links Using a Photocaged Nucleobase

Abstract

The missing linking: BCNU is a chemotherapy drug that generates an ethylene bridge between N(1) of deoxyguanosine and N(3) of deoxycytidine. No synthesis of a DNA containing this moiety has been reported until now. A new strategy uses a photocaged nucleobase that, when released, generates a highly reactive intermediate which cross-links the opposing DNA strand in a manner analogous to BCNU.

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

“…This is usually unacceptable from a preparative perspective. Alternatively, elegant syntheses of structurally-defined cross-linked duplexes have been devised (Balkrishnen et al, 1996; Carrette et al, 2013; Gao and Orgel, 1999; Gruppi et al, 2014; Haque et al, 2014; Harwood et al, 2000; Hentshel et al, 2012; Hong et al, 2006; Manoharan et al, 1999; Mukherjee et al, 2014; Nakatani et al, 2002; Nishimoto et al, 2013; Noll et al, 2001; O'Flaherty et al, 2013; Pujari et al, 2014; Schärer, 2005; Tomás-Gamasa et al, 2014; Ye et al, 2013), but many of these multi-step organic reaction sequences may not be practical in many of the biochemical and materials science laboratories with interests in cross-linked DNA. In the work described here, we sought a simple, benchtop procedure for the synthesis of DNA duplexes containing a site-specific interstrand cross-link that employs inexpensive commercially available enzymes, chemicals, and oligodeoxynucleotides.…”

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

“…[3b,6] As a result, laborious purification is required to obtain duplexes containing a single, structurally-defined ICL. Alternatively, elegant multistep chemical syntheses of cross-linked duplexes have been developed, [7] but these methods may not be practicable in some biochemistry and biology laboratories where the study of cross-link repair takes place.…”

“…This protocol mirrors the conditions developed by Hayatsu and coworkers for preparation of the N 4 -amino-2’-deoxycytidine nucleoside [10] and adapted by Gao and Orgel for use with 2’-deoxyoligonucleotides. [7c] The 5’- 32 P-labeled, Ap-containing oligonucleotide E was prepared by treatment of the corresponding 5’- 32 P-labeled 2’-deoxyuridine-containing oligonucleotide D with the enzyme uracil DNA glycosylase (UDG, Scheme 2B). [11] Efficient formation of the Ap-containing oligonucleotide E was confirmed by treatment of the DNA with warm piperidine to generate the faster-migrating 3’-phosphate cleavage product F (Figure 3, lane 3).…”

“…The covalent-reversible nature of the dC*-Ap cross-link is strongly reminiscent of the “post-it type” interstrand cross-link recently described by Carell and coworkers that employs reversible imine formation. [7a] …”

“…This type of covalent, thermally-reversible cross-link may find applications in both biochemistry and materials science. [7a,15,16] …”