A modified thymine for the synthesis of site-specific thymine-guanine DNA interstrand crosslinks

Abstract

DNA interstrand crosslinks (ICLs) are highly cytotoxic lesions formed by a variety of important anti-tumor agents. Despite the clinical importance of ICLs, the mechanisms by which these lesions are repaired in mammalian cells have so far remained elusive. One of the obstacles in the study of ICL repair has been the limited availability of suitable methods for the synthesis of defined site-specific ICLs. We report here the synthesis of a site-specific ICL containing an ethylene-bridged G-T base pair based on the incorporation of a crosslink precursor containing a selectively reactive group on one strand using solid-phase synthesis. 3-(2-chloroethyl)thymidine was incorporated into oligonucleotides and underwent ICL formation upon annealing to a complementary strand by reacting with the base opposite to the modified T residue. A strong preference for ICL formation with a G residue opposite the reactive T was observed. Detailed characterization of the reaction product revealed that the alkylation reaction occurred with the O-6 group of G and a mechanism accounting for this preference is proposed. These G-T crosslinks introduced here will be useful for studies of ICL repair.

Synthesis of DNA Interstrand Cross‐Links Using a Photocaged Nucleobase

“…An alternative approach to ICL synthesis involves the incorporation of stable ICL precursors into singlestranded oligonucleotides using standard DNA synthesis. [18][19][20][21][22][23][24][25][26] Following hybridization to a complementary sequence, ICL formation can be initiated in the modified duplex. Alzeer and Schärer used this approach to generate a O 6 (dG)-ethyl-N 3 (dT) ICL by incorporating N 3 -(2-chloroethyl)thymine into oligonucleotides.…”

New synthetic substrates of mammalian nucleotide excision repair system

“…Many previous investigations with artificial NER substrates have been performed using circular structures (phage or plasmid DNA) bearing lesions in the target position or statistically distributed in the DNA molecule (8,15,29,30). The primary evaluation of the substrate properties of recently suggested photoreactive lesions Fap-dC (-dU) introduced in pBSK II DNA was described (16).…”

“…The products of NER-specific excision in HeLa cell extract were also not observed using linear 120mer DNA containing well-defined damage, namely, AAF-dG (9). AAF-dG was efficiently repaired by NER system in vitro only when it was introduced into circular DNA (9,15). As it was aforementioned, the other activities in the cell extracts, such as a non-specific nucleases and DNA-binding proteins of high affinity to DNA, particularly Ku 70–80 antigen, the protein, high content of which is so typiсal for cancer cells extracts, can impede detection of the NER reaction for ldsmDNA (23,31,32).…”

Using synthetic DNA interstrand crosslinks to elucidate repair pathways and identify new therapeutic targets for cancer chemotherapy

“…Although CENU ICL-containing oligonucleotides have been generated by treatment of oligonucleotides with BCNU [39], strategies for the efficient site-specific generation of this adducts on a larger scale have so far remained elusive. A number of BCNU-like adducts have been synthesized either by incorporation of a precursor (a chloroethyl modified thymine) that can form an ICL with a guanine residue [40] or by incorporation of crosslinked nucleotide dimer into DNA using solid-phase synthesis (Figure 1C) [41–43]. This later strategy allowed the synthesis of bridges with different lengths and different structures.…”