Synthesis and Characterization of DNA Containing anN¹-2′-Deoxyinosine-ethyl-N³-thymidine Interstrand Cross-Link: A Structural Mimic of the Cross-Link Formed by 1,3-Bis-(2-chloroethyl)-1-nitrosourea

Abstract

Interstrand cross-links, which are generated by chemotherapeutic treatment with bis-alkylating agents, exert their therapeutic effect by connecting the nucleobases of adjacent DNA strands together and represent some of the most threatening forms of damage suffered by genomic DNA. However, one of the reasons for treatment failure using these agents is due to enhanced repair of this DNA damage. The pursuit of understanding the repair of interstrand cross-links by repair systems has necessitated the synthesis of sufficient quantities of such damaged DNA. We report the synthesis of a site-specific interstrand cross-linked duplex containing an ethylene-bridged N (1)-2'-deoxyinosine- N (3)-thymidine base pair prepared by solution and solid-phase synthesis as a mimic for the lesion formed by the therapeutic agent 1,3-bis-(2-chloroethyl)-1-nitrosourea using both a phosphoramidite and a bis-phosphoramidite approach. UV thermal denaturation experiments revealed that this cross-linked duplex was stabilized by 52 degrees C relative to the noncross-linked control, and circular dichroism studies indicated little deviation from a B-form structure compared to a duplex that contained a G-C base pair at the same position. Molecular models of the cross-linked duplex that were geometry optimized using the AMBER forcefield also suggest that this lesion induces minimal distortion in B-form DNA. This modified oligonucleotide will be useful for studies related to the investigation of interstrand cross-linked DNA repair.

Synthesis and characterization of an O6-2′-deoxyguanosine-alkyl-O6-2′-deoxyguanosine interstrand cross-link in a 5′-GNC motif and repair by human O6-alkylguanine-DNA alkyltransferase

“…First, synthesis was performed using a polystyrene rather than controlled-pore glass (CPG) solid-support due to the incompatibility of TEA·3HF with the latter 5. As an added precaution, the cyanoethyl protecting groups were removed using TEA as it has been observed that prolonged fluoride treatment using TBAF could lead to chain cleavage 29.…”

Furan‐Oxidation‐Triggered Inducible DNA Cross‐Linking: Acyclic Versus Cyclic Furan‐Containing Building Blocks—On the Benefit of Restoring the Cyclic Sugar Backbone

“…The interest of ICL oligonucleotides lies in the possibility to generate substrates for a detailed study of cross-link repair in order to elucidate the mechanistic background for this phenomenon. Indeed, isolation of defined ICL adducts from natural sources in sufficient quantities for mechanistic studies remains a major problem [16] and treatment of DNA with external cross-linking reagents [17][18][19][20][21][22] typically yields complex mixtures of mono-adducts, and inter-and intrastrand cross-links with the desired ICLs are usually represented as minor fractions. [23] A variety of applications in the chemical biology area further justify the ongoing and intense research for efficient and highly sequence-specific cross-linked oligonucleotides.…”

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.…”