Synthesis and characterization of DNA duplexes containing an N ³T-ethyl-N³T interstrand crosslink in opposite orientations

DNA duplexes containing an ethyl interstrand crosslink that bridges the N³ atoms of thymidines on the opposite strands have been synthesized using an approach that combines conventional solid phase oligonucleotide synthesis and the selective removal of protecting groups of a crosslinked thymidine dimer. This approach allows for the assembly of a crosslinked duplex directly on the solid support. Duplexes that contain a N³T-ethyl- N³T interstrand crosslink in a staggered orientation at either a -TA- or -AT-step in a duplex have been prepared. When placed in an -AT- step of a duplex the effect was stabilizing relative to the non-crosslinked control duplex (ΔT_m = +24°C) and this crosslinked duplex was found to efficiently form multimers in the presence of T4 ligase. In the case of the -TA- crosslinked duplex the stabilizing effect was less pronounced (ΔT _m= +6°C) and likewise did not undergo self ligation under identical conditions. Molecular modeling studies suggested that the -AT- containing lesion had little deviation in structure relative to the non-crosslinked duplex DNA control, whereas the -TA- crosslinked duplex exhibited significant buckling of the base pairs flanking the lesion.