Synthesis of building blocks and oligonucleotides containing {T}O4-alkylene-O4{T} interstrand cross-links

This protocol describes the preparation of O(4)-thymidine-alkylene-O(4)-thymidine dimer bis-phosphoramidites and precursors for incorporation into DNA sequences to produce site-specific DNA interstrand cross-links. Linkers are introduced at the 4-position of thymidine by reacting the sodium salt of a diol with a pyrimidinyl-convertible nucleoside to produce mono-adducts, which then undergo reaction with a stoichiometric equivalent of a pyrimidinyl-convertible nucleoside under basic conditions to form O(4)-thymidine-alkylene-O(4)-thymidine dimers. Bis-phosphoramidites are incorporated into oligonucleotides by solid-phase synthesis, and mild conditions for deprotection and cleavage from the solid support are employed to prevent degradation of the thymidine modifications. Purification of these cross-linked oligonucleotides is performed by denaturing polyacrylamide gel electrophoresis. This approach allows for the preparation of cross-linked DNA substrates in quantities and purity sufficient for a wide range of biophysical experiments and biochemical studies as substrates to investigate DNA repair pathways.