title: Synthesis of building blocks and oligonucleotides containing {T}O4-alkylene-O4{T} interstrand cross-links.
abstract: 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.
authors: Department of Chemistry and Biochemistry, Concordia University, Montréal, Québec, Canada.
authors: Derek K
authors: DK
authors: O'Flaherty
authors: Francis P
authors: FP
authors: McManus
authors: Anne M
authors: AM
authors: Noronha
authors: Christopher J
authors: CJ
authors: Wilds
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry*; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000737:chemistry*
chemical_list: D003432:Cross-Linking Reagents; D009841:Oligonucleotides
keywords: DNA damage; DNA interstrand cross‐link; DNA repair; chemically modified oligonucleotide; oligonucleotide synthesis; solid‐phase synthesis