Synthesis of building blocks and oligonucleotides with {T}N3-alkylene-N3{T} cross-links

Gang Sun, Anne M. Noronha, Paul S. Miller, Christopher J. Wilds

Research output: Contribution to journalArticle

Abstract

This unit describes two methods to directly prepare oligonucleotide duplexes containing an N3thymidine-alkylene-N3thymidine inter-strand cross-link. The inter-strand crosslink can be engineered into the duplex with a number of possible orientations. Both methods require the preparation of a protected thymidine dimer where the N3 atoms of the two nucleosides are covalently attached by an alkyl linker. This linker is prepared starting from a protected diol using two successive alkylation reactions under basic conditions to accomplish the alkylation selectively at the N3 atom of the nucleoside. The chain length of the cross-link can be varied based on the selection of the diol used in the dimer synthesis. The solid-phase mono-phosphoramidite approach involves oligonucleotide synthesiswith 3'-O-phosphoramidites, on-column removal of a 3′-O-tertbutyldimethylsilyl protecting group, and continued oligonucleotide synthesis with 5′-Ophosphoramidites. The bis-phosphoramidite approach does not require synthesis with 5′-O-phosphoramidites. At the end of synthesis using either method, the N3thymidine-alkylene- N3thymidine inter-strand cross-linked oligonucleotides can be removed from the solid-support and purified using standard techniques (ion-exchange HPLC) in yields sufficient for various structural studies and repair assays.

Original languageEnglish (US)
Article number5.11
JournalCurrent Protocols in Nucleic Acid Chemistry
Issue numberSUPPL.51
DOIs
StatePublished - Dec 1 2012

Keywords

  • Chemical synthesis
  • DNA repair
  • Inter-strand cross-link
  • Oligonucleotide synthesis
  • Phosphoramidite
  • Solid-phase synthesis

ASJC Scopus subject areas

  • Biochemistry
  • Organic Chemistry

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