UNIT 5.11 Synthesis of Building Blocks and Oligonucleotides with {T}N3-Alkylene-N3{T} Cross-Links

  1. Gang Sun1,
  2. Anne M. Noronha1,
  3. Paul S. Miller2,
  4. Christopher J. Wilds1

Published Online: 1 DEC 2012

DOI: 10.1002/0471142700.nc0511s51

Current Protocols in Nucleic Acid Chemistry

Current Protocols in Nucleic Acid Chemistry

How to Cite

Sun, G., Noronha, A. M., Miller, P. S. and Wilds, C. J. 2012. Synthesis of Building Blocks and Oligonucleotides with {T}N3-Alkylene-N3{T} Cross-Links. Current Protocols in Nucleic Acid Chemistry. 51:5.11:5.11.1–5.11.17.

Author Information

  1. 1

    Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada

  2. 2

    Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland

Publication History

  1. Published Online: 1 DEC 2012


This unit describes two methods to directly prepare oligonucleotide duplexes containing an N3thymidine-alkylene-N3thymidine inter-strand cross-link. The inter-strand cross-link 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 synthesis with 3′-O-phosphoramidites, on-column removal of a 3′-O-tert-butyldimethylsilyl protecting group, and continued oligonucleotide synthesis with 5′-O-phosphoramidites. 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. Curr. Protoc. Nucleic Acid Chem. 51:5.11.1-5.11.17. © 2012 by John Wiley & Sons, Inc.


  • inter-strand cross-link;
  • phosphoramidite;
  • oligonucleotide synthesis;
  • chemical synthesis;
  • solid-phase synthesis;
  • DNA repair