Terminal alkyne-containing phosphoramidite and solid support were prepared and used for the synthesis of 5′- and 3′-alkyne-modified oligonucleotides. The copper-catalyzed Huisgen [3+2] cycloaddition reaction in water-DMSO was used for the efficient preparation of perylene diimide-oligonucleotide conjugates suitable for constructing various types of PDI-modified DNA duplexes. © 2007 Elsevier Ltd. All rights reserved.