Synthesis of three pyrene-modified nucleosides was accomplished using the CuAAC reaction. Hereby, pyrene is attached either to the 5'-position of thymidine or to the 2'-position of 2'-deoxyuridine through triazolemethylene linkers, or to the 2'-position of 2'-deoxyuridine through a more rigid triazole linker. The three nucleosides were incorporated into oligonucleotides, and these were combined in different duplexes and other secondary structures, which were analyzed by thermal stability and fluorescence studies. The three monomers were found to have different impacts on the nucleic acid complexes. Hence, pyrene attached to the 5'-position shows a tendency for intercalation into the duplex as indicated by a general decrease in fluorescence intensity followed by an increase in duplex thermal stability. Pyrene attached to the 2'-position through a rigid triazole linker also shows a tendency for intercalation but with decrease in duplex stability, whereas the pyrene attached to the 2'-position through a triazolemethylene linker is primarily situated in the minor groove as indicated by an increase in fluorescence but here in most cases leading to increase in duplex stability. All three pyrene nucleotides lead to thermal stabilization of bulged duplexes and three-way junctions. In some cases when two pyrenes were introduced into the core of these complexes, the formation or disappearance of a fluorescence excimer band can be used to indicate the hybridization process. Hereby these oligonucleotides can act as specific recognition probes.