Fluorescent nucleic acids were prepared utilizing the polymerase extension (PEX) reaction to incorporate fluorescent molecules. 2'-Deoxyuridine triphosphate (dUTP) derivatives possessing pyrene molecules as fluorophores were synthesized using the aqueous-phase Sonogashira coupling between 5-Iodo-dUTP and acetylene-linked pyrene molecules. The incorporation of the pyrene (Py)-labeled deoxyuridine triphosphates (PyU) into DNA by polymerase was evaluated by polyacrylamide gel electrophoresis, demonstrating that the PyU can work as a good substrate for the PEX reaction. The fluorescent properties of the functionalized DNA prepared by the PEX reaction were characterized by steady-state fluorescence measurements. The Py-conjugated DNA showed typical emission spectra of the pyrene, and the DNA with two pyrene molecules connected to each other by a diethylene glycol linker exhibited a broadened emission attributed to the electronic interaction between the Py molecules.Nucleic acids modified with functional groups/molecules are of significant interest as potential applications in chemical biology, diagnostic tools or nanotechnology and material science.1-3 To expand their applications as versatile tools, various kinds of oligodeoxynucleotide modified with functional molecules have been prepared by a general synthetic approach using the corresponding phosphoroamidite units during solid-phase DNA synthesis and the combination with post-synthetic oligonucleotides modification. 4,5 As an alternative approach to the conventional DNA synthesis for the preparation of the functionalized oligonucleotides, an enzymatic method using the polymerase extension (PEX) reaction has attracted much attention because of the facile preparation of longer oligonucleotides from DNA templates and multiple incorporation of functional groups/molecules into DNA based on the sequence of the templates. 6,7 Certain kinds of deoxynucleotide triphosphate (dNTP) derivatives possessing a fluorescent reporter group at an appropriate position on the nucleobase can work as effective substrates for the enzymatic incorporation by polymerase and are used as fluorescent probes for the detection of the hybridization and amplification of DNA. 3,[8][9][10][11][12] Pyrene fluorophores with unique fluorescent properties, capable of generating an excited dimer with a characteristic green light, were conjugated to oligonucleotides for the structural study of the folding and development of single nucleotide polymorphisms (SNPs). In this study, we demonstrated the preparation of the deoxyuridine triphosphate derivatives labelled with the pyrene (Py) fluorophore through an ethylene glycol linker, and evaluation of the enzymatic incorporation of the triphosphate by DNA polymerase and the emission properties of the Py-incorporated DNA.Multiple incorporation of the pyrene molecules into DNA by the polymerase extension (PEX) reaction is schematically shown in Figure 1. We have chosen pyrene (Py) as the fluorescent molecule because pyrene is known to show a strong e...

Deoxyuridine triphosphate derivatives modified with pyrene was synthesized to functionalize DNA with fluorescent molecules based on the template DNA sequence. Incorporation of pyrene-labeled deoxyuridine triphosphates into DNA by DNA polymerase was investigated by using reverse-phase HPLC and polyacrylamide gel electrophoresis. The fluorescent properties of functionalized DNA were characterized by the steady-state fluorescence measurements.