High-density functionalization and cross-linking of DNA: "click" and "bis-click" cycloadditions performed on alkynylated oligonucleotides with fluorogenic anthracene azides.

Abstract

High density functionalization of DNA with ethynyl and octadiynyl side chains followed by CuAAC "click labeling" with 9-azidomethylanthracene was performed. Alkynyl DNA was also cross-linked with fluorogenic 9,10-bis-azidomethylanthracene employing the "bis-click" reaction. By this means the fluorescence of the anthracene moiety was imparted to the virtually nonfluorescent DNA. Phosphoramidites of 8-aza-7-deaza-2'-deoxyadenosine with short and long linker arms in a steric nondemanding 7-position were utilized in solid phase oligodeoxynucleotide synthesis. High density alkynylated DNA-without anthracene residues-was found to be of comparable stability with both long and short linker arms. High density anthracene functionalized DNA became less stable with the short linker compared to that with the long linker connectivity. Interstrand cross-linked homodimers constructed from alkynylated oligonucleotides with fluorogenic 9,10-bis-azidomethylanthracene were hybridized with complementary strands to form double helices. They are more stable when the linker was located at a terminus than in a central position. Short linker anthracene adducts were destabilizing compared to long linker adducts. The fluorogenic anthracene residues not only have a significant effect on the duplex stability, but also impart fluorescence to the species. Fluorescence of cross-linked double helices with long linker connectivity was quenched when the cross-link was in a terminal position and was dequenched when the linker was connecting the two double helices at the center of the molecule. The fluorescence of the anthracene cross-linked double helices was strongly increased (dequenched) when the correct base pair was formed, while no change occurred upon mismatch formation.