ArticleSchwechheimer C, Doll L, Wagenknecht HA.
Curr Protoc Nucleic Acid Chem. 2018 03;72(1):4.80.1-4.80.13.
Fluorescence molecular imaging is widely used to visualize and observe different biomolecules, in particular DNA and RNA, in vivo and in real time. Typically, DNA strands are tagged with only one fluorophore, and, in the case of molecular beacons, an additional quencher is conjugated, which bears the risk of false-positive or false-negative results because only fluorescence intensities at one fluorescence wavelength (color) are compared. To address this drawback, the concept of "DNA/RNA traffic lights," which is characterized by a fluorescence color change due to energy transfer between two dyes, was developed by our working group. For these DNA and RNA systems, the oligonucleotides are post-synthetically labeled, specifically after solid-phase synthesis by chemical means, with a fluorescent dye using copper(I)-catalyzed cycloaddition at the 2' position of single uridines. In order to functionalize oligonucleotides with several different labels, an on-resin method is required to ensure the necessary selectivity. This unit describes two different CuAAC ("click") approaches-in solution (post-synthetic) and on solid phase (during synthesis)-for the attachment of fluorophores to the 2' position of DNA. © 2018 by John Wiley & Sons, Inc.