Scope and Limitations of Typical Copper-Free Bioorthogonal Reactions with DNA: Reactive 2'-Deoxyuridine Triphosphates for Postsynthetic Labeling

Abstract

Four triphosphates of 2'-deoxyuridine that carried the following bioorthogonally reactive groups were synthesized by organic-chemical methods. Two triphosphates with tetrazines and one with a cyclopropene moiety were designed for Diels-Alder reactions with inverse electron demand, and one triphosphate with a tetrazole core was designed for the "photoclick" cycloaddition. These triphosphates were not only successfully applied for oligonucleotide preparation by standard DNA polymerases, including Hemo KlenTaq, Vent, and Deep Vent, but also bypassed for full length primer extension products. Fluorescent labeling of the primer extension products was achieved by fluorophores with reactive counterparts and analyzed by polyacrylamide gel electrophoresis mobility shifts. The tetrazine-oligonucleotide conjugates were reacted with carboxymethylmonobenzocyclooctyne- and bicyclononyne-modified fluorophores. The yield of these postsynthetic reactions could significantly be improved by a more stable but still reactive nicotinic acid-derived tetrazine and by changing the key experimental conditions, mainly the pH of 7.2 and the temperature of 45-55 °C. The cyclopropene-oligonucleotide conjugate could be successfully labeled with a tetrazine-modified rhodamine in very good yields. The "photoclick" cycloaddition between tetrazole-oligonucleotide conjugates and a maleimide-modified dye worked quantitatively. The combination of primer extension, bypass, and bioorthogonal modification works also for double and triple labeling using the cyclopropene-modified 2'-deoxyuridine triphosphate.