Click Chemistry for Rapid Labeling and Ligation of RNA

Abstract

The copper(I)-promoted azide-alkyne cycloaddition reaction (click chemistry) is shown to be compatible with RNA (with free 2'-hydroxyl groups) in spite of the intrinsic lability of RNA. RNA degradation is minimized through stabilization of the Cu(I) in aqueous buffer with acetonitrile as cosolvent and no other ligand; this suggests the general possibility of "ligandless" click chemistry. With the viability of click chemistry validated on synthetic RNA bearing "click"-reactive alkynes, the scope of the reaction is extended to in-vitro-transcribed or, indeed, any RNA, as a click-reactive azide is incorporated enzymatically. Once clickable groups are installed on RNA, they can be rapidly click labeled or conjugated together in click ligations, which may be either templated or nontemplated. In click ligations the resultant unnatural triazole-linked RNA backbone is not detrimental to RNA function, thus suggesting a broad applicability of click chemistry in RNA biological studies.

Incorporation Efficiency of 5'-Azido-5'-Deoxyguanosine into 5'-Terminus of RNA for Preparation of Azido-Functionalized RNA

“…9 Differently from the literature, 9 RNA degradation was minimized in the present study through stabilization of the Cu I in aqueous buffer with DMSO as cosolvent and no other ligand, suggesting the general possibility of ligandless click chemistry. The ability to utilize click chemistry directly to label RNA would offer an important consideration for handling RNA, providing an orthogonal method that could enable more facile labeling of oligonucleotides and expanding the application of click chemistry approach beyond use with synthetic RNA to any natural RNA.…”

Preparation of modified long-mer RNAs and analysis of FMN binding to theypaAaptamer fromB. subtilis

“…In addition to enzymatic protocols for ligation of chemically synthesized fragments, chemical protocols were developed, [40][41][42][43] whereby the cupper-catalyzed azide-alkyne coupling (Click ligation) 26,29,34,44 stands out as most promising. A drawback of Click ligation is the need for terminal functionalization of fragments to be ligated (5′-azide and 3′-alkyne or vice versa), and the generation of a triazole linkage in the nucleic acid backbone.…”

“…Nevertheless, Click ligation has been used for fragment joining in a number of applications. 26,29,34,[40][41][42][43][44] Among those, also functional RNAs, such as hairpin and hammerhead ribozymes, were prepared. 29 However, to the best of our knowledge, we here show the first example of a Click ligated natural aptamer as long as 129 nucleotides that retains full functionality in binding of its cognate ligand, in spite of the two triazole backbone linkages.…”

“…It has been discussed in the literature that the azide is not stable at RNA synthesis conditions, in particular, suffering from the applied P(III) chemistry. 33,34 Therefore, in order to evaluate the applicability of our 3′-azido functionalized support, we used it for the synthesis of a short model oligonucleotide bearing a 3′-terminal azide. The 3′-functionalized oligomer was successfully Click-ligated to a short 5′-alkyne modified oligonucleotide, demonstrating that the 3′-azido functionality was preserved under the conditions of chain assembly.…”

“…5 and 6). Click ligation was performed basically following the protocols of El-Sagheer and Brown 29 and Paredes and Das, 34 although we varied conditions in terms of reaction with or without DNA splint, and changed buffer composition, more appropriate for DNA splint digestion right after successful ligation (see Materials and Methods). Particular care was taken to work oxygen-free in order to allow Cu(I) as catalyst to be formed.…”