Click chemistry for rapid labeling and ligation of RNA.
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.
Chemistry Department, Carnegie Mellon University, 4400 Fifth Avenue Pittsburgh, PA 15213, USA.
Eduardo
E
Paredes
Subha R
SR
Das
D000097:Acetonitriles / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D057930:Click Chemistry; D004247:DNA / Q000737:chemistry / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D007700:Kinetics; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012997:Solvents / Q000737:chemistry; D013194:Staining and Labeling; D013379:Substrate Specificity
D000097:Acetonitriles; D001386:Azides; D012997:Solvents; D012313:RNA; D004247:DNA; D012321:DNA-Directed RNA Polymerases; C032159:acetonitrile