Fast and Efficient Postsynthetic DNA Labeling in Cells by Means of Strain-Promoted Sydnone-Alkyne Cycloadditions.

Sydnones are highly stable mesoionic 1,3-dipoles that react with cyclooctynes through strain-promoted sydnone-alkyne cycloaddition (SPSAC). Although sydnones have been shown to be valuable bioorthogonal chemical reporters for the labeling of proteins and complex glycans, nucleic acids have not yet been tagged by SPSAC. Evaluation of SPSAC kinetics with model substrates showed fast reactions with cyclooctyne probes (up to k=0.59 M-1 s-1 ), and two different sydnones were effectively incorporated into both 2'-deoxyuridines at position 5, and 7-deaza-2'-deoxyadenosines at position 7. These modified nucleosides were synthetically incorporated into single-stranded DNAs, which were successfully postsynthetically labeled with cyclooctyne probes both in vitro and in cells. These results show that sydnones are versatile bioorthogonal tags and have the premise to become essential tools for tracking DNA and potentially RNA in living cells.

Fluorogenic and Bioorthogonal Modification of RNA Using Photoclick Chemistry.

A bromoaryltetrazole-modified uridine was synthesized as a new RNA building block for bioorthogonal, light-activated and postsynthetic modification with commercially available fluorescent dyes. It allows "photoclick"-type modifications by irradiation with light (300 nm LED) at internal and terminal positions of presynthesized RNA with maleimide-conjugated fluorophores in good yields. The reaction was evidenced for three different dyes. During irradiation, the emission increases due to the formation of an intrinsically fluorescent pyrazoline moiety as photoclick product. The fluorogenecity of the photoclick reaction was significantly enhanced by energy transfer between the pyrazoline as the reaction product (poor emitter) and the photoclicked dye as the strong emitter. The RNA-dye conjugates show remarkable fluorescent properties, in particular an up to 9.4 fold increase of fluorescence, which are important for chemical biology and fluorescent imaging of RNA in cells.

Postsynthetic Modifications of DNA and RNA by Means of Copper-Free Cycloadditions as Bioorthogonal Reactions.

Bioorthogonal chemistry has mainly been developed for proteins and carbohydrates. The chemistry of nucleic acids is different, and bioorthogonal labeling strategies that were successfully applied for proteins and carbohydrates cannot be simply transferred to DNA and RNA. Cycloadditions play a central role for bioorthogonal chemistry with nucleic acids. In vivo postsynthetic labeling of DNA and RNA requires copper-free variants of cycloaddition chemistry to achieve "bio"orthogonality that can be applied even in living cells. Currently, there are three major types of copper-free cycloadditions available for nucleic acids: (i) the ring-strain-promoted azide-alkyne cycloadditions, (ii) the "photoclick" 1,3-dipolar cycloadditions, and (iii) the Diels-Alder reactions with inverse electron demand. In principle, bioorthogonally reactive building blocks for postsynthetic modifications of nucleic acids by cycloaddition can be prepared by three different ways: (i) The organic synthesis of DNA and RNA applies phosphoramidites as building blocks for solid-phase automated chemistry. (ii) The biochemical preparation of DNA and RNA by primer extension (PEX) and PCR applies triphosphates as building blocks together with DNA/RNA polymerases, and works in aqueous buffer. (iii) DNA and RNA is labeled by the intrinsic metabolism in cells using bioorthogonally reactive nucleosides. In contrast to proteins and carbohydrates, for which metabolic labeling strategies are well developed, there are only a few examples in the literature for metabolic labeling of nucleic acids. In this review, we summarize the currently available DNA and RNA building blocks, both phosphoramidites and nucleotide triphosphates, for copper-free and bioorthogonal postsynthetic modification strategies.