The Efficiency of Metabolic Labeling of DNA by Diels-Alder Reactions with Inverse Electron Demand: Correlation with the Size of Modified 2'-Deoxyuridines.

A selection of four different 2'-deoxyuridines with three different dienophiles of different sizes was synthesized. Their inverse electron demand Diels-Alder reactivity increases from k2 = 0.15 × 10-2 M-1 s-1 to k2 = 105 × 10-2 M-1 s-1 with increasing ring strain of the dienophiles. With a fluorogenic tetrazine-modified cyanine-styryl dye as reactive counterpart the fluorescence turn-on ratios lie in the range of 21-48 suitable for wash-free cellular imaging. The metabolic DNA labeling was visualized by a dot blot on a semiquantitative level and by confocal fluorescence microscopy on a qualitative level. A clear correlation between the steric demand of the dienophiles and the incorporation efficiency of the modified 2'-deoxyuridines into cellular DNA was observed. Even 2'-deoxyuridines with larger dienophiles, such as norbornene and cyclopropene, were incorporated to a detectable level into the nascent genomic DNA. This was achieved by an optimized way of cell culturing. This expands the toolbox of modified nucleosides for metabolic labeling of nucleic acids in general.

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.

Labelling of DNA and RNA in the cellular environment by means of bioorthogonal cycloaddition chemistry.

Labelling of nucleic acids as biologically important cellular components is a crucial prerequisite for the visualization and understanding of biological processes. Efficient bioorthogonal chemistry and in particular cycloadditions fullfill the requirements for cellular applications. The broadly applied Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC), however, is limited to labellings in vitro and in fixed cells due to the cytotoxicity of copper salts. Currently, there are three types of copper-free cycloadditions used for nucleic acid labelling in the cellular environment: (i) the ring-strain promoted azide-alkyne cycloaddition (SPAAC), (ii) the "photoclick" 1,3-dipolar cycloadditions, and (iii) the Diels-Alder reactions with inverse electron demand (iEDDA). We review only those building blocks for chemical synthesis on solid phase of DNA and RNA and for enzymatic DNA and RNA preparation, which were applied for labelling of DNA and RNA in situ or in vivo, i.e. in the cellular environment, in fixed or in living cells, by the use of bioorthogonal cycloaddition chemistry. Additionally, we review the current status of orthogonal dual and triple labelling of DNA and RNA in vitro to demonstrate their potential for future applications in situ or in vivo.