Double FIT hybridization probes - towards enhancing brightness, turn-on and specificity of RNA detection.

Efficient fluorogenic hybridization probes combine high brightness and specificity of fluorescence signaling with large turn-on of fluorescence. Herein, we present an approach to enhance signaling by combining two identical fluorescence base surrogates in FIT2 probes. Provided there is a suitable positioning of dyes, target-bound FIT2 probes emit brighter than mono dye probes, while dye-dye contact in the single stranded state provides opportunities for decreasing background fluorescence. The probes were used to explore the single nucleotide-specific detection of a C → U edited RNA of the glycine receptor (GlyR). We observed strong self-quenching upon single base mismatched hybridization of FIT2 probes, which helped in distinguishing edited from unedited RNA target in cell lysates.

Chemo-biological mRNA imaging with single nucleotide specificity.

Unambiguous imaging of C → U edited mRNA calls for a method that distinguishes a locally high concentration of unbound probe or single nucleotide mismatched target from a locally low concentration of matched mRNA target. To address this issue, we combine FIT probes as a "chemical" detection system with the "biological" MS2 technique. Ratio measurements provide a convenient parameter to discriminate the edited from the unedited state of mRNA encoding for GlyR α2 in HEK cells.

Terminal alkenes as versatile chemical reporter groups for metabolic oligosaccharide engineering.

The Diels-Alder reaction with inverse electron demand (DAinv reaction) of 1,2,4,5-tetrazines with electron rich or strained alkenes was proven to be a bioorthogonal ligation reaction that proceeds fast and with high yields. An important application of the DAinv reaction is metabolic oligosaccharide engineering (MOE) which allows the visualization of glycoconjugates in living cells. In this approach, a sugar derivative bearing a chemical reporter group is metabolically incorporated into cellular glycoconjugates and subsequently derivatized with a probe by means of a bioorthogonal ligation reaction. Here, we investigated a series of new mannosamine and glucosamine derivatives with carbamate-linked side chains of varying length terminated by alkene groups and their suitability for labeling cell-surface glycans. Kinetic investigations showed that the reactivity of the alkenes in DAinv reactions increases with growing chain length. When applied to MOE, one of the compounds, peracetylated N-butenyloxycarbonylmannosamine, was especially well suited for labeling cell-surface glycans. Obviously, the length of its side chain represents the optimal balance between incorporation efficiency and speed of the labeling reaction. Sialidase treatment of the cells before the bioorthogonal labeling reaction showed that this sugar derivative is attached to the glycans in form of the corresponding sialic acid derivative and not epimerized to another hexosamine derivative to a considerable extent.