Bodipy-Labeled Nucleoside Triphosphates for Polymerase Synthesis of Fluorescent DNA

New fluorescent nucleosides and nucleoside triphosphate (dNTPs) analogs bearing the F-Bodipy fluorophore linked through a short, flexible nonconjugate tether were synthesized. The Bodipy-labeled dNTPs were substrates for several DNA polymerases which incorporated them into DNA in primer extension, nicking enzyme amplification reaction, and polymerase chain reaction. The fluorescence of F-Bodipy is not quenched upon incorporation in DNA and can be detected both in solutions and on gels

A Bifunctional Noncanonical Amino Acid: Synthesis, Expression, and Residue-Specific Proteome-wide Incorporation

Mapping of weak and hence transient interactions between low-abundance interacting molecules is still a major challenge in systems biology and protein biochemistry. Therefore, additional system-wide acting tools are needed to determine protein interactomics. Most important are reagents that can be applied at any kind of protein interface and the possibility to enrich cross-linked fragments with high efficiency. In this study, we report the synthesis of a novel noncanonical amino acid that features a diazirine group for ultraviolet cross-linking as well as an alkyne group for labeling by click chemistry. This bifunctional amino acid, called PrDiAzK, may be inserted into almost any protein interface with minimal structural perturbation using genetic code expansion. We demonstrate that PrDiAzK can be site-selectively incorporated into proteins in both bacterial and mammalian cell cultures, and we show that PrDiAzK allows protein labeling as well as cross-linking. In addition, we tested PrDiAzK for proteome-wide incorporation via stochastic orthogonal recoding of translation, implying potential applications in system-wide mapping of protein–protein interactions in the future

A Universal Nucleoside with Strong Two-Band Switchable Fluorescence and Sensitivity to the Environment for Investigating DNA Interactions

With the aim of developing a new tool to investigate DNA interactions, a nucleoside analogue incorporating a 3-hydroxychromone (3HC) fluorophore as a nucleobase mimic was synthesized and incorporated into oligonucleotide chains. In comparison with existing fluorescent nucleoside analogues, this dye features exceptional environmental sensitivity switching between two well-resolved fluorescence bands. In labeled DNA, this nucleoside analogue does not alter the duplex conformation and exhibits a high fluorescence quantum yield. This probe is up to 50-fold brighter than 2-aminopurine, the fluorescent nucleoside standard. Moreover, the dual emission is highly sensitive to the polarity of the environment; thus, a strong shielding effect of the flanking bases from water was observed. With this nucleoside, the effect of a viral chaperone protein on DNA base stacking was site-selectively monitored