Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy.
Gerti BeliuAndreas J KurzAlexander C KuhlemannLisa Behringer-PliessMara MeubNatalia WolfJürgen SeibelZhen-Dan ShiMartin SchnermannJonathan B GrimmLuke D LavisSören DooseMarkus SauerPublished in: Communications biology (2019)
Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine is the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.
Keyphrases
- single molecule
- electron transfer
- living cells
- atomic force microscopy
- fluorescence imaging
- aqueous solution
- fluorescent probe
- energy transfer
- highly efficient
- computed tomography
- photodynamic therapy
- quantum dots
- single cell
- amino acid
- high throughput
- cancer therapy
- positron emission tomography
- copy number
- molecularly imprinted
- pet imaging
- optical coherence tomography
- mass spectrometry