FLUORESCENCE MICROSCOPY Strategic blinking

For decades chemists have focused on increasing the brightness of fluorophores. In super-resolution microscopy, however, fluorophores that preferentially exist in a non-fluorescent state, but occasionally re-arrange into a fluorescent form, can give better results

Cell permeable, fluorescent dye

The invention pertains to a near-infrared fluorescent dye that is cell permeable and can be attached to selected proteins in living cells. The dye has the general formula (I) or its or its corresponding spirolactone (II) wherein Y is chosen from the group consisting of Si, Ge and Sn R0 is -COO- or COOH R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15 and R16 are substituents, including hydrogen, independently from each other. The dye (i) absorbs and emits light at wavelengths above 600 nm (ii) possesses high photostability (iii) has high extinction coefficients and high quantum yields (iv) can be derivatized with different molecules and (v) is membrane-permeable and shows mini not mal background binding to biomolecules and biomolecular structures

Direct transfer of extended groups from synthetic cofactors by DNA methyltransferases

Christian Dalhoff, Gražvydas Lukinavičius, Saulius Klimas̆auskas & Elmar Weinhol

Reduced Dyes Enhance Single-Molecule Localization Density for Live Superresolution Imaging

Cell-permeable rhodamine dyes are reductively quenched by NaBH4 into a non-fluorescent leuco-rhodamine form. Quenching is reversible, and their fluorescence is recovered when the dyes are oxidized. In living cells, oxidation occurs spontaneously, and can result in up to ten-fold higher densities of single molecule localizations, and more photons per localization as compared with unmodified dyes. These two parameters directly impact the achievable resolution, and we see a significant improvement in the quality of live-cell point-localization super-resolution images taken with reduced dyes. These improvements carry over to increase the density of trajectories for single-molecule tracking experiments

Selective cross-linking of interacting proteins using self-labeling tags

We have designed molecules that permit the selective cross-linking (S-CROSS) of interacting proteins in cell lysates and the sensitive detection of the trapped complexes through in-gel fluorescence scanning. S-CROSS requires the expression of the putative interacting proteins as fusion to CLIP-tag or SNAP-tag, two protein tags that can be specifically labeled with synthetic probes. Bifunctional molecules that contain the substrates of the two tags connected via a fluorophore are used to selectively cross-link interacting proteins in cell lysate. The amount of trapped complex can be then quantified after SDS gel electrophoresis by in-gel fluorescence scanning. On the basis of a detailed kinetic analysis of the cross-linking reaction, we showed that the cross-linking efficiency can be used as an indicator of interaction between two proteins, allowing thereby the unambiguous identification of interacting protein pairs. We validated our approach by confirming a number of interactions through selective cross-linking and showed that it permits the quantitative and simultaneous analysis of multiple homotypic and heterotypic protein complexes and the differentiation between strong and weak protein-protein interactions

Targeted labeling of DNA by methyltransferase-directed transfer of activated groups (mTAG)

Methyltransferases catalyze highly specific transfers of methyl groups from the ubiquitous cofactor S-adenosyl-l-methionine (AdoMet) to various biopolymers like DNA, RNA, and proteins. Here we describe the first synthetic analogue of AdoMet with an activated side chain carrying a primary amino group that permits efficient methyltransferase-directed functionalization of DNA and subsequent amine-specific chemoligations with various reporter groups. The demonstrated two-step sequence-specific labeling of natural DNA offers a facile way to query the methylation status of the target sites and envisions numerous applications in functional studies and medical diagnostics

Targeted Photoswitchable Probe for Nanoscopy of Biological Structures

We introduce a photoswitchable O6-benzylguanine derivative and demonstrate its use for super-resolution microscopy of SNAP-tagged proteins based on single fluorophore localization. Stochastic Optical Reconstruction Microscopy (STORM) reveals SNAP-tagged microtubule structures with ~25 nm resolution. The described probe in combination with the versatile SNAP-tag labeling opens new possibilities for imaging biological structures at the nanoscale