Biology subjects

Menche, T.

Publications and source records attributed to Menche, T..

2 recordsLinked to original sources

bioRxiv2025

A high-affinity split-HaloTag for live-cell protein labeling

We introduce a high-affinity split-HaloTag comprised of a short peptide tag (Hpep, 14 residues) and a large, inactive fragment (cpHalo{Delta}3). Hpep binds to cpHalo{Delta}3 spontaneously with nanomolar affinity, enabling subsequent labeling with fluorescent HaloTag ligands. The small size of Hpep facilitates cloning-free endogenous protein tagging using CRISPR/Cas9 and the complementation of Hpep-tagged proteins can be achieved in live cells through co-expression with cpHalo{Delta}3 and in fixed cells through incubation with cpHalo{Delta}3. The approach is compatible with advanced microscopy techniques such as expansion microscopy and live-cell STED imaging. Additionally, variants of Hpep that modulate the spectral properties of labeled fluorophores enable simultaneous imaging of two different Hpep-tagged proteins via fluorescence lifetime microscopy. In summary, our high-affinity split-HaloTag is a robust and versatile tool for live-cell imaging and diverse applications in chemical biology.

biochemistry↗

bioRxiv2024

Self-quenched fluorophore-DNA labels for super-resolution fluorescence microscopy

Protein labeling through transient and repetitive hybridization of short, fluorophore-labeled DNA oligonucleotides has become widely applied in various optical super-resolution microscopy methods. The main advantages are multi-target imaging and molecular quantification. A challenge is the high background signal originating from the presence of unbound fluorophore-DNA labels in solution. Here, we report self-quenching of fluorophore dimers conjugated to DNA oligonucleotides as a general concept to reduce the fluorescence background. Upon hybridization, the fluorescence signal of both fluorophores is fully restored. Here, we expand the toolbox of fluorophores suitable for self-quenching and report their spectra and hybridization equilibria. We apply self-quenched fluorophore-DNA labels to stimulated emission depletion (STED) microscopy and single-molecule localization microscopy (SMLM) and report improved imaging performances.

biophysics↗