🧵Prepint alert! Optimizing Multifunctional Fluorescent Ligands for Intracellular Labeling | tinyurl.com/3n55hvsc. With Jason Vevea, Ed Chapman, and @so-lets-kilab70.bsky.social, we combined dye chemistry, HaloTag, microscopy and cell biology to make protein purification and manipulation tools. - ThreadSky

dyechemist.bsky.social • 26 days ago

🧵Prepint alert! Optimizing Multifunctional Fluorescent Ligands for Intracellular Labeling | http://tinyurl.com/3n55hvsc. With Jason Vevea, Ed Chapman, and @so-lets-kilab70.bsky.social, we combined dye chemistry, HaloTag, microscopy and cell biology to make protein purification and manipulation tools.

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rhodamine110.bsky.social•24 days ago

Our multifunctional ligands to BRD4 and histone H3.3:

so-lets-kilab70.bsky.social•24 days ago

Euchromatin is gonna have some FOMO 🙌

dyechemist.bsky.social•26 days ago

Self-labeling tags like HaloTag are great for labeling proteins with synthetic molecules. However, creating multifunctional ligands is tricky due to the competing demands of cell permeability vs. functionality.

dyechemist.bsky.social•26 days ago

We discovered that dyes with low lactone–zwitterion equilibrium constants and high distribution coefficients (logD) are great for making cell-permeable multifunctional ligands!

dyechemist.bsky.social•26 days ago

We could use this to guess apriori cell-impermeable (yellow) and cell-permeable (red and magenta) biotin-containing rhodamine ligands! We used imaging to verify this for HaloTag fusions at the cell surface, mitochondria, ER, and nucleus.

dyechemist.bsky.social•26 days ago

These live-cell permeable biotins are great for affinity purification. Here, Jason Vevea figured out their in-cell labeling performance and purified mitochondria! This works so much better than the commercial biotin-HaloTag ligand (this doesn't work actually!)

dyechemist.bsky.social•26 days ago

There’s More! Using the same Si-rhodamine dye, we created JQ1-containing fluorescent ligands to manipulate the localization of BRD4. @so-lets-kilab70.bsky.social showed rapid translocation of BRD4 from euchromatin to HaloTag sites at the nucleolus.

dyechemist.bsky.social•26 days ago

Even Cooler: When BRD4 was translocated to constitutive heterochromatin, we observed increased transcriptional activity (histone H3.3 accumulation as a proxy). This shows that multifunctional fluorescent ligands can functionally alter chromatin in living cells!

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