Optimizing multifunctional fluorescent ligands for intracellular labeling

TL;DR

This paper introduces new fluorescent ligands that can enter cells and manipulate proteins, enabling experiments like purifying mitochondria or moving nuclear proteins.

Contribution

The paper presents a general framework for designing cell-permeable multifunctional ligands using rhodamine-based dyes.

Findings

Multifunctional ligands based on Si-rhodamines efficiently enter cells and enable affinity purification or translocation.

BRD4 translocation to heterochromatin increases transcriptional activity in cells.

Ligand cell permeability correlates with the lactone–zwitterion equilibrium constant and logD7.4 values.

Abstract

Understanding cellular processes requires tools to measure and manipulate biomolecules in living systems. Self-labeling tags, such as the HaloTag, enable the attachment of synthetic molecules to specific proteins inside cells. Creating ligands for these systems with more than one chemical motif remains challenging due to competing demands between cell permeability and functionality. We found that multifunctional ligands based on certain rhodamines efficiently entered cells and enabled affinity purification of mitochondria or translocation of nuclear proteins; the performance of these molecules could be verified by fluorescence microscopy. These compounds are useful for a variety of biological experiments, and our general framework will allow the design of other multifunctional ligands to study living systems.

Enzyme-based self-labeling tags enable the covalent attachment of synthetic molecules to proteins inside living cells. A frontier of this field is designing cell-permeable multifunctional ligands that contain fluorophores in combination with affinity tags or pharmacological agents. This is challenging since attachment of additional chemical moieties onto fluorescent ligands can adversely affect membrane permeability. To address this problem, we examined the chemical properties of rhodamine-based self-labeling tag ligands through the lens of medicinal chemistry. We found that the lactone–zwitterion equilibrium constant (KL–Z) of rhodamines inversely correlates with their distribution coefficients (logD7.4), suggesting that ligands based on dyes exhibiting low KL–Z and high logD7.4 values, such as Si-rhodamines, would efficiently enter cells. We designed cell-permeable multifunctional HaloTag ligands with a biotin moiety to purify mitochondria or a JQ1 appendage to translocate BRD4 within the nucleus. We found that translocation of BRD4 to constitutive heterochromatin in cells leads to apparent increases in transcriptional activity. These fluorescent reagents enable affinity capture and translocation of intracellular proteins in living cells, and our general design concepts will facilitate the design of multifunctional chemical tools for biology.

Figures