Luciferase-induced photoreductive uncaging of small-molecule effectors

Eric Lindberg,Nicolas Winssinger,Marcello Anzola,Simona Angerani

doi:10.1038/s41467-018-05916-9

Abstract

Bioluminescence resonance energy transfer (BRET) is extensively used to study dynamic systems and has been utilized in sensors for studying protein proximity, metabolites, and drug concentrations. Herein, we demonstrate that BRET can activate a ruthenium-based photocatalyst which performs bioorthogonal reactions. BRET from luciferase to the ruthenium photocatalyst is used to uncage effector molecules with up to 64 turnovers of the catalyst, achieving concentrations >0.6 μM effector with 10 nM luciferase construct. Using a BRET sensor, we further demonstrate that the catalysis can be modulated in response to an analyte, analogous to allosterically controlled enzymes. The BRET-induced reaction is used to uncage small-molecule drugs (ibrutinib and duocarmycin) at biologically effective concentrations in cellulo.

Highlights

Bioluminescence resonance energy transfer (BRET) is extensively used to study dynamic systems and has been utilized in sensors for studying protein proximity, metabolites, and drug concentrations
We introduce LUciferase-based Photocatalysis Induced via Nucleic acid template (LUPIN), wherein the photocatalyst is conjugated to a nucleic acid (PNA) to create a high effective concentration of substrate capitalizing on the fast chemical transformation of nucleic acid-templated reactions at low substrate concentrations (Fig. 1c)
LUCID is a dynamic platform with three components fused together: a SNAP protein to conjugate a synthetic linker containing the dye–drug adduct, NLuc for bioluminescence, and a receptor protein that binds the drug. This construct responds to a drug by changing the proximity of the fluorophore to NLuc, and a LUCID developed for methotrexate (MTX) was shown to respond with an EC50 of 85 μM

Summary

Introduction

Bioluminescence resonance energy transfer (BRET) is extensively used to study dynamic systems and has been utilized in sensors for studying protein proximity, metabolites, and drug concentrations. We introduce LUPIN (luciferase-based photocatalysis induced via nucleic acid template), wherein the photocatalyst is conjugated to a nucleic acid (PNA) to create a high effective concentration of substrate capitalizing on the fast chemical transformation of nucleic acid-templated reactions at low substrate concentrations (Fig. 1c).

Results

Conclusion