A genetically targetable near-infrared photosensitizer

Abstract

Upon illumination, photosensitizer molecules produce reactive oxygen species (ROS) that can be utilized for functional manipulation of living cells, including protein inactivation, targeted damage introduction, and cellular ablation. Photosensitizers used to date have been either exogenous, resulting in delivery and removal challenges, or genetically encoded proteins that form or bind a native photosensitizing molecule, resulting in a constitutively active photosensitizer inside the cell. By binding a heavy-atom substituted fluorogenic dye with a genetically encoded Fluorogen Activating Protein (FAP), we demonstrate an ‘on-demand’ activated photosensitizer that produces singlet oxygen and fluorescence only when FAP-bound and activated with near infrared light. This Targeted and Activated Photosensitizer (TAPs) approach enables protein inactivation and targeted cell killing in cultured cells and rapid targeted lineage ablation in living larval and adult zebrafish. The near-infrared excitation and emission of this FAP-TAPs photosensitizer module provides a new spectral range for photosensitizer proteins, useful for imaging, manipulation and cellular ablation deep within living organisms.

Spectral properties of the zebrafish visual motor response

“…Our data indicate that this should be straightforward. For example, the excitation spectrum of dL5/MG2I [14] is almost entirely non-overlapping with the green light spectrum used in the present study. This suggests that visual motor responses could be elicited using the 514nm green channel, both before and after chemoptogenetic ablation driven by far red light at 660nm.…”

“…Consequently, there is no information on the spectral sensitivity of the VMR. In addition to helping identify the receptors mediating the response, this information will be important for exploiting approaches that are currently being developed for light-dependent ablation of genetically-targeted neuronal groups [14, 18]. In order to employ the VMR as a functional endpoint in neuro-ablation studies using these reagents, it will be critical to elicit the response using light wavelengths that do not overlap with the excitation spectra of photosensitizers.…”

Fluorogenic Protein‐Based Strategies for Detection, Actuation, and Sensing

“…However, longer wavelength photoablation probes allow for greater tissue penetration for use in other complex organisms or tissue samples. To overcome the limitation of blue light excitation, FAP‐based systems utilizing malachite green derivatives substituted with heavy atoms have been developed . The resulting system is excited at 666 nm, which is more adapted for deep tissue imaging.…”

Subcellular Redox Targeting: Bridging in Vitro and in Vivo Chemical Biology

“…Similar experiments have been performed in D rerio . 155 This system employed a f luorescence a ctivating p rotein— t argeted and a ctivated p hotosensitizer (FAP-TAP), a domain that binds a specific otherwise inefficient fluorophore, enhancing its fluorescence. The protein/fluorophore pair was optimized in vitro to produce high levels of singlet oxygen, when exposed to near-IR light.…”