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.

We designed novel 4'-modified 2'-deoxy-2'-fluorouridine (2'-F U) analogues with the aim to improve nuclease resistance and potency of therapeutic siRNAs by introducing 4'-C-methoxy (4'-OMe) as the alpha (C4'α) or beta (C4'β) epimers. The C4'α epimer was synthesized by a stereoselective route in six steps; however, both α and β epimers could be obtained by a nonstereoselective approach starting from 2'-F U. H NMR analysis and computational investigation of the α-epimer revealed that the 4'-OMe imparts a conformational bias toward the North-East sugar pucker, due to intramolecular hydrogen bonding and hyperconjugation effects. The α-epimer generally conceded similar thermal stability as unmodified nucleotides, whereas the β-epimer led to significant destabilization. Both 4'-OMe epimers conferred increased nuclease resistance, which can be explained by the close proximity between 4'-OMe substituent and the vicinal 5'- and 3'-phosphate group, as seen in the X-ray crystal structure of modified RNA. siRNAs containing several C4'α-epimer monomers in the sense or antisense strands triggered RNAi-mediated gene silencing with efficiencies comparable to that of 2'-F U.

Summary Agonist-promoted G-protein coupled receptor (GPCR) endocytosis and recycling plays an important role in many signaling events in the cell. However, the approaches that allow fast and quantitative analysis of such processes still remain limited. Here we report an improved labeling approach based on the genetic fusion of a Fluorogen Activating Protein (FAP) to a GPCR and binding of a sulfonated analog of the malachite green (MG) fluorogen to rapidly and selectively label cell surface receptors. Fluorescence microscopy and flow cytometry demonstrate that this dye does not cross the plasma membrane, binds with high affinity to a dL5** FAP-GPCR fusion construct, activating tagged surface receptors within seconds of addition. The ability to rapidly and selectively label cell surface receptors with a fluorogenic genetically encoded tag allows quantitative imaging and analysis of highly dynamic processes like receptor endocytosis and recycling.

Chemical modification is a prerequisite of oligonucleotide therapeutics for improved metabolic stability, uptake and activity, irrespective of their mode of action, i.e. antisense, RNAi or aptamer. Phosphate moiety and ribose C2′/O2′ atoms are the most common sites for modification. Compared to 2′-O-substituents, ribose 4′-C-substituents lie in proximity of both the 3′- and 5′-adjacent phosphates. To investigate potentially beneficial effects on nuclease resistance we combined 2′-F and 2′-OMe with 4′-Cα- and 4′-Cβ-OMe, and 2′-F with 4′-Cα-methyl modification. The α- and β-epimers of 4′-C-OMe-uridine and the α-epimer of 4′-C-Me-uridine monomers were synthesized and incorporated into siRNAs. The 4′α-epimers affect thermal stability only minimally and show increased nuclease stability irrespective of the 2′-substituent (H, F, OMe). The 4′β-epimers are strongly destabilizing, but afford complete resistance against an exonuclease with the phosphate or phosphorothioate backbones. Crystal structures of RNA octamers containing 2′-F,4′-Cα-OMe-U, 2′-F,4′-Cβ-OMe-U, 2′-OMe,4′-Cα-OMe-U, 2′-OMe,4′-Cβ-OMe-U or 2′-F,4′-Cα-Me-U help rationalize these observations and point to steric and electrostatic origins of the unprecedented nuclease resistance seen with the chain-inverted 4′β-U epimer. We used structural models of human Argonaute 2 in complex with guide siRNA featuring 2′-F,4′-Cα-OMe-U or 2′-F,4′-Cβ-OMe-U at various sites in the seed region to interpret in vitro activities of siRNAs with the corresponding 2′-/4′-C-modifications.