Small Fluorogenic Amino Acids for Peptide-Guided Background-Free Imaging

Abstract

The multiple applications of super-resolution microscopy have prompted the need for minimally invasive labeling strategies for peptide-guided fluorescence imaging. Many fluorescent reporters display limitations (e.g., large and charged scaffolds, non-specific binding) as building blocks for the construction of fluorogenic peptides. Herein we have built a library of benzodiazole amino acids and systematically examined them as reporters for background-free fluorescence microscopy. We have identified amine-derivatized benzoselenadiazoles as scalable and photostable amino acids for the straightforward solid-phase synthesis of fluorescent peptides. Benzodiazole amino acids retain the binding capabilities of bioactive peptides and display excellent signal-to-background ratios. Furthermore, we have demonstrated their application in peptide-PAINT imaging of postsynaptic density protein-95 nanoclusters in the synaptosomes from mouse brain tissues.

Keywords: Fluorescence; Microscopy; Probes; Proteins; Super-Resolution.

Figure 1

Chemical synthesis of nitrobenzodiazole amino acids. The table summarizes the optical properties of all amino acids. Relative quantum yields (QY) were determined in DMSO using rhodamine B as a standard.

Figure 2

a) Chemical synthesis of lipid analogues of the benzoselenadiazole amino acid 5. b) Representative spectra (from 3 experiments) of compound 13 (10 μM) in phosphate buffer saline (PBS, black) or in PBS containing phosphatidylcholine (PC)‐based liposomes (red). Excitation wavelength: 500 nm. c) Wash‐free microscopy images of liposomes after incubation with compound 13 or BODIPY‐FL at the indicated concentrations. d) Representative histograms of signal‐to‐background (S/B) ratios in PC liposomes after imaging with BODIPY‐FL or compound 13 (both 1 μM). Data as means±SD (n=8).

Figure 3

a) Schematic illustration of a complementary peptide imaging pair using the amino acid 5 as the direct binding reporter. The peptide 5‐101A is regarded as the “imager peptide” and the peptide biotin‐101B as the “anchor peptide”. b) Synthetic scheme for the preparation of the peptide 5‐101A using standard protocols in SPPS. For synthetic details, see Figure S7 and Supporting Information. c) Representative fluorescence microscopy images showing the binding between 5‐101A (100 nM, green) and biotin‐101B (100 nM) in the presence of AlexaFluor647 (AF647)‐streptavidin (17 nM, red). Merge fluorescence signals displayed in yellow. Similar contrast was observed between fluorescence images with 5‐101A (wash‐free) and fluorescence images with AF647‐streptavidin (after 3 washes with PBS).

Figure 4

a) Chemical structure of the PSD95‐binding peptide 16 highlighting the replacement of Ile in the original sequence by amino acid 5. b) Representative fluorescence microscopy images of PSD95‐HaloTag within a synaptosome labeled with SiR‐HaloTag ligand (40 μM, top: diffraction limited image) and peptide 16 (500 nM, bottom: peptide‐PAINT image). Scale bar: 500 nm. c) Montage of frames, starting from the top left frame and going from left to right and top to bottom with 1 second separation between frames, from time‐lapse fluorescence microscopy of a single synaptosome after incubation with peptide 16 (Movie S2 in Supporting Information). Scale bar: 500 nm. d) Longitudinal plot of fluorescence emission of peptide 16 within the synaptosome shown in c. The peaks “ON” indicate the binding of peptide 16 to the PDZ domain and the troughs “OFF” indicate the dissociation of peptide 16 from the PDZ domain. e) Representative fluorescence microscopy images from different synapse subtypes as defined by the number of PSD95 nanoclusters per postsynaptic density (PSD). The left panels display peptide‐PAINT SR microscopy images of PSD95 in synaptosomes, and the right panels display the same images after nearest neighbor (NN) analyses, which feature high local density in the protein nanoclusters. Scale bar: 500 nm. f, g) Representative histograms of the mean local density (top, determined by counting the number of neighbors within a radius of each molecule scaled to the mean density in its synaptosome) and the effective map resolution for each synaptosome (bottom).