Photon Biosciences LLC

A Biosinpired Genetically-expressible Dual-modality Peptide-based Probe for Super-resolution Correlative Light and Electron Microscopy - 1 Abstract 2 the Overall Goal of This Project Is to Rationally Engineer a New Genetically Expressible Lanthanide-based 3 Photostable Phosphorescent Probe With Time-integrated Single Molecule Brightness and High Electron Density 4 for Super Resolution Correlative Light and Electron Microscopy (CLEM) to Enable Elucidation of Cellular and 5 Detailed Subcellular Structures at the Pseudoatomic Level. Imaging/monitoring Single Molecules With Nanoscale 6 Spatiometric Resolution Within Cells Is an Essential Step for Investigating the Details of Cellular Processes. Super- 7 Resolution and Electron Microscopy Techniques Can Achieve the Resolution Required for Those Purposes and to 8 Those Ends, Fluorescent Probes Such as Fluorescent Small Molecules, Fluorescent Proteins (FPS), and Quantum 9 Dots (QDS) Have Become Essential Tools for Research and Medical Diagnostics. 10 Despite the Variety of Probes That Exist, There Is an Unmet Need for a Genetically Encoded, Dual-modality Probe 11 Suitable for Super-resolution Correlated Light and Electron Microscopy (clem). Such a Probe Would Have Time- 12 Integrated Single-molecule Brightness Necessary for Super-resolution Light Microscopy and Be Electron Dense 13 Enough to Allow Visualization/localization by Electron Microscopy, Especially Cryogenic Electron Tomography 14 (cet). to Meet Those Needs, Photon Biosciences Is Engineering a Novel and Innovative Enabling Bioinspired 15 Peptide-based Probe That Has the Time-integrated Single-molecule Brightness and Electron Density Necessary 16 for Super-resolution Clem. the Objective of This Phase I Project Is to Rationally Engineer a Probe to Have ≥25 17 LN3+-PHOSPHORESCENT Centers, Which We Hypothesize Will Have the Phosphorescence and Electron Density 18 Necessary to Surpass Current Single-modality Clem Probes and Then Show the Probe’s Versatility by Using It in 19 Cryo-luminescence Microscopy to Guide Focused Ion Beam (FIB) Milling of Pseudomonas Aeruginosa PAO1 20 Expressing a Crispr-cas Genome-edited Engineered Construct of Our Innovative Peptide Probe Fused to the 21 S15 Ribosomal Protein at Native Levels as a Proof-of-concept for Super-resolution Clem. Aim 1 Is to Optimize 22 Our Initial Peptide Probe via Rational Engineering for High Electron Density and Brightness. Aim 2 Is to Develop a 23 Multimeric Probe From Its Precursor by Concatenation and Increase Its Electron Density and Brightness Further. 24 Aim 3 Is to Demonstrate Proof of Concept by Cryo-luminescence Microscopy. Aim 4 Is to Demonstrate Further 25 Proof-of-concept by Using the Probe for Probe-guided Fib Milling and Tomography. Successful Completion of 26 This Project Will Result in an Electron Dense, Time-integrated Single Molecule Brightness, Genetically Expressible 27 Dual Modality Probe for Super-resolution Clem.