PCNA as Protein‐Based Nanoruler for Sub‐10 nm Fluorescence Imaging

Abstract

Super‐resolution microscopy has revolutionized biological imaging enabling direct insight into cellular structures and protein arrangements with so far unmatched spatial resolution. Today, refined single‐molecule localization microscopy methods achieve spatial resolutions in the one‐digit nanometer range. As the race for molecular resolution fluorescence imaging with visible light continues, reliable biologically compatible reference structures will become essential to validate the resolution power. Here, PicoRulers (protein‐based imaging calibration optical rulers), multilabeled oligomeric proteins designed as advanced molecular nanorulers for super‐resolution fluorescence imaging are introduced. Genetic code expansion (GCE) is used to site‐specifically incorporate three noncanonical amino acids (ncAAs) into the homotrimeric proliferating cell nuclear antigen (PCNA) at 6 nm distances. Bioorthogonal click labeling with tetrazine‐dyes and tetrazine‐functionalized oligonucleotides allows efficient labeling of the PicoRuler with minimal linkage error. Time‐resolved photoswitching fingerprint analysis is used to demonstrate the successful synthesis and DNA‐based points accumulation for imaging in nanoscale topography (DNA‐PAINT) is used to resolve 6 nm PCNA PicoRulers. Since PicoRulers maintain their structural integrity under cellular conditions they represent ideal molecular nanorulers for benchmarking the performance of super‐resolution imaging techniques, particularly in complex biological environments.

Fluorescence coding techniques for RNA detection

“…This approach allows for imaging with a resolution of up to 25 nm by precisely manipulating the binding and dissociation of DNA. 89,90 The study presents ten unique rectangular DNA origami structures that closely resemble the numerical digits from 0 to 9. The fluorescence switching between on and off states is achieved by two complementary DNA molecules: the target DNA, which is complementary to the DNA sequence of the chemical being tested, and the probe DNA, which has a fluorescent dye attached.…”