Chiral Single Molecule Localization Microscopy (chiralSMLM)

Chiral fingerprints are unique to a molecule and convey information related to binding sites, conformation, and its immediate chemical environment. Deciphering this information is expected to aid a better understanding of the biological processes (protein trafficking, kinetics, and aggregation) in a cellular environment at the scale of a single molecule. Here, we present a new optical technique called chiralSM LM , to selectively detect chiral enantiomers (both left-handed and right-handed) of single molecules and generate a super-resolved map to understand the role of chirality associated with the biological processes in a cell. Accordingly, calibration, characterization, and optimization of the system are carried out using a polarized light source (linear polarized, left and right circularly polarized) and by recording known structures (Actin filaments) in a cell. The system is employed to carry out cell transfection studies on two different disease models (Hemagglutinin protein for Influenza type-A and NS3 protein for Dengue type-2) to understand the role of molecular chirality on disease specific biological processes leading to clustering. Single-molecule cluster analysis revealed that left-handed Dendra2-HA and Dendra2-NS3 molecules have a larger footprint, suggesting the role of chiral molecules in promoting cluster formation. In addition, the presence of left-handed molecules at the cluster-periphery is perplexing. This is interesting, as it demonstrates the active role of single molecule handedness (left or right) during protein clustering in a transfected cell. The new classification of single molecules purely based on their chiral nature is expected to advance single-molecule imaging and provide new insights in disease biology. Summary & Significance The chirality of single protein molecules is crucial to its functioning. This provides a new perspective on understanding biological functions, based solely on the chiral nature of proteins, and deciphers its role in a cellular processes. We developed a chiralSM LM microscopy system to access the role of chirality on the clustering behavior of viral proteins (HA for Influenza type-A and NS3 in Dengue type-2) in a cell. Hence, the technique offers a new approach to quantify chirality aided molecular interactions in disease biology.