Fluorescent Polymer-AS1411-Aptamer Probe for dSTORM Super-Resolution Imaging of Endogenous Nucleolin.

Abstract

Nucleolin is a multifunctional protein involved in essential biological processes. To precisely localize it and unravel its different roles in cells, fluorescence imaging is a powerful tool, especially super-resolution techniques. Here, we developed polymer-aptamer probes, both small and bright, adapted to direct stochastic optical reconstruction microscopy (dSTORM). Well-defined fluorescent polymer chains bearing fluorophores (AlexaFluor647) and a reactive end group were prepared via RAFT polymerization. The reactive end-group was then used for the oriented conjugation with AS1411, a DNA aptamer that recognizes nucleolin with high affinity. Conjugation via strain-promoted alkyne/azide click chemistry (SPAAC) between dibenzylcyclooctyne-ended fluorescent polymer chains and 3'-azido-functionalized nucleic acids proved to be the most efficient approach. In vitro and in cellulo evaluations demonstrated that selective recognition for nucleolin was retained. Their brightness and small size make these polymer-aptamer probes an appealing alternative to immunofluorescence, especially for super-resolution (10-20 nm) nanoscopy. dSTORM imaging demonstrated the ability of our fluorescent polymer-aptamer probe to provide selective and super-resolved detection of cell surface nucleolin.