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Super-Resolved Protein Imaging Using Bifunctional Light-Up Aptamers.

Franziska Grün¹, Niklas van den Bergh^1,2, Maja Klevanski³

¹Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, 69120, Heidelberg, Germany.

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Summary

This summary is machine-generated.

Researchers developed bifunctional aptamers for protein imaging using single-molecule localization microscopy (SMLM). These aptamers enable super-resolution imaging of proteins with high precision and photostability, outperforming traditional antibody methods.

Keywords:

Fluorescent light-up aptamer Point accumulation for imaging in nanoscale topography (PAINT)Protein imaging Protein-binding aptamer Super-resolution imaging

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Bimolecular Fluorescence Complementation

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Published on: April 15, 2011

Area of Science:

Molecular and Cellular Biology
Biophysics
Microscopy

Background:

Single-molecule localization microscopy (SMLM) requires efficient protein labeling methods with small tags and suitable photophysical properties for visualizing biomolecular organization and interactions.
Fluorescent light-up aptamers (FLAPs) offer potential for SMLM, with RhoBAST showing promise due to its brightness, photostability, fluorogenicity, and rapid exchange kinetics.

Purpose of the Study:

To expand the utility of RhoBAST, a fluorescent light-up aptamer, for protein imaging by conjugating it with protein-binding aptamers.
To demonstrate the effectiveness of these bifunctional aptamers for high- and super-resolution imaging of proteins, including GFP-tagged proteins, using SMLM techniques.

Main Methods:

Fusion of RhoBAST with various protein-binding aptamers to create bifunctional aptamers for protein targeting.
Application of RhoBAST-PAINT, an SMLM technique utilizing the fluorogenic dye SpyRho, for super-resolution imaging in mammalian cell lines and primary neurons.
Comparison of bifunctional aptamer performance against standard antibody-based immunofluorescence protocols.

Main Results:

Demonstrated versatility of bifunctional aptamers using diverse protein-binding aptamers and FLAPs for protein imaging.
Achieved high- and super-resolution imaging of GFP-tagged proteins by fusing RhoBAST with the GFP-binding aptamer AP3.
Bifunctional aptamers showed superior performance compared to antibodies, being 7-fold smaller and exhibiting enhanced bleaching resistance.

Conclusions:

The developed bifunctional aptamers effectively enable super-resolution protein imaging via SMLM, expanding the application of RhoBAST.
This approach offers a powerful alternative to antibody-based methods, providing smaller probes with improved photostability for cellular imaging.