Tracking single molecules and interfering with their activity can be instrumental to understanding how living cells work. With the improvement in the resolution of microscopy methods, more of the cell is now accessible to the eye. In this context, choosing the right probe for imaging or manipulation experiments in living cells is more critical than ever.

Probes that label intracellular proteins should be specific for their target and not interfere with the protein's function, localization or expression. They should also be small enough so that they can access proteins in tiny corners of the cell. Genetically encoded probes are preferable, so they can be easily introduced in specific cells of interest or targeted to organelles.

Small, genetically encoded probes that bind to specific proteins are gaining users. Credit: Katie Vicari

To meet these challenges, researchers are increasingly turning to old friends such as intrabodies and aptamers. These probes have already received much attention for their putative pharmaceutical value, but they are now being used to develop biosensors and probes for basic biological studies.

Intrabodies are small, recombinant, antibody-like proteins that bind to specific antigens. They can be obtained from animals that naturally produce very small single-domain antibodies, such as camels or sharks, or they can be engineered from larger mammalian antibodies or other proteins such as fibronectin. Aptamers, even smaller probes, are either oligonucleotide or peptide based, and are created through engineering and selection.

The genes encoding for these 'mini-binders' can be fused to other genetically encoded elements or proteins to monitor and perturb cellular components and processes. In recent work, intrabodies have been tagged with fluorescent proteins to track proteins in small subcellular structures (Neuron 78, 971–985, 2013). They have also been fused to protein-degradation machinery to target the protein of interest for degradation under controlled conditions (Nat. Struct. Mol. Biol. 19, 117–121, 2011) or used as a scaffold to bring different molecular components together and drive gene expression (Cell 154, 928–939, 2013). Intrabodies that detect specific conformational states of the target protein can be used as biosensors to probe dynamic conformational changes with high spatiotemporal resolution in living cells (Nature 495, 534–538, 2013).

Further optimization of these probes for cellular applications will make their use easier and more wide-spread. But given their unique properties, these small binders are no doubt promising tools for biological experiments.