Beyond DNA: new probes for PAINT super-resolution microscopy

Abstract

In the last decade, point accumulation for imaging in nanoscale topography (PAINT) has emerged as a versatile tool for single-molecule localization microscopy (SMLM). Currently, DNA-PAINT is the most widely used, in which a transient stochastically binding DNA docking-imaging pair is used to reconstruct specific characteristics of biological or synthetic materials on a single-molecule level. Slowly, the need for PAINT probes that are not dependent on DNA has emerged. These probes can be based on (i) endogenous interactions, (ii) engineered binders, (iii) fusion proteins, or (iv) synthetic molecules and provide complementary applications for SMLM. Therefore, researchers have been expanding the PAINT toolbox with new probes. In this review, we provide an overview of the currently existing probes that go beyond DNA and their applications and challenges.

Fig. 1. An overview of the probes discussed in this paper. In the middle, the general idea behind PAINT approaches is illustrated. The four main categories for probes are: endogenous probes, engineered probes, fusion-based probes and synthetic probes. Schematics created with Biorender.com.

Fig. 2. Schematic overview of the considerations that have to be made while designing or choosing a PAINT probe. (A) The affinity of the probe towards its target. Too weak will give no signal or the acquisition time is long and too strong will not result in single molecule binding. (B) The choice of fluorophores is important. The color and photophysical properties are of importance as is the buffer conditions in which is being imaged. (C) For imaging in cells, a choice between live and fixed cells has to be made. Furthermore, the permeability and the stability of the probe should be optimized. During imaging, the pool of probes should be replenished and the lasers should not be toxic to live cells. Schematics created with Biorender.com.

Fig. 3. Three examples of endogenous probes as discussed above. (A) IRIS uses (fractions of) endogenous transient interaction partners to the target of interest, e.g. the lifeAct fragment coupled to a fluorophore for PAINT acquisition of actin filaments. (B) Motor-PAINT uses purified active kinesins to reconstruct the microtubule cytoskeleton and directly infer the orientation of the microtubules. (B) Glyco-PAINT and uPAINT are live-cell imaging methods that can be used to track the motion of receptors on the cell membrane. The probe binds to the receptor with a certain k_on, moves along with the receptor, until it is released again (k_off). Schematics created with Biorender.com.

Fig. 4. Two examples of engineered probes. (A) High affinity aptamers can be adapted by mutations in the sequence, resulting in probes with a lower affinity, but with the same specificity. (B) FDSAs are antibody fragments that are rationally engineered by introducing point mutations in the binding site. This results in antibody fragments with a lower affinity towards their target, while the specificity is maintained. Schematics created with Biorender.com.

Fig. 5. Schematic examples of two fusion-protein approaches. (A) A combination of a HaloTag on the protein of interest and a transiently binding fluorescent ligand opens up the possibility to do PAINT on both live and fixed samples. (B) By directly fusing a fluorophore to a transiently binding molecule, that is in turn binding to a peptide fused to the protein of interest, fixation compatible peptide-PAINT can be performed. Schematics created with Biorender.com.

Fig. 6. Schematic examples of synthetic probes. (A) iPAINT uses polyethylene glycol (PEG) to characterize interfaces by non-invasive binding. (B) FmocFF and Thioflavin T use a similar principle, in which a single component of a dynamic system is fluorescently labeled and used for imaging of the dynamics and characteristics of a material. Schematics created with Biorender.com.

Marrit M. E. Tholen

Roderick P. Tas

Yuyang Wang

Lorenzo Albertazzi