Lucinskaite, E. | Search bioRxiv

bioRxiv2024

Reduced non-specific binding of super-resolution DNA-PAINT markers by Shielded DNA-PAINT labeling protocols

The DNA-based single molecule super-resolution imaging approach, DNA-PAINT, can achieve nanometer resolution of single targets. However, the approach can suffer from significant non-specific background signals originating from non-specifically bound DNA-conjugated DNA-PAINT secondary antibodies as shown here. Using dye-modified oligonucleotides the location of DNA-PAINT secondary antibody probes can easily be observed with widefield imaging prior to beginning a super-resolution measurement. This reveals that a substantial proportion of DNA probes can accumulate, non-specifically, within the nucleus, as well as across the cytoplasm, of cells. Here, Shielded DNA-PAINT labeling is introduced, a method using partially or fully double-stranded docking strand sequences, prior to labeling, in buffers with increased ionic strength to greatly reduce non-specific interactions in the nucleus as well as the cytoplasm. This new labeling approach is evaluated against various conditions and it is shown that applying Shielded DNA-PAINT can reduce non-specific events [~]5 fold within the nucleus. This marked reduction in non-specific binding of probes during the labeling procedure is comparable to results obtained with unnatural left-handed DNA albeit at a fraction of the cost. Shielded DNA-PAINT is a straightforward adaption of current DNA-PAINT protocols and enables nanometer precision imaging of nuclear targets with low non-specific background.

biophysics↗

bioRxiv2023

Analysis of RyR2 distribution in HEK293 cells and mouse cardiac myocytes using 3D MINFLUX microscopy

The cardiac type 2 ryanodine receptor (RyR2) is a large homotetramer of a [~]560 kD subunit and is the molecular pathway through which the majority of Ca2+ enters the cytosol during cardiac activation. It constitutes the molecular basis of the process of calcium-induced calcium release where activation of RyR2s can be locally regenerative giving rise to local release events termed Ca2+ sparks. Accordingly, the molecular distribution of RyR2 in cardiac myocytes has been of great interest. Here we present the first purely optical data of RyR2 distribution with sub-molecular resolution by applying 3D MINFLUX fluorescence super-resolution microscopy. We demonstrate that by using single-domain antibodies (sdABs) against fluorescent protein domains in engineered RyR2 fluorescent protein fusions we can determine the location of individual RyR2 subunits with high precision ([~]3 nm) in all directions. Combining MINFLUX with DNA-PAINT, to maximize detection efficiency, we measured in situ labeling efficiencies using NPC structures as reference and regularly achieved efficiencies around 50%, which would translate to RyR2 detection efficiencies close to 95%, i.e. the probability that at least one subunit is detected, if target accessibility is similar. Using this approach, we detect dense and extended RyR2 expression in HEK cells with some clusters spanning several micrometres in extent and containing several hundred RyR2s. Similarly, myocytes from PA-RFP RyR2 mice contained large clusters containing many tens of RyR2s. The new data also suggests a resolution to apparent discrepancies between previous data from electron microscopy and super-resolution data. The methodology developed here will be critical to reveal the full complexity of RyR2 and related Ca2+ handling proteins in 3D as well as their relationship to contractile function. Our new approaches should be applicable to other multi-subunit complexes in cardiac muscle and other cell types.

physiology↗