MIiSR: Molecular Interactions in Super-Resolution Imaging Enables the Analysis of Protein Interactions, Dynamics and Formation of Multi-protein Structures
Fig 2
Validation of Spatial Association Analysis (SAA) using defined-length DNA oligomers.
(A) SAA histogram plotting the distance between non-complementary 30-mer DNA oligomers 5’ labeled with Cy3 and Cy5. The vertical dotted line indicates the Colocalization Distance Criterion (CDC), the separation distance below which molecules are potentially interacting. To determine if the degree of colocalization is significant the positions of the Cy5-labeled molecules were randomized using a Monte Carlo approach and the SAA measurement repeated (SRP). (B) SAA histogram plotting the distance between annealed, complementary 30-mer DNA oligomers 5’ labeled with Cy3 and Cy5. (C) Measured and predicted intermolecular distances between Cy3 and Cy5 on differing length complementary DNA oligos 5’ labeled with Cy3 and Cy5 deposited on coverslips at equimolar concentration. N/C indicates the measured distance of non-complementary Cy3 and Cy5 labeled oligos deposited at the same concentration. (D) Impact of partial intermolecular interactions on SAA analysis, determined by annealing complementary 30-mer DNA oligomers 5’ labeled with Cy3 and Cy5 at differing ratios. SRP = Simulated Random Positions. A-B: Representative histograms, C-D: Data are presented as mean ± SEM. n = 3, minimum of 5 images per experiment. n.s. = not significantly different, paired t-test, † = p < 0.05 compared to SRP, * = p < 0.05 compared to Cy5:Cy3 at a 1:1 ratio.