April 2, 2020
by Thomas Porter
Since our last meeting, we've been working on a variety of problems with the DNA cages. The non-specific binding problems have been all but eliminated by incubating the DNA cages with 1% BSA in PBS for 1 hour before cell labeling. Cell labeling of actin, which is an intracellular target, was also achieved. And, that signal was erased with about 40% efficiency over the course of 20 minutes. The erase efficiency increased to about 60-80% (depending on the area) after the samples were washed. These results are very promising, since there had been some uncertainty about the DNA cages being able to get inside fixed cells.
We also completed some initial work with tissue. We were able to confirm that the solutions used in tissue labeling don’t disrupt the DNA cage functionalities. The DNA cages are able to penetrate tissue slices. The erase solution does not damage these slices. All good news.
I am pursuing the original QD-DNA-gold nanoparticle linking and delinking strategy for achieving stochastic blinking behavior in labelled cells. The QD-DNA conjugates have been prepared using a phytochelatin-3 (PC3) coating with single-stranded DNA conjugated via copper free click chemistry. In my previous post, I reported optimizing QD storage conditions by keeping the QDs under argon in an air-free solution to avoid oxidation.
This month, my focus was on gold nanoparticle (AuNP) - DNA conjugation. Previously, I had been getting disappointingly large aggregates of AuNPs after the conjugation reaction, discovered by using dynamic light scattering, which measures nanoparticle diffusion speed in a solution to estimate the nanoparticle radius. Large aggregates of AuNPs could give inconsistent photoswitching behavior because the ratio of fluorescence donor (QDs) to quencher (AuNPs) would be highly variable within a given sample as well as from sample to sample. And, aggregated particles are just not feasible for use in biological imaging applications. This last month, by tuning reaction timings and salt concentrations, I was able to greatly reduce aggregation during the conjugation step. In future experiments (when labs open up again someday...), I hope to apply these new insights on QD-DNA and AuNP-DNA conjugates to finally test photoswitching behavior.