Not too long after publishing version 1 of the SmartFlare paper at Science Open, we received a couple of very helpful reviews. We’ve been working on the points made by the reviewers to improve the manuscript for version 2 (coming very shortly!). Below are some thoughts on a couple of the comments and a quick update on some new data in the works.
Update: we have submitted a Letter to PNAS as a response to the paper discussed below. It is currently available as a preprint.
The Mirkin group recently published the latest paper to use Spherical Nucleic Acid (SNA) technology that we’ve come to know and love as Smart/Nano Flares. In an interesting twist, instead of having target oligos bind to the nanoparticle (displacing fluorescent reporter sequences), the reporter sequences are now complimentary to the target oligos, labelling them directly. In theory this allows for dynamic labelled, tracking and quantitation of mRNA in cells.
As mentioned before, one of the things that has been bugging me about the SmartFlares is that they’re not where anyone expects them. EMD expect them in the cytosol (we don’t see them there), I expected them in endosomes and they weren’t really there either. So what could this mystery compartment be?
Throughout all of the fluorescent microscopy experiments, we’ve been seeing SmartFlare fluorescence in punctate structures. Using these techniques, it’s very difficult to say with certainty that the SmartFlares are within vesicles or (for example) aggregated/agglomerated in the cytosol. As this is pertinent to the current investigation, we took cells loaded with SmartFlares to the electron microscope which has the resolution to discern membrane structures as well as the gold particles.
We’ve so far, not seen any response of the VEGF SmartFlares to treatment of our cells with DMOG. Combined with the punctate distribution of the signal, we have no evidence that the SmartFlares make it to the cytosol in 18 hours. As we’re not sure if the problem lies in the SmartFlares or the delivery, I microinjected the SmartFlares directly into the cells thus bypassing the need for endosomal uptake.
In a recent post, I was musing about why the SmartFlares weren’t in the swollen chloroquine-induced vacuoles. Out of interest more than anything, I did a quick experiment to see what would happen if I pre-labelled the endocytic pathway with one coloured dextran, then added in a second with the chloroquine.