StickyFlares

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.

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Probing for Caveosomes

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?

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TEM of VEGF SmartFlares after uptake

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.

2015-07-14-TEM01 Continue reading

Discussion Points from SmartFlare Group Meeting

I recently discussed the SmartFlare project at a joint Sée and Lévy Group Meeting, partially extolling the virtues of Open Science but mostly focussing on the data acquired so far and some of the proposed experiments.

By Andreas E. Neuhold[CC BY 3.0], via Wikimedia Commons
Regarding the SmartFlare project, the three big questions that formed the basis of the talk were:

  • Why do only some cells take up SmartFlares?
  • Where are the SmartFlares?
  • Why are the VEGF and Scrambled SmartFlares fluorescent in puncta?

Below are some of the interesting points that were made during the meeting. Replies at the time (as best as I can remember them) in green:

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Interpretation of chloroquine results

The last post highlighted some of the results of using chloroquine to investigate the localisation and functionality of the SmartFlares. This post will deal solely with the interpretation of those results.

2015-06-10-chloroquine

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Effect of Chloroquine on SmartFlare Distribution and Intensity

In order to investigate why the SmartFlares seem to be fluorescent in puncta within the cells, we designed an experiment to try to inhibit endosomal maturation, thus (hopefully) preventing SmartFlare processing and release of fluorescence.

2015-06-02-Endosomal_Maturation

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