New data on SmartFlare – do they detect mRNA?

Almost exactly a year ago, I wrote a post regarding my concerns with SmartFlare, supposedly a novel method for live imaging of RNA in cells.

In a nutshell, SmartFlare are gold nanoparticles covered in oligos specific to a certain mRNA of interest. Supposedly, cells internalize these particles and, once the mRNA hybridize to the oligo, a complementary fluorecently labeled oligo is being unquenchhed and “flares”, indicating the present of said mRNA.

You can read about my concerns in that older post, but apparently I wasn’t the only one concerned about their validity.

Raphaël Lévy from U. of Liverpool (UK) was concerned as well. He endeavored into an open science project to try and answer his concerns (which is why I allow myself to openly review his paper).

Raphael’s concerns were that these gold nanoparticles are maintained in endosomes and do not reach the “cytoplasm” where mRNAs reside. Since he assumes mRNAs are abent from endosomes, any fluorescent signal that we detect is an artefact.

He used several imaging approaches to test his hypothesis: electron microscopy (EM), confocal microscopy, photothermal microscopy
(I had to read about it, there’s no explanation in the text what it measures, really) and immunofluoresence.

Unfortunately, Raphael did not perform any assesment of mRNA levels, not by RT-PCR, nor FISH. I think that for a paper that is supposed to test the mRNA detection capacity of SmartFlare, one needs to confirm that the mRNA to be detected is present in this particular system, that it responds to the treatment as published and that it does or does not co-localize with the SmartFlare.  This is my main complaint about this paper, but not the only one.

Here are some of my other concerns/problems with this paper:

1. The paper starts by EM examination of gold particles uptake by the cells to examine particle size. Next, they use confcal microscopy to detect uptake. They mention that only ~1/4 of the cells uptake the particles. Is this common? Is it a matter of concentration? What is the range of uptake (particles/cell)?  Is it from 1 to 1000? Or do you see some cells with dozens of spots and all others have zero? [One should have the numbers from the EM studies] What would explain that?
2. Based on the punctate images, they assume that these are endosomes. They then go through a series of imaging experiments to show that:

• EM images which show the particles surounded by membranes. But I am not sure what I see. My advice – label all components in the image (plasma membrane, endosomes, mitochondria, etc…).
• co-localization with fluorescent dextran (which, I assume, is taken into endosomes). I’m not sure I understant what that number represents. Is it  % overlap? The test says “rarely overlap” but to my eyes this is clearly not the case. It appears to fairly ovelap.
• co-localization with transferrin-containing vesicles or lysosomes. Though it is clear that there’s no co-loc with the transferrin, to my eyes there’s high co-loc with lysosomes. Yet the text reads “little overlap”.
• Anyway, I suggest having a higher mag or zoomed-in image for all, and a clearer expression of co-loc levels.
• If the authors are right and its not endosomse or lysosomes, then what are these vesicles?  The authors do not suggest any alternative or ways to test that.

3.  To test if SmartFlare responds to changing mRNA levels, they treated the cells with a drug which, supposedly, induces an increase of VEGF mRNA levels (VEGF is the mRNA tested here).

• First of all, the authors do not show that VEGF is indeed expressed in these cells (HeLa) under their growth conditions.
• Second, they do not show that these cells respond to the drug as previously published. They quote 3 papers which showed a range of 2-10 fold increase (none of the cells that were examined were HeLa cells), each paper uses a different concentration of this drug. (Raphael does not mention the concentration and duration of treatment). These are important controls.
• Third, we know today that not all cells in the culture are equal. Some respond faster, some respond to higher levels than others. Since only 1/4 of the cells internalize SmartFlare (with even or odd distribution?), it is possible that, by chance, most of the cells (how many were imaged?) that Raphael examined are those which responded poorly to the drug.
• The best way would be to image SmartFlare with FISH in these cells. This wil give you a) expression levels of VEGF; b) is there a correlation between expresion level and SmartFlare fluorescence; and c) enable co-localization analysis of the mRNA and SmartFlare fluorescence (is the mRNA in these vesicles?).

4. There is another option why there’s no increase in SmartFlare signal upon increase in mRNA levels – the system is already saturated. This is a matter of mathematics: on the one side, how many mRNA molecules/cell (dozens? hundreds? thousands?). On the other hand – how many particles/cell and how many oligos/particle? Do these numbers match?

• I would do the reciprocal experiment – to reduce (even to zero) the VEGF mRNA level (e.g. by siRNA, knock/out). Then, see if SmartFlare fluorescence is reduced, in correlation to mRNA levels in a particular cell.

5. Throughout the text, Raphael assumes that mRNA molecules are absent from endosomes. Is that proven? Since there’s a whole field of study that shows mRNAs in exosomes (extra-cellular vesicles derived from the multivesicular body), I can assume that at least some mRNAs are indeed encapsulated in vesicles inside cells. This, of course, needs to be tested on a case by case basis (VEGF in this case).

 

A minor point:

In the introduction (and then the discussion) Raphael mentions in vitro and in situ methods to detect mRNAs, then goes on to say how great it is if we had a system to visualize mRNA in live cells. Well, we do, more than one. We have MS2-like systems that allow live tracking of mRNAs at the single molecule level. There are also GFP-mimic RNA aptamers (Spinach, Broccoli, RNA-Mango) which enable visualization of RNA in live cells. So SmartFlare is only the 3rd option.

 

Overall, the only thing that was convincing, to me, was that SmartFlares are encapsulated in some kind of vesicles. I am not concinved that they do not detect mRNA inside.

David Mason,, Gemma Carolan,, Marie Held,, Joan Comenge,, & Raphael Levy (2015). The Spherical Nucleic Acids mRNA Detection Paradox ScienceOpen Research DOI: 10.14293/S2199-1006.1.SOR-CHEM.AZ1MJU.v1