The Spherical Nucleic Acids mRNA Detection Paradox

We are publishing today “The Spherical Nucleic Acids mRNA Detection Paradox“, the outcome of an open science project which started with an Hns student last year. In the last 12 months, we have reported in quasi real-time our experiments, protocols and analyses in an open science notebook and shared the data on our repository. The data are also stored at FigShare (e.g. Electron Microscopy results).

In addition to being exciting scientifically (he says!), this has been an experiment in how do science in the open using the tools of the 21st century to share information and solicit feedback. It is therefore fitting to publish it on a platform that challenges conventional modes of peer review.

We have chosen ScienceOpen where publication happens immediately (a couple of hours from submission to publication), followed by open peer review. In the coming weeks and months, I hope that many scientists will provide their expert evaluation of our article. In particular, Chad Mirkin will be invited to provide a review.

This article is important to all scientists who are using nanoparticles for imaging and sensing inside living cells. It should also be particularly relevant to past, current and prospective customers of the SmartFlares. Here is the abstract:

From the 1950s onwards, our understanding of the formation and intracellular trafficking of membrane vesicles was informed by experiments in which cells were exposed to gold nanoparticles and their uptake and localisation, studied by electron microscopy.  In the last decade, building on progress in the synthesis of gold nanoparticles and their controlled functionalisation with a large variety of biomolecules (DNA, peptides, polysaccharides), new applications have been proposed, including the imaging and sensing of intracellular events. Yet, as already demonstrated in the 1950s, uptake of nanoparticles results in confinement within an intracellular vesicle which in principle should preclude sensing of cytosolic events. To study this apparent paradox, we focus on a commercially available nanoparticle probe that detects mRNA through the release of a fluorescently-labelled oligonucleotide (unquenching the fluorescence) in the presence of the target mRNA. Using electron, fluorescence and photothermal microscopy, we show that the probes remain in endocytic compartments and that they do not report on mRNA level. We suggest that the validation of any nanoparticle-based probes for intracellular sensing should include a quantitative and thorough demonstration that the probes can reach the cytosolic compartment.

The paper will be typeset in the next few days and open peer review will be open from that point. Comments are already possible. Thank you to Dave Mason, Gemma Carolan, Joan Comenge and Marie Held for their contributions to this work.



  1. Raphaël, it is great to see that your work is taking shape. A few comments:
    1) A suggestion for readability: I recommend that each fluorescence image be labeled in the corner with the name of the probe being used, particularly in the overlays, where the labels would best be in the same color as the pseudocolor.
    2) The legend to Figure 4 seems to indicate that 4B is stained with a secondary antibody in the phrase “…secondary antibody (B,E,H)”, but the first sentence indicates that the fluorescence in 4B is the fluorescent dextran, no secondary antibody involved (hence my comment 1 above to clarify such ambiguities).
    3) Regarding the cells lacking SmartFlare signal in Fig. 4D and 4G that are labeled with antibody in 4E and 4H, some of the SmartFlare fluorescence may be lost during the post-fixation process. The similar degree of labeling in 4A as in 4B, which I interpret to be pre-fixation, would support this. I don’t think that would change your conclusion about the degree of colocalization though (unless the SmartFlare signal moves from one location to another, which seems unlikely).
    4) You should check the degree of colocalization with mitochondria, in addition to the endocytic pathway compartments that you show. The Mirkin StickyFlare article found the signal to be predominantly mitochondrial in HeLa cells, which they interpreted to mean that the target mRNA is associated with mitochondria. However, if your hypothesis is correct that the cyanine dye-labeled oligonucleotides detach from the gold nanoparticles while still in the endocytic compartments, the Cy-oligos may be translocated to another location, such as mitochondria, as has been noted in the literature from 3 different labs (PMID 20147460, 21600074, 23946765).
    5) The lack of a change in VEGF SmartFlare signal between untreated and DMOG-treated cells in Fig. 3C-E is a key observation. The failure of SmartFlare to detect changes in expression of target mRNAs indicates that SmartFlare is detecting something other than the target mRNAs. I think it is very important that you nail down this observation by demonstrating the degree of DMOG-induced VEGF mRNA expression in your system in addition to referring to the literature. A qRT-PCR measurement of VEGF mRNA would be easiest and sufficient; RNA-FISH would be ideal (but it may not be feasible to do SmartFlare and RNA-FISH in the same samples due to loss of the SmartFlare oligo as I mentioned in comment #3).

    – Luke Armstrong


    1. Thanks Luke for your comments and advice. We are considering those, together with Gal’s, and hopefully some more via the ScienceOpen publication interface soon (we will certainly add the qRT-PCR data in the revised version).


  2. Dear Raphaël,
    Very interesting story! Thanks for sharing the results. We made similar observations: punctate fluorescence, fluorescent scrambled flare etc. We used different cell lines like CHO, Hela, HEK293. The result was always the same and the support from Millipore ridiculous.
    Did you tried other methods to image RNA in living cells? What about RNA-aptamers like Spinach or Malachite Green? Any experience on those?

    Thanks and cheers,

    Liked by 1 person

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