Discussion Points from Honours Project Report

With the Undergraduate project reports now marked, Gemma has kindly agreed to post her project report on the blog. You can find a pdf of the report here.

As most of the methods and results are already on the blog, a slightly abridged and edited version of the discussion section is replicated below:

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In this study we have used the commercially available oligonucleotide functionalised gold nanoparticles known as SmartFlare RNA detection probes to try and detect VEGF mRNA expression within live HeLa cells. These probes are sold as being able to enter cells without addition agents, are non-toxic to cells and are able to gain access to the cytosol to detect specific mRNA in live cells, however we have not found this to be the case in the experiments we have carried out.

A consistent observation seen throughout the experiments that raises many questions is the punctate signals for all three SmartFlares. The punctate nature of the signals, which suggests that the SmartFlares are contained within endosomes, is unexpected in the sense that SmartFlares are meant to escape these compartments, gain access the cytosol where if mRNA is detected a diffuse fluorescent signal is produced.

To investigate if the SmartFlares are becoming entrapped within endosomes, we carried out immunostaining experiments. The antibodies selected were anti-LAMP1 to mark lysosomes and anti-transferrin receptor to mark the recycling compartments. The imaging results were consistent with ones obtained previously with the live experiments as the cells show varied uptake of the SmartFlares and the signals are mostly punctate.

Interestingly there is not complete co-localisation of the SmartFlares with FITC-dextran, which shows that they are not always found within the same compartments. This could suggest that they are being taken up separately from the FITC-dextran or there is possibly another mode of uptake causing them to be in different compartments.

This problem of possible containment within endosomes has been investigated before by the group of Chad Mirkin where they used confocal immunofluorescence to characterize the subcellular localization of spherical nucleic acids (SNAs), which are the basis of SmartFlares. The results of this experiment actually concluded that the SNAs appear to be endocytosed and become sorted into early endosomes (see Choi et al, 2013). The report goes on to conclude a success with their experiments stating that the SNAs provide a platform for various intracellular, in vivo application, but interestingly they do not discuss how the SNAs could escape endosomes which would be key for many intercellular uses (ibid).

An interesting observation was also seen for the three SmartFlares as they all showed similar signal intensities. Signal is expected for the Cy5-Uptake Control SmartFlare as it is designed to constantly fluoresce to show uptake of the SmartFlares. Also a signal can be expected for the Cy3-VEGF SmartFlares as these should fluoresce if mRNA is detected, but because of the punctate signal this suggests that VEGF mRNA is also contained within endosomes where hybridization should not take place. However a signal of similar intensity to the Cy5-Uptake Control and Cy3-VEGF SmartFlares was not anticipated for the Cy5-Scramble Control SmartFlares as these are designed to have no endogenous mRNA targets. This suggests that the oligonucleotides or reporter molecules are becoming cleaved or dissociating from the SmartFlares during the endocytic process.

DMOG treatment was carried out on cell samples of each SmartFlare. As DMOG is a PHD inhibitor which leads to the stabilization of HIF-1α it is expected to cause an increase in the levels of VEGF mRNA, which should create an increased signal for the Cy3-VEGF SmartFlare. To confirm that DMOG treatment causes an increase in VEGF mRNA quantitative PCR was carried out. The results showed that a one-day treatment of 500 nM DMOG caused a 13-fold increase in VEGF mRNA compared to normoxic cells. A similar effect was expected with the Cy3-VEGF SmartFlares however this did not appear to be the case. With the Cy5-Scramble Control being a negative control it’s signal should have been lower than the other SmartFlares, but a similar signal is seen.

In, conclusion, our experiences in using Merck Millipore’s SmartFlare RNA detection probes indicate that the probes do not work as advertised with all of our images showing varied uptake and punctate signal for the SmartFlares suggesting that they are not escaping endosomes. A very beneficial next step would be to investigate if endosomal processing is causing the punctate signals we have seen. It would also be interesting to see if the SmartFlares could be directly microinjected into the cells to see if they do then work as advertised, detecting the mRNA and giving a diffuse signal.

Authors: Gemma Carolan & Dave Mason