I am Gemma and I’m currently an honours student working with Raphaël as my project supervisor. This first blog post is an introduction to my project which aims to investigate how SmartFlares gain access to the cytosol.
SmartFlares are a commercially available adaptation of the NanoFlare technology developed by Dr. Chad Mirkin. These are a 13 nm gold nanoparticle core which are densely functionalised with specific 18 base-pair thiolated oligonucleotides. The flare part of the particle comes from a short cyanine dye-terminated reporter sequence that is hybridised to the oligonucleotides close enough to the gold core to be fluorescently quenched. The idea is that the particles can enter cells, detect and hybridise to a specific mRNA where they release their reporter flare signalling detection.
The SmartFlare we have ordered is specific to VEGF mRNA. We have chosen this mRNA as it can be upregulated in response to dimethyloxaloylglycine (DMOG) so we can evaluate the changes in the signal produced with or without DMOG treatment. There are also two other types of SmartFlare we have purchased that are controls. Uptake controls are designed with the fluorescent reporter conjugated to the particle via a non-hybridised oligonucleotide sequence but far enough from the gold core so that it will constantly fluoresce. The scramble control is designed to not detect anything so it should not become fluorescent and this can give a reading for background noise.
Our experiments will initially include characterisation of the SmartFlares, such as testing their colloidal stability, treating with DTT to assess the fluorescence of the reporters and dynamic light scattering to evaluate the size. To investigate the uptake we will take approaches with live and fixed cell imaging with cell compartment staining to see if/how the SmartFlares enter the cytosol. These experiments will be carried out with and without DMOG to evaluate changes in the signal. We will also compare the SmartFlare signals with the expected biological response measured via a quantitative polymerase chain reaction on VEGF with/without DMOG.
We aim to report the experimental design and results on a regular basis in the next few weeks on this blog.