Imaging the localisation of SmartFlares – experimental design

As discussed in a previous post, the punctate distribution of all of the SmartFlares (SFs) is both unexpected and curious. In order to better understand where the SFs are localised in the cell, we would like to do a fairly routine co-staining experiment.

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Use of dimethyloxalylglycine (DMOG)

The SmartFlare that we have chosen for this project is specific to vascular endothelial growth factor (VEGF) mRNA.

VEGF was selected as the target because it’s transcript becomes increased in a cell as part of a characterized response to hypoxia with the aid of hypoxia inducible factor-1 (HIF-1). Active HIF-1 is a heterodimer of α and β subunits, levels of HIF-1α are kept low by oxygen-dependent prolyl hydroxylases (PHDs). When a cell becomes hypoxic, PHD levels are reduced so more HIF-1α is present to dimerise with HIF-1β forming the active heterodimer that can act as the VEGF transcription factor.

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Imaging of VEGF, Uptake Control and Scramble SmartFlares

Despite the reported stability of SmartFlares (which according to the brochure is up to a year at room temperature after reconstitution), we have until now, not opened and reconstituted the VEGF and Scrambled SFs, wanting to make sure that our biological system was in place before we did so.

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TEM of SmartFlare Uptake Control

As with any reagent, it’s important to know what you’re dealing with. The size of the gold nanoparticles at the centre of a SmartFlare will dictate the number of oligonucleotides that can be bound to the surface. Likewise, if the GNPs are aggregated, that will affect the way they interact with cells. In order to visualise the SmartFlares, lab member Joan Comenge took a sample of the uptake control to the Transmission Electron Microscope (TEM).

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UV-vis, DLS and zeta potential of SmartFlare Uptake control

SmartFlare Cy5 Uptake control were resuspended in 1 mL of nuclease free de-ionised water on the 26/02/2015. The UV-vis spectrum was measured on a SpectraMax PluS Spectrophometer (Molecular Devices) using a quartz cuvette. The DLS and zeta potential were measured with a ZetaSizer Nano (Malvern). The peak diameter was at 18 nm (distribution by number). The Zeta potential mean value is -39 mV but the quality of the zeta potential measurement is poor (this normally is due to a too diluted or impure sample).

UV-vis
UV-vis absorbance spectrum of SmartFlare Uptake control (one day after resuspension)
Dynamic Light Scattering of SmartFlare Uptake control
Dynamic Light Scattering of SmartFlare Uptake control (one day after resuspension)
zeta potential
Zeta potential of SmartFlare Uptake control (one day after resuspension)

PS: Thank you Joan for your help with the DLS and zeta potential measurements.

Authors: Raphaël Lévy & Joan Comenge