Evaluation of SmartFlare probe applicability for verification of RNAs in early equine conceptuses, equine dermal fibroblast cells and trophoblastic vesicles

Abstract

Live cell RNA imaging has become an important tool for studying RNA localisation, dynamics and regulation in cultured cells. Limited information is available using these methods in more complex biological systems, such as conceptuses at different developmental stages. So far most of the approaches rely on microinjection of synthetic constructs into oocytes during or before fertilisation. Recently, a new generation of RNA-specific probes has been developed, the so named SmartFlare probes (Merck Millipore). These consist of a central 15-nm gold particle with target-specific DNAs immobilised on its surface. Because of their central gold particle, SmartFlare probes are detectable by transmission electron microscopy. The aim of the present study was to investigate the uptake and distribution of SmartFlare probes in equine conceptuses at developmental stages suitable for embryo transfer (Days 6-10), equine trophoblast vesicles and equine dermal fibroblast cell cultures, and to determine whether differences among these cell types and structures exist. Probe uptake was followed by transmission electron microscopy and fluorescence microscopy. Although the embryonic zona pellucida did not reduce uptake of the probe, the acellular capsule fully inhibited probe internalisation. Nanogold particles were taken up by endocytosis by all cell types examined in a similar manner with regard to time and intracellular migration. They were processed in endosomal compartments and accumulated within lysosomal structures after longer incubation times. In conclusion, the SmartFlare probe is applicable in equine conceptuses, but its use is limited to the developmental stages before the formation of the embryonic capsule.

Re-Evaluating the Spherical-Nucleic-Acid Technology

“…Indeed, we and others noticed that the bulk of NanoFlare fluorescence is punctate and at least partially localizes to endosomes/lysosomes. 10,13 In two recent publications (not cited by Yeo et al), it was shown that NanoFlares do not detect intracellular mRNA level and that the cell-associated fluorescent signals correlate with the applied concentrations of NanoFlares. 5,10 A third independent confirmation of the lack of correlation between mRNA levels and NanoFlare fluorescence has now been published.…”

“…9 Similar observations were made by us 10 and by others. [11][12][13] Endosomal entrapment is a problem for two reasons. First, only particles that have escaped from the endosome can detect cytosolic mRNAs.…”

Re-Evaluating the Spherical-Nucleic-Acid Technology

“…8 Similar observations were made by us 9 and by others. [10][11][12] Endosomal entrapment is a problem for two reasons. First, only particles that have escaped from the endosome can detect cytosolic mRNAs.…”

“…Indeed, we and others noticed that the bulk of NanoFlare fluorescence is punctate and at least partially localizes to endosomes/lysosomes. 9,12 In two recent publications (not cited by Yeo et al), it was shown that NanoFlares do not detect intracellular mRNA level and that the cell-associated fluorescent signals correlate with the applied concentrations of NanoFlares. 5,9 A third independent confirmation of the lack of correlation between mRNA levels and NanoFlare fluorescence has now been published.…”

On-Demand Intracellular Delivery of Single Particles in Single Cells by 3D Hollow Nanoelectrodes

“…The spherical nucleic acids were found to be trapped in vesicles after endocytosis and thus failed to reflect intracellular RNA transcripts levels. 16,17 It was reported that only 1–2% of the endocytosed nanoparticles could escape from the endosomal vesicles into the cytosol. 15 Additionally, nanoparticle aggregates could distort molecular behavior in mechanistic studies, such as those of intracellular transport by motor proteins.…”