Fluorescence resonance energy transfer (FRET) between a quantum dot (QD) and the pH-sensitive fluorescent protein
mOrange has been used to develop a fluorescent pH-indicator that is bright and photostable enough for applications in
fluorescence imaging, including the tracking of molecules through endocytic pathways. As the molar extinction
coefficient of mOrange increases with pH, the ratio of the mOrange emission to the QD emission (FA/FD) increases
sharply, producing greater than 10-fold increases in the FA/FD ratio between pH 4.5 and 7.5. This probe has been
thoroughly characterized and it intracellular imaging potential explored.
Efficient Fluorescence (or Förster) Resonance Energy Transfer (FRET) pairs between fluorescent proteins and quantum
dots (QDs) have a significant potential for ultrasensitive biochemical assays in disease detection and diagnosis. We have
developed such FRET pairs using commercially available QDs as donors and fluorescent protein as acceptor, with
polyhistidine-chelation as the means of bioconjugation. In this study we compared two brands of QDs with different
surface coatings and found that the FRET pair containing EviTags from Evident Technology produced a higher FRET
efficiency due to the shorter donor-acceptor distance. The polyhistidine binds directly to the ZnS capping layer of the
EviTags, whereas the carboxyl QDots from Invitrogen, although having a higher quantum yield, require the addition of
Ni2+ to the solution in order to facilitate chelation-mediated binding to outer surface of the polymer coating. These
findings have significant implications to QD-based FRET assay design.
The intracellular localization and specific organelle association of mRNA may reflect essential functions, stages, and stability of mRNA. We report the direct visualization of subcellular localization of K-ras and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNAs in live HDF cells using molecular beacons together with membrane-permeabilization and peptide-based delivery. Unexpectedly, we found that both K-ras and GAPDH mRNAs colocalize with mitochondria. Extensive control studies are performed, including the use of fluorescence in-situ hybridization (FISH), negative-control beacons, and the detection of colocalization of 28S ribosomal RNA with the rough endoplasmic reticulum (ER), suggesting that the mRNA localization and colocalization patterns observed in our study are true and specific. Our observation reveals intriguing subcellular associations of mRNA with organelles such as mitochondria, which may provide new insight into the transport, dynamics, and functions of mRNA and mRNA-protein interactions.
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