One way to visualize nucleic acids in intact cells is by leveraging the Staudinger reaction. This scheme begins with a rhodamine dye that has been quenched by an azide group. The dye is conjugated to an oligo complementary to one half of a target nucleotide sequence. The other half of the sequence is recognized by an oligo probe carrying a phosphine. If both oligos anneal to the target, the phosphine reacts with the azide to reduce it to an amine, thereby unquenching the rhodamine signal. This templated Staudinger reaction has been harnessed in the past to detect highly expressed mRNAs, but in a recently published article in Chemical Science, Gorska et al. translate the technique to miRNA detection. It is becoming increasingly clear that miRNAs play important roles in oncogenesis, and methods to reveal their presence and abundance within human cells would simplify efforts to elucidate mechanisms by which they regulate critical physiological functions. Fluorescence in situ hybridization might seem to be an appropriate tool, but the small size of miRNAs means that they can diffuse out of cells during incubation and wash steps, at least without extra crosslinking. The templated Staudinger reaction is speedy enough to avoid this problem; in addition, the requirement for simultaneous annealing of both the phosphine- and azidorhodamine-conjugated probes reduces the opportunity for false-positive signals. As a model target, Gorska et al. selected miR-21, one of the miRNAs most often overexpressed in solid tumors. Both oligos were synthesized as peptide nucleic acids with guanidinium modifications, which facilitates cell entry. Optimal responses were evident with a 7-mer phosphine-conjugated probe and an 8-mer probe bearing bis-azidorhodamine (a four-fold improvement over the original, single-azide–quenched fluorophore). After a relatively brief fixation protocol (3.7% formaldehyde for 20 minutes) true target and single-nucleotide mismatches were easily distinguished in cells. In cancer cell lines with known differences in miR-21 expression, a good correlation was observed between RT-PCR quantitation of miR-21 expression and intracellular rhodamine fluorescence. Pilot studies with suspension cells showed that the technique, unlike in situ hybridization, works with analysis by flow cytometry, and the method was also readily extended to detection of the cervical cancer–related miR-31.
Gorska et al. 2011. Rapid fluorescence imaging of miRNAs in human cells using templated Staudinger reaction. Chem Sci. 2:1969-1975.It's No Myth
There have been so many protein expression systems over the years that it's not unreasonable to imagine divine inspiration would be needed for further improvements. Perhaps that's why Bandaranayake et al., writing in Nucleic Acids Research, have named their new platform “Daedalus”, after the skillful craftsman of Greek mythology. The motivation for this work rests on the well-known limitations of yeast and insect cells in faithfully performing the full array of posttranslational modifications available in mammalian cells. However, even popular mammalian producer cell lines have limitations, most notably the typical requirement to select for high-yield clones. Daedalus circumvents this problem by transducing producer cells with lentiviral vectors containing a ubiquitous chromatin opening element (UCOE), which helps maintain the vector integration site in transcriptionally active form. Typically, a UCOE can occupy 20% of the cargo space available in a lentiviral vector, but Daedalus uses a new 0.7-kb UCOE, leaving room for incorporation of sequences that can code for proteins up to nearly 70 kDa in size. Despite being downsized, the svelte UCOE variant was capable of maintaining consistent, high-level expression for at least 1 month; indeed, although the expression cassette had an IRES-GFP downstream of the gene of interest, sorting for the brightest cells was not required for efficient expression. Like many mammalian expression systems, Daedalus harnesses the secretory pathway, sidestepping the need for cell lysis and allowing repeated harvesting from cells maintained in culture. Use of serum-free medium simplifies target protein purification to just one size-exclusion chromatography step. Interestingly, the serum-free-adapted HEK293 cells employed in the system proved easier to transduce than the more commonly used CHO producer line. To show off Daedalus’ capabilities, the authors tested a panel of 14 proteins, including lipocalins, membrane proteins, cytokines, and a cytoplasmic protein, adding signal peptides for secretion as needed. Of these, 12 could be expressed, garnering yields of 20 to 100 mg/L in 100 mL cultures. Since the authors developed Daedalus to produce properly glycosylated proteins for crystallography, they also demonstrated the suitability of their platform for producing decigram quantities of a protein for structural studies, in this case going from vector transduction to structure determination in a jaw-dropping 18 days.
Bandaranayake et al. Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors. Nucleic Acids Res. [Epub ahead of print, September 12, 2011; doi:10.1093/nar/gkr706].