Ribonucleic acid (RNA) plays an important role in many cellular physiological processes. In protein synthesis, messenger RNA (mRNA) is the template for translation, whereas transfer RNA (tRNA) and ribosomal RNA (rRNA) are responsible for transport and catalytic polymerization of amino acids, respectively. The dynamic expression of these RNAs directly affects cell functions such as cell division, cell differentiation, and circadian rhythm. Therefore, it is important to examine these RNAs to assess gene function and understand cellular mechanisms.
Fluorogenic aptamers are low-cost and easy-to-use endogenous RNA detection tools for studying RNA interactions, folding, and localization. Fluorogenic aptamers are RNA sequences that bind otherwise non-fluorescent dyes and activate their fluorescence while bound to RNAs. By tagging an RNA with a fluorogenic aptamer, the fluorescence can be encoded genetically into the RNA, and then the endogenously expressed tagged RNA can be detected by fluorescence microscopy. Only the RNA-fluorophore complex is fluorescent and the fluorescent signal is from the labeled RNA, whereas the unbound fluorophore is nonfluorescent.
Spinach and Broccoli are fluorogenic RNA aptamers that bind the small-molecule fluorophore 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) and activate its fluorescence. DFHBI is an otherwise non-fluorescent small molecule that mimics the fluorophore found in green fluorescent protein (GFP). Its variants include DFHBI-1T, BI, OBI, and DFHO. DFHBI and its variants are cell permeable and have negligible toxicity in living cells.
|DFHBI||3, 5-difluoro-4-hydroxybenzylidene imidazolinone||1 mg; 5 mg; 10 mg|
|DFHBI-1T||4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4H)-one||1 mg; 5 mg; 10 mg|
|DFHO||3,5-difluoro-4-hydroxybenzylidene imidazolinone-2-oxime||1 mg|
DFHBI is a mimic of the chromophore of green fluorescent protein (GFP). Upon bound to Spinach aptamer, the highly fluorescent state of DFHBI is turned on and the fluorescence can be detected at an emission wavelength of 501 nm. DFHBI can be used to label any genetically encoded Spinach RNA aptamer family, including Spinach, Spinach2 and Broccoli. Tagged RNAs in living cells can be visualized using standard fluorescence microscopy. In addition, tagged RNAs could also be selectively detected by DFHBI in total RNA gel electrophoresis.
Fluorescence spectra of Spinach/DFHBI complex in pH 7.4 buffer
DFHBI-1T is a non-fluorescent dye containing a 1,1,1-trifluoroethyl substituent on the imidazolone ring of the DFHBI fluorophore. When bound to Spinach aptamers or Spinach-derived aptamers such as Spinch2 and Broccoli, DFHBI-1T is converted to a hyperfluorescent state and its fluorescence can be detected at an emission wavelength of 505 nm. Compared to DFHBI, DFHBI-1T has lower background fluorescence and exhibits stronger overall brightness in living cells.
Fluorescence spectra of Spinach2/DFHBI-1T complex in pH 7.4 buffer
BI is a DFHBI derivative with high binding affinity to Broccoli aptamers and photostability. It can be used for single-molecule mRNA imaging in living mammalian cells. BI is negligible toxicity and can be used to label any genetically encoded broccoli RNA tag without affecting biological function in living cells.
The optimized BI fluorescence properties enable live single-molecule imaging of Broccoli-tagged mRNA transcripts in mammalian cells. Compared to MS2-tagged mRNA, Broccoli-tagged mRNA puncta show similar size and puncta diameters fall within a single Gaussian curve.
Fluorescence spectra of Broccoli/BI complex in pH 7.4 buffer
OBI, a derivative of BI, is a "plug-and-play" fluorophore that binds either Broccoli or Red Broccoli to generate red fluorescence. Conversely, Red Broccoli binds BI to emit green fluorescence. The "plug and play" capability of OBI allows the spectral properties of fluorogenic aptamers and fluorophores to be altered according to specific experimental needs.
Red fluorescent RNA based tags and probes improve the accuracy of intracellular imaging, because green fluorescent probes suffer from the cellular green background fluorescence derived from endogenous vitamins and cofactors. The Red Broccoli/OBI complex can be easily detected in living mammalian cells. Compared to Red Broccoli/DFHO complex, the Red Broccoli/OBI complex exhibits increase in overall brightness and markedly red-shifted excitation wavelength. In cells, OBI binds Red Broccoli with high affinity, making it resistant to thermal unfolding. OBI exhibits enhanced stability to Red Broccoli, and the Red Broccoli/OBI system can be used to monitor cellular mRNA or metabolite dynamics more accurately in real-time.
Fluorescence spectra of Red Broccoli/OBI complex in pH 7.4 buffer
DFHO is a non-fluorescent dye that is structurally similar to the chromophore in red fluorescent protein (RFP). Upon binding to Corn aptamer, DFHO is converted to a highly fluorescent state that can be detected at 545 nm emission wavelength. DFHO is cellular permeable with negligible toxicity in living cells and can label any genetically encoded Corn RNA tag. When bound to Corn, DFHO is highly photostable compared to DFHBI, DFHBI-1T and mVenus. The photostability of DFHO was demonstrated in the imaging and quantitative measurement of cellular Pol III promoter activity in living cells. In addition, Broccoli derivative aptamers can bind to and activate DFHO, turning on its fluorescence.
A. Fluorescence spectra of Corn/DFHO complex. B. Fluorescence spectra of Red and Orange Broccoli/DFHO complex.
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