Spinach2 RNA aptamer, Broccoli RNA aptamer [1][2]
The target of DFHBI-1T is not a protein or enzyme, but specific RNA aptamers. Its fluorescence is "turned on" by binding to specific RNA sequences. The primary RNA targets it recognizes include the series of fluorescent RNA aptamers such as Spinach, Spinach2, iSpinach, and Broccoli. Upon binding to these aptamers, the intramolecular rotation of DFHBI-1T is restricted, leading to a highly fluorescent state. Specifically, the Broccoli-DFHBI-1T complex exhibits an excitation/emission wavelength of 472 nm/507 nm, while the Spinach2-DFHBI-1T complex shows 482 nm/505 nm.

Guidelines (The following protocol is recommended. It serves as a guide and should be modified according to your specific experimental needs.)
1.1 Preparation of Stock Solution [1]
Prepare a 50 mM stock solution of DFHBI-1T in anhydrous DMSO.
Note: It is recommended to aliquot the stock solution and store at -20°C or -80°C, protected from light.
1.2 Preparation of Working Solution Dilute the stock solution with serum-free culture medium to obtain a 20 μM working solution of DFHBI-1T.
Note: The concentration of the working solution should be adjusted according to the specific experimental conditions and prepared fresh immediately before use.

2. Staining Procedure
1) Seed cells expressing Spinach2-labeled RNA into appropriate culture dishes or plates and culture under standard cell culture conditions.
2) Remove the old culture medium and gently wash the cells twice with pre-warmed PBS to remove residual serum and cell debris.
3) Add the 20 μM DFHBI-1T working solution to the cells, ensuring complete coverage of the cell monolayer.
4) Incubate the cells with the dye for 10 minutes at 37°C in a humidified incubator with 5% CO₂.
5) After incubation, remove the dye-containing medium and wash the cells three times with pre-warmed PBS to remove unbound DFHBI-1T.
6) Replace the PBS with fresh cell culture medium or imaging buffer.
7) Immediately image the cells using a fluorescence microscope equipped with a GFP filter cube to detect the fluorescence of the Spinach2-DFHBI-1T complex.

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When bound to Spinach2, DFHBI-1T exhibited a 35 nm red shift in the excitation peak and a slight red shift in the emission peak compared to DFHBI. The Spinach2-DFHBI-1T complex also exhibited an overall increase in brightness, reflecting a slight increase in extinction coefficient and an increase in quantum yield. [1]
The photophysical properties of the Spinach2-DFHBI-1T complex were: maximum absorption at 426 nm, maximum excitation at 472 nm, maximum emission at 507 nm, extinction coefficient of 29,600 M⁻¹ cm⁻¹, quantum yield of 0.94, brightness of 28 (relative to Spinach2-DFHBI), Kd of 560 nM, and melting temperature (Tm) of 37°C. [1]
The photophysical properties of the Broccoli-DFHBI-1T complex were: maximum absorption at 469 nm, maximum excitation at 472 nm, maximum emission at 507 nm, extinction coefficient of 29,600 M⁻¹ cm⁻¹, quantum yield of 0.94, brightness of 28 (relative to Spinach2-DFHBI), Kd of 480 nM, and melting temperature (Tm) of 48°C. [2]
In vitro folding analysis showed that Broccoli exhibited improved folding compared to Spinach2 in the presence of low magnesium concentrations. [2]
DFHBI-1T exhibited lower background fluorescence than DFHBI when incubated with cells. DFHBI-1T showed reduced nonspecific fluorescence activation by cells and in media compared to DFHBI. [1]

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In COS7 cells expressing (CGG) 60-Spinach2, DFHBI-1T (20 μM; 10 min) increases fluorescein in comparison to DFHBI (20 μM)[1]. Broccoli-DFHBI-1T has an ex/em of 472 nm/507 nm, and spinach-2-DFHBI-1T has an ex/em of 482 nm/505 nm[2].

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In in vitro assays, DFHBI-1T demonstrates significantly enhanced fluorescence performance compared to the first-generation DFHBI. When bound to the Spinach2 aptamer, DFHBI-1T exhibits higher molar extinction coefficients and quantum yields, with a peak excitation of 482 nm and a peak emission of 505 nm, making its spectral properties compatible with standard GFP filter sets. Importantly, compared to the Spinach2-bound DFHBI, DFHBI-1T exhibits stronger specific fluorescence and lower background fluorescence, resulting in a significantly better signal-to-noise ratio.

In COS7 cells expressing (CGG)₆₀-Spinach2, replacing DFHBI (20 μM) with DFHBI-1T (20 μM) resulted in foci that were approximately 60% brighter when imaged using a GFP filter cube. Cells treated with DFHBI-1T exhibited lower background fluorescence than cells treated with the same concentration of DFHBI. [1]
In COS7 cells expressing (CGG)₆₀-Spinach2, cells treated with DFHBI-1T exhibited readily detectable intranuclear foci when imaged with a GFP filter cube, with increased specific fluorescence signal and significantly lower background fluorescence compared to DFHBI. The S5-Spinach2 signal was readily detectable without background subtraction when using DFHBI-1T. [1]
In E. coli expressing tBroccoli or tSpinach2, DFHBI-1T (40 μM) was used for fluorescence imaging. tBroccoli-expressing cells were approximately twice as fluorescent as tSpinach2-expressing cells. [2]
In HEK293T cells expressing 5S-tBroccoli or 5S-tdBroccoli, DFHBI-1T (20 μM) treatment resulted in clearly visible cytoplasmic RNA foci without added magnesium. tdBroccoli was 70% brighter than tBroccoli as measured by flow cytometry. [2]
In HEK293T cells expressing 5S-Broccoli or 5S-dBroccoli (without tRNA scaffold), DFHBI-1T (20 μM) treatment resulted in readily detectable fluorescence by flow cytometry and fluorescence microscopy. [2]

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At the living cell level, DFHBI-1T demonstrates excellent RNA imaging capabilities. Studies have shown that in COS7 cells expressing (CGG)₆₀-Spinach2, treatment with 20 μM DFHBI-1T for 10 minutes significantly enhances the fluorescence signal, showing superior performance to DFHBI under the same conditions. This probe is cell-permeable and enables real-time dynamic imaging of RNA in living cells, including the detection of circular RNAs (tricRNAs) generated from tRNA introns and their subcellular localization. Flow cytometry analysis demonstrates that DFHBI-1T staining, combined with fluorescence-activated cell sorting (FACS), can effectively distinguish cells expressing the target RNA from control groups.

DFHBI-1T is an RNA aptamer-activated fluorescent probe that requires binding to RNA to exhibit fluorescent properties. A core protocol for cell-free binding detection is as follows: In a cell-free system, mix purified RNA aptamers (such as Broccoli or Spinach2) with DFHBI-1T in binding buffer (typically containing 40 mM HEPES pH 7.4, 100 mM KCl, 1 mM MgCl₂) and incubate at room temperature or 37°C for 5-30 minutes. Measure fluorescence intensity at specific wavelengths using a fluorescence spectrophotometer (for Broccoli-DFHBI-1T, use Ex/Em = 472/507 nm; for Spinach2-DFHBI-1T, use 482/505 nm). Binding affinity can be determined by varying DFHBI-1T concentrations, with Kd values typically in the nanomolar to low micromolar range.

Live-cell imaging of Spinach2 fusion RNAs: COS7 cells expressing (CGG)₆₀-Spinach2 were cultured in media containing 20 μM DFHBI-1T for 10 minutes. Images were acquired using a GFP filter cube with a 100 ms exposure. Fluorescence intensity was quantified from 10 foci and normalized to DFHBI brightness. [1]
Flow cytometry analysis of HEK293T cells: Cells transfected with plasmids expressing 5S fused to aptamers (tBroccoli, tdBroccoli, or tSpinach2) were treated with 20 μM DFHBI-1T and analyzed in two channels: green (ex = 488 nm, em = 525 ± 50 nm) and red (ex = 561 nm, em = 610 ± 20 nm). mCherry was used as a transfection control. Where indicated, cells were pretreated with 5 mM MgSO₄. [2]
Fluorescence microscopy of HEK293T cells: Cells were pretreated with 20 μM DFHBI-1T, 5 μg/mL Hoechst 33258, and 0.3 M sucrose, and where indicated with 5 mM MgSO₄. Exposure times were 0.5 s for green fluorescence and 200 ms for mCherry and Hoechst. Scale bar, 10 μm. [2]
Fluorescence microscopy of E. coli: Bacteria expressing tSpinach2, tBroccoli, or tdBroccoli were attached to poly-d-lysine coated glass-bottom dishes, preincubated with 200 μM DFHBI-1T, and imaged under a fluorescent microscope for 100 ms. Cells were imaged in PBS (without magnesium). Scale bar, 2 μm. [2]
Quantification of bacterial fluorescence: Fluorescence signal from bacterial cells was measured in suspension on a plate reader. Error bars indicate standard deviations (n = 3). [2]

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A typical experimental protocol for live-cell RNA imaging is as follows: Culture cells expressing the target RNA aptamer (such as Broccoli or Spinach2) in confocal dishes to appropriate density. Thirty minutes prior to imaging, replace the culture medium with imaging medium (e.g., phenol-red-free Fluorobrite) containing 20 μM DFHBI-1T, supplemented with 25 mM HEPES and 1% FBS to maintain cell conditions. Incubate the cells at 37°C with 5% CO₂ for 30 minutes before imaging. Acquire images using a confocal microscope equipped with a 488 nm laser; the Broccoli-DFHBI-1T complex has an emission peak at 507 nm (green channel). Set up cells not expressing the aptamer as a negative control to subtract background autofluorescence.

Information describing in vivo animal experiments for DFHBI-1T is relatively limited in the publicly available literature. As this probe is primarily used in basic cell biology research, protocols for in vivo animal experiments typically involve delivering genes carrying the target RNA aptamer sequence into animals via viral vectors (such as AAV) or plasmids (e.g., local injection). After confirming target gene expression, DFHBI-1T is administered to the animal (routes may include local injection, intraperitoneal injection, or intravenous injection, with dosages requiring optimization). Subsequently, fluorescence signals in target tissues are detected under anesthesia using a live imaging system or an endoscope equipped with fluorescence imaging capabilities.

It is known that this probe has good cell membrane permeability and rapidly enters living cells to bind with intracellular RNA aptamers. The compound has a relatively small molecular weight (320.21) and certain lipophilic properties. In terms of solubility, DFHBI-1T has a solubility of 100 mg/mL (312.30 mM) in DMSO, making it convenient for preparing stock solutions. Formulation protocols for animal experiments have been reported: sequentially diluting the stock solution with 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline yields a homogeneous suspension at 5.5 mg/mL, suitable for oral or intraperitoneal administration.

Existing research indicates that DFHBI-1T exhibits negligible cytotoxicity in living cells, making it an ideal probe for long-term live-cell RNA imaging. Supplier data shows that the purity of DFHBI-1T exceeds 98%. Unlike many fluorescent dyes, DFHBI-1T is inherently non-fluorescent, with its fluorescence activation occurring only upon specific binding to the target RNA. This mechanism prevents non-specific background fluorescence and interference with normal cellular metabolism. However, detailed safety and toxicological data, as well as long-term exposure risks, have not been systematically reported, and this compound is intended for research use only.

[1]. Plug-and-play fluorophores extend the spectral properties of Spinach. J Am Chem Soc. 2014 Jan 29;136(4):1198-201.

[2]. Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution. J Am Chem Soc. 2014 Nov 19;136(46):16299-308.

DFHBI-1T ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4H)-one) is a fluorophore that binds to and is activated by Spinach2 and Broccoli RNA aptamers. It is a structural mimic of the GFP fluorophore (4-hydroxybenzylidene-imidazolinone, HBI). [1][2]
The compound was synthesized by replacing the methyl substituent at the N-1 position of the imidazolone ring in DFHBI with a 1,1,1-trifluoroethyl substituent. This modification resulted in a 35 nm red shift in the excitation peak and a slight red shift in the emission peak when bound to Spinach2, as well as an overall increase in brightness. [1]
DFHBI-1T exhibits lower background fluorescence than DFHBI when incubated with cells, likely due to reduced nonspecific fluorescence activation by cellular components and in media. This allows specific signals from Spinach2-tagged RNAs to be detected without background subtraction. [1]
DFHBI-1T is used as a "plug-and-play" fluorophore that allows the spectral properties of Spinach2 to be altered based on specific experimental needs. It is compatible with GFP filter cubes commonly used in fluorescence microscopes. [1]
The Broccoli-DFHBI-1T complex exhibits a melting temperature (Tm) of 48°C, which is higher than that of Spinach2-DFHBI-1T (37°C), indicating improved thermostability. [2]
DFHBI-1T is used for imaging Broccoli-tagged RNAs in both bacterial and mammalian cells. Broccoli does not require a tRNA scaffold for folding or imaging and shows reduced dependence on magnesium for fluorescence compared to Spinach2. [2]

C13H9F5N2O2

320.214780569077

320.058

1539318-36-9

DFHBI-2T;1539318-40-5

101889712

Light yellow to yellow solid powder

1.49±0.1 g/cm3(Predicted)

327.9±52.0 °C(Predicted)

152.1±30.7 °C

0.0±0.7 mmHg at 25°C

1.527

2.96

1

8

2

22

505

0

CC1=N/C(=C\C2=CC(=C(C(=C2)F)O)F)/C(=O)N1CC(F)(F)F

AWYCLBWNRONMQC-WMZJFQQLSA-N

InChI=1S/C13H9F5N2O2/c1-6-19-10(12(22)20(6)5-13(16,17)18)4-7-2-8(14)11(21)9(15)3-7/h2-4,21H,5H2,1H3/b10-4-

(5Z)-5-[(3,5-Difluoro-4-hydroxyphenyl)methylene]-3,5-dihydro-2-methyl-3-(2,2,2-trifluoroethyl)-4H-imidazol-4-one

DFHBI-1T; DFHBI1T; 1539318-36-9; (Z)-5-(3,5-Difluoro-4-hydroxybenzylidene)-2-methyl-3-(2,2,2-trifluoroethyl)-3,5-dihydro-4H-imidazol-4-one; (5Z)-5-[(3,5-Difluoro-4-hydroxyphenyl)methylene]-3,5-dihydro-2-methyl-3-(2,2,2-trifluoroethyl)-4H-imidazol-4-one; DFHBI 1T; DF-HBI-1T; DF HBI 1T

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~156.15 mM)

Solubility in Formulation 1: 5.5 mg/mL (17.18 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 55.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)