Spinach2 RNA aptamer, Broccoli RNA aptamer [1][2]

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].

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]

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]

[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.)