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Broccoli aptamer
Timeline
A 49-nt aptamer, Broccoli, which binds and activates the fluorescence of (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1Himidazol-5(4H)-one was identified.[1]
A natural threeway junction structure to generate alternative scaffold that used to create cassettes containing up to four Broccoli units and enables stable aptamer expression in cells was designed.[2]
A study of the performance of distinct RNA Spinach and Broccoli aptamer sequences in isolation or inserted into the small subunit of the bacterial ribosome.[6]
Split-Broccoli, which represents the first functional split aptamer system to operate in vivo, was designed.[7]
The development of an aptamer-initiated fluorescence complementation method for RNA imaging by engineering a GFP-mimicking turn-on RNA aptamer, Broccoli, into two split fragments that could tandemly bind to target mRNA was reported.[8]
A dynamic systems for molecular computing which can be used to monitor real-time processes in cells and construct biocompatible logic gates was designed.[9]
An expression system in which Broccoli can be introduced for achieving rapid RNA circularization, resulting in RNA aptamers with high stability and expression levels was designed.[10]
Computational screening was used to identify a DFHBI derivative that binds Broccoli with higher affinity and leads to markedly higher fluorescence in cells compared to previous ligands.[11]
A new fluorophore which binds Red Broccoli with high affinity and makes Red Broccoli resistant to thermal unfolding was developed.[12]
A straightforward and cost-effective platform for assessing transcription in vitro which used the “Broccoli” RNA aptamer as a direct, real-time fluorescent transcript readout was developed.[13]
Broccoli was introduced to serve as the substate of activated CRISPR-Cas13a to monitor the presence of pathogen RNAs.[14]
Description
Jaffrey, S. R. et al. reported Broccoli, a 49-nt aptamer which binds and activates the fluorescence of (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1Himidazol-5(4H)-one in their article published in 2002. Broccoli shows robust green fluorescence in cells, with increased fluorescence relative to other RNA aptamers like Spinach2. This enhanced brightness makes it more easily detectable in imaging applications. It has a high folding efficiency in vitro, similar to Spinach2, but with reduced dependence on magnesium for proper folding. This allows Broccoli to function well in the cellular environment where magnesium concentrations may vary. Later, in a work published in 2025 by Huang, L. et al., the structure of the Broccoli-DFHBI-1T complex was determined by X-ray diffraction.[1,16].
SELEX
In their work published in 2014, Jaffrey, S. R. et al. used SELEX to isolate RNA aptamer Broccoli from a nucleic acid library containing about 1014 sequences after 6 rounds of selection process. In each round, the RNA library is incubated with the target molecule. RNA sequences that bind to the target are separated and then amplified via RT-PCR to create a new library for the next round of selection. After several rounds, RNA sequences with high affinity for the target are enriched.[1].
Structure
2D representation
This aptamer is an optimized sequence. The secondary structure diagram was informed by atomic-resolution structural studies of the Broccoli–DFHBI-1T complex. In this structure, Broccoli immobilises the ligand within a binding pocket formed between G-quadruplex's π-π stacking interactions and adjacent nucleotides. Its main part is just one stem loop with many bulges. The Broccoli aptamer was named by Jaffrey, S. R. et al. in the article. Here we utilized RiboDraw to complete the figure, based the 3D structure information.[16].
5'-GGGACGGUCGGGUCCAGAUAUUCGUAUCUGUCGAGUAGAGUGUGGGCUC-3'
3D visualisation
In 2025, Huang, L. et al. analyzed the structure of the Broccoli-DFHBI-1T complex by X-ray diffraction. The core structure of the Broccoli aptamer comprises a stem-loop, with its ligand immobilised within a binding pocket formed via π-π stacking interactions between a G-quadruplex and adjacent nucleotides. The sequence design of this structure incorporates a mutagenesis strategy involving engineered GU base pairs. The PDB ID of this structure is 8K7W (2.24 Å).[16].
Additional available structures that have been solved and detailed information are accessible on Structures page.
Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 8K7W by X-ray crystallography. DFHBI-1T (shown in sticks) is labeled in magenta. Right: The hydrogen bonds of binding sites of the aptamer bound with DFHBI-1T or other nucleotides surround small molecules.
Additional available structures that have been solved and detailed information are accessible on Structures page.
(Clicking the "Settings/Controls info" to turn Spin off)
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Binding pocket
Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 8K7W by X-ray crystallography. DFHBI-1T (shown in sticks) is labeled in magenta. Right: The hydrogen bonds of binding sites of the aptamer bound with DFHBI-1T or other nucleotides surround small molecules.
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Ligand information
SELEX ligand
Dissociation constant was calculated by performing a titration of 50 nM RNA with increasing concentration of DFHBI-1T and then fitted the resulting data points with the curve based on the Hill equation. ——-From Source
| Name | Ligand | Affinity |
|---|---|---|
| Broccoli aptamer | DFHBI-1T | 360 nM |
Structure ligand
DFHBI-1T is a membrane-permeable RNA aptamers-activated fluorescence probe. DFHBI-1T binds to RNA aptamers (Spinach, Spinach2, iSpinach, and Broccoli) and causes specific fluorescence and lower background fluorescence. DFHBI-1T is used to image RNA in live cells.----From MedChemExpress
PubChem CID: a unique identifier for substances in the PubChem database.
CAS number: a global registry number for chemical substances.
Drugbank: a comprehensive database with detailed information on drugs and drug targets.
| Name | PubChem CID | Molecular Formula | Molecular Weight | CAS | Solubility | Drugbank ID |
|---|---|---|---|---|---|---|
| DFHBI-1T | 101889712 | C13H9F5N2O2 | 320.21 g/mol | 1539318-36-9 | 100 mg/mL in DMSO | NA |
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Similar compound(s)
We screened the compounds with great similarity to by using the ZINC database and showed some of the compounds' structure diagrams. For some CAS numbers not available, we will supplement them with Pubchem CID. For another compound, we used a similar compound query method from the PubChem database.
ZINC ID: a compound identifier used by the ZINC database, one of the largest repositories for virtual screening of drug-like molecules.
PubChem CID: a unique identifier for substances in the PubChem database.
CAS number: a global registry number for chemical substances.
| ZINC ID | Name | CAS | Pubchem CID | Structure |
|---|---|---|---|---|
| ZINC1857790839 | DFHBI-1T | 1539318-36-9 | 101889712 |  |
| ZINC2051913110 | DFHBI | 1241390-29-3 | 70808995 |  |
| ZINC2048528770 | DFHBI-2T | 1539318-40-5 | 129080921 |  |
| ZINC2054344318 | DMHBI | 1241390-25-9 | 68787434 |  |
| DMHBI+ | 442072768 |  |
||
| DMHBO+ | 2322286-80-4 | 156022789 |  |
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| DMHBI-lmi |  |
|||
| ZINC174814 | DMABI | 21889-13-4 | 89546 |  |
| ZINC492560597 |  |
References
[1] Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution.Filonov, G. S., Moon, J. D., Svensen, N., & Jaffrey, S. R.
Journal of the American Chemical Society, 136(46), 16299–16308. (2014)
[2] In-gel imaging of RNA processing using broccoli reveals optimal aptamer expression strategies..
Filonov, G. S., Kam, C. W., Song, W., & Jaffrey, S. R.
Chemistry & biology, 22(5), 649–660. (2015)
[3] RNA imaging with dimeric Broccoli in live bacterial and mammalian cells.
Filonov, G. S., & Jaffrey, S. R.
Current protocols in chemical biology, 8(1), 1–28. (2016)
[4] Fluorescent RNA aptamers as a tool to study RNA-modifying enzymes..
Svensen, N., & Jaffrey, S. R.
Cell chemical biology, 23(3), 415–425. (2016)
[5] Quadruplex-flanking stem structures modulate the stability and metal ion preferences of RNA mimics of GFP..
Ageely, E. A., Kartje, Z. J., Rohilla, K. J., Barkau, C. L., & Gagnon, K. T.
ACS chemical biology, 11(9), 2398–2406. (2016)
[6] Use of baby Spinach and Broccoli for imaging of structured cellular RNAs..
Okuda, M., Fourmy, D., & Yoshizawa, S.
Nucleic acids research, 45(3), 1404–1415. (2017)
[7] A fluorescent split aptamer for visualizing RNA-RNA assembly in vivo..
Alam, K. K., Tawiah, K. D., Lichte, M. F., Porciani, D., & Burke, D. H.
ACS synthetic biology, 6(9), 1710–1721. (2017)
[8] In situ spatial complementation of aptamer-mediated recognition enables live-cell imaging of native RNA transcripts in real time..
Wang, Z., Luo, Y., Xie, X., Hu, X., Song, H., Zhao, Y., Shi, J., Wang, L., Glinsky, G., Chen, N., Lal, R., & Fan, C.
Angewandte Chemie, 57(4), 972–976. (2018)
[9] Broccoli fluorets: split aptamers as a user-friendly fluorescent toolkit for dynamic RNA nanotechnology..
Chandler, M., Lyalina, T., Halman, J., Rackley, L., Lee, L., Dang, D., Ke, W., Sajja, S., Woods, S., Acharya, S., Baumgarten, E., Christopher, J., Elshalia, E., Hrebien, G., Kublank, K., Saleh, S., Stallings, B., Tafere, M., Striplin, C., & Afonin, K. A.
Molecules, 23(12), 3178. (2018)
[10] Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts..
Litke, J. L., & Jaffrey, S. R.
Nature biotechnology, 37(6), 667–675. (2019)
[11] Fluorophore-promoted RNA folding and photostability enables imaging of single broccoli-tagged mRNAs in live mammalian cells..
Li, X., Kim, H., Litke, J. L., Wu, J., & Jaffrey, S. R.
Angewandte Chemie, 59(11), 4511–4518. (2020)
[12] Imaging intracellular S-adenosyl methionine dynamics in live mammalian cells with a genetically encoded red fluorescent RNA-based sensor..
Li, X., Mo, L., Litke, J. L., Dey, S. K., Suter, S. R., & Jaffrey, S. R.
Journal of the American Chemical Society, 142(33), 14117–14124. (2020)
[13] Revisiting T7 RNA polymerase transcription in vitro with the Broccoli RNA aptamer as a simplified real-time fluorescent reporter..
Kartje, Z. J., Janis, H. I., Mukhopadhyay, S., & Gagnon, K. T.
The Journal of biological chemistry, 296, 100175. (2021)
[14] Light-up RNA aptamer signaling-CRISPR-cas13a-based mix-and-read assays for profiling viable pathogenic bacteria..
Zhang, T., Zhou, W., Lin, X., Khan, M. R., Deng, S., Zhou, M., He, G., Wu, C., Deng, R., & He, Q.
Biosensors & bioelectronics, 176, 112906. (2021)
[15] Engineering fluorophore recycling in a fluorogenic RNA aptamer..
Li, X., Wu, J., & Jaffrey, S. R.
Angewandte Chemie, 60(45), 24153–24161. (2021)
[16] A general strategy for engineering GU base pairs to facilitate RNA crystallization..
Ren, Y., Lin, X., Liao, W., Peng, X., Deng, J., Zhang, Z., Zhan, J., Zhou, Y., Westhof, E., Lilley, D. M. J., Wang, J., & Huang, L.
Nucleic acids research, 53(3), gkae1218. (2025)