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DFHBI-1T 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[1].
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].
Detailed information are accessible on SELEX page.
Structure
The 2D structure of the figure is based on the article by online secondary structure prediction tool to draw. The Figure shows the secondary structure prediction of the Broccoli aptamer sequence. This aptamer is an optimized sequence. 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[1].
5'-GAGACGGUCGGGUCCAGAUAUUCGUAUCUGUCGAGUAGAGUGUGGGCUC-3'
Ligand information
SELEX 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.
MedChemExpress: an entry number for the classification of chemicals by the well-known chemical manufacturing company MedChemExpress.
| Name | Molecular Formula | Molecular Weight | CAS | Solubility | PubChem | MedChemExpress |
|---|---|---|---|---|---|---|
| DFHBI-1T | C13H9F5N2O2 | 320.21 g/mol | 1539318-36-9 | 100 mg/mL in DMSO | 101889712 | HY-110251 |
 |
 |
| Name | Sequence | Ligand | Affinity |
|---|---|---|---|
| DFHBI-1T | 5'-GAGACGGUCGGGUCCAGAUAUUCGUAUCUGUCGAGUAGAGUGUGGGCUC-3' | DFHBI-1T | 360 nM |
Similar compound(s)
We screened the compounds with great similarity 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.
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 |  |
| NA | DMHBI+ | NA | 442072768 |  |
| NA | DMHBO+ | 2322286-80-4 | 156022789 |  |
| NA | DMHBI-lmi | NA | NA |  |
| ZINC174814 | DMABI | 21889-13-4 | 89546 |  |
| ZINC492560597 | NA | NA | NA |  |
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)