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Pepper aptamer
Timeline
Crystal structure of RNA aptamer Pepper and dye ligand HBC and its six analogues and mechanism of dye ligand recognition[2]
A new optogenetic tool in mammalian cells (mOptoT7) can induce Pepper RNA aptamer for RNA visualization[5]
The CRISPR palette system was developed by binding the Pepper RNA aptamer into a single gRNA to successfully visualize multicolor RNA in live cells[6]
RNA origami scaffolds facilitate cryo-EM characterization of a Broccoli–Pepper aptamer FRET pair[7]
A universal orthogonal imaging platform for livingcell RNA detection using Pepper-HBC620 (red) and Squash-DFHBI-1T (green)[10]
A fluorescent aptamer reporter assay for RNA methylation sensitivity was developed using the A-pepper RNA aptamer[11]
Description
In 2019, Yang, Y et al. used the SELEX method to select aptamers that bind to HBC, identifying an RNA aptamer named 'Pepper' with its HBC complex dubbed 'Pepper530'. In 2021, Ren, A et al. determined the high-resolution X-ray crystal structures of Pepper-HBC and Pepper-HBC-like fluorophore complexes. In 2022, Piccirilli, J. A et al. employed antibody-assisted crystallography to reveal the structures of Pepper bound to HBC530 and HBC599 at 2.3 Å and 2.7 Å resolutions, respectively. Despite variations in space groups and lattice packing, the overall structures reported by both Piccirilli, J. A et al. and Yang, Y et al. show strong similarity. The analysed structures by Piccirilli, J. A et al. underscore the functional significance of the observed 3D architecture. HBC530 and HBC599 are analogues of the fluorescent molecule HBC[1,2,3].
SELEX
In vitro selection experiments involve constructing a single-stranded (ssDNA) library that contains two 26-base random stretches, separated by a 12-base fixed sequence and flanked by constant regions at the 5' and 3' ends for PCR amplification and in vitro transcription. This ssDNA library was then used in a PCR reaction to gently amplify the library and create double-stranded DNA (dsDNA) templates for in vitro RNA synthesis. Phenol-chloroform extraction and ethanol precipitation were used to purify the RNA transcripts. RNA binding to HBC was achieved through affinity chromatography. After eight rounds, the complementary DNA was cloned into the pGEM-T Easy vector (Promega) and sequenced[1].
Detailed information are accessible on SELEX page.
Structure
2D representation
In 2021, Ren, A et al. obtained multiple different sequences through SELEX and designed a 43 nt sequence after comparing the information of these sequences. The 43 nt aptamer is characterized by a sequence that forms a distinct secondary structure, as illustrated in the subsequent diagrams. Here we utilized RiboDraw to complete the figure, based the 3D structure information[2].
5'-GGCGCACUGGCGCUGCGCCUUCGGGCGCCAAUCGUAGCGUGUCGGCGCC-3'
3D visualisation
Ren, A. et al. determined the structures of complexes formed by the Pepper aptamer bound with its cognate HBC or HBC-like fluorophores at high resolution using X-ray crystallography. The structure of the Pepper-HBC complex was refined to a high resolution of 1.64 Å. The PDB ID for this structure is 7EOH[2].
Additional available structures that have been solved and detailed information are accessible on Structures page.
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Piccirilli, J.A. et al. determined the structures of complexes formed by the Pepper aptamer bound with its cognate HBC530 or HBC599 at high resolution using X-ray crystallography. The structure of the Pepper aptamer binding to both HBC530 and HBC599 is identical, yet the binding ligands differ. BL3-6 Fab serves as a molecular chaperone protein for assisting in crystallization. The structure of the Pepper-HBC599 complex was refined to a high resolution of 2.7 Å. The PDB ID for this structure is 7U0Y[3].
Additional available structures that have been solved and detailed information are accessible on Structures page.
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Binding pocket
Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 7EOH with 1.64 Å resolution, as determined by X-ray crystallography. (4-((2-hydroxyethyl)(methyl)amino)-benzylidene)-cyanophenyl-acetonitrile (HBC) (shown in sticks) is labeled in magenta. Right: The hydrogen bonds of binding sites of the aptamer bound with HBC.
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Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 7U0Y with 2.7 Å resolution, as determined by X-ray crystallography. HBC599 (shown in sticks) is labeled in magenta. Right: The hydrogen bonds of binding sites of the aptamer bound with HBC599.
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Ligand information
SELEX ligand
To determine the dissociation rate constant (Kd) of the Pepper-HBC complex, Pepper RNA was synthesised with biotin labelling at the 3' end and coupled to streptavidin-coated sepharose beads (GE Healthcare). Two-photon fluorescence images were captured immediately after the beads were transferred from a buffer containing HBC to a HBC-free buffer. The quantitative fluorescence data were fitted to the exponential decay formula (y = y0 + ae^(-bx)). (4-((2-hydroxyethyl)(methyl)amino)-benzylidene)-cyanophenylacetonitrile (HBC). All other compounds are analogues of HBC[1].
Structure ligand
HBC is a synthetic dye of green fluorescent protein (GFP) fluorescent groups, with structurally rigid electron acceptors and strong electron donors. HBC is used to detect RNA localization.-----From MedChemExpress
PubChem CID: a unique identifier for substances in the PubChem database.
CAS number: a global registry number for chemical substances.
MedChemExpress: a leading global compound supplier, offering over 200 compound libraries with approximately 26 million compounds for drug discovery and research.
| Name | PubChem CID | Molecular Formula | Molecular Weight | CAS | Solubility | MedChemExpress ID |
|---|---|---|---|---|---|---|
| HBC | 145712177 | C19H17N3O | 303.4g/mol | 156840-13-0 | 125 mg/mL (in DMSO) | HBC |
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Similar compound(s)
We screened the compounds with great similarity to HBC 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 |
|---|---|---|---|---|
| ZINC1857624275 | 3-[4-[2-Cyanoethyl(methyl)amino]phenyl]-2-(4-methoxyphenyl)prop-2-enenitrile | NA | 305296 |  |
| ZINC1735964 | (E)-3-[4-[2-cyanoethyl(methyl)amino]phenyl]-2-(4-methoxyphenyl)prop-2-enenitrile | NA | 20835609 |  |
| ZINC1735963 | (Z)-3-[4-[2-cyanoethyl(methyl)amino]phenyl]-2-(4-methoxyphenyl)prop-2-enenitrile | NA | 6285076 |  |
| ZINC5011924 | (E)-3-[4-[2-cyanoethyl(methyl)amino]phenyl]-2-(4-ethylphenyl)prop-2-enenitrile | NA | 20835258 |  |
| ZINC1735967 | (Z)-3-[4-[2-cyanoethyl(methyl)amino]phenyl]-2-(4-ethylphenyl)prop-2-enenitrile | NA | 5921549 |  |
| ZINC408583021 | (Z)-2-(4-methoxyphenyl)-3-(4-pyrrolidin-1-ylphenyl)prop-2-enenitrile | NA | 125594441 |  |
| ZINC1733626 | (E)-3-[4-(dimethylamino)phenyl]-2-(4-methoxyphenyl)prop-2-enenitrile | NA | 20835477 |  |
| ZINC1733625 | 3-(4-(Dimethylamino)phenyl)-2-(4-methoxyphenyl)acrylonitrile | NA | 6089169 |  |
| ZINC305827293 | 5-cyano-N-[4-[2-hydroxyethyl(methyl)amino]phenyl]pyridine-2-carboxamide | NA | 109989812 |  |
References
[1] Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs.Chen, X., Zhang, D., Su, N., Bao, B., Xie, X., Zuo, F., Yang, L., Wang, H., Jiang, L., Lin, Q., Fang, M., Li, N., Hua, X., Chen, Z., Bao, C., Xu, J., Du, W., Zhang, L., Zhao, Y., Zhu, L., Loscalzo, J., Yang, Y. Zhao, Y., Zhu, L., Loscalzo, J., Yang, Y.
Nature Biotechnology, 37(11) , 1287-1293. (2019)
[2] Structure-based investigation of fluorogenic Pepper aptamer.
Huang, K., Chen, X., Li, C., Song, Q., Li, H., Zhu, L., Yang, Y., Ren, A.
Nature Chemical Biology, 17(12) , 1289-1295. (2021)
[3] Structural basis for fluorescence activation by Pepper RNA.
Rees, H. C., Gogacz, W., Li, N.-S., Koirala, D., Piccirilli, J. A.
ACS chemical biology, 17(7) , 1866-1875. (2022)
[4] Inert Pepper aptamer-mediated endogenous mRNA recognition and imaging in living cells.
Wang, Q., Xiao, F., Su, H., Liu, H., Xu, J., Tang, H., Qin, S., Fang, Z., Lu, Z., Wu, J., Weng, X., Zhou, X.
Nucleic Acids Research, 50(14) , e84. (2022)
[5] Implementation of a novel optogenetic tool in mammalian cells based on a split t7 RNA polymerase.
Dionisi, S., Piera, K., Baumschlager, A., Khammash, M.
ACS synthetic biology, 11(8) , 2650-2661. (2022)
[6] Multi-color RNA imaging with CRISPR-Cas13b systems in living cells.
Yang, L.-Z., Gao, B.-Q., Huang, Y., Wang, Y., Yang, L., Chen, L.-L.
Cell Insight, 1(4) , 100044. (2022)
[7] RNA origami scaffolds facilitate cryo-EM characterization of a Broccoli-Pepper aptamer FRET pair.
Sampedro Vallina, N., McRae, E. K. S., Hansen, B. K., Boussebayle, A., Andersen, E. S.
Nucleic Acids Research, 51(9) , 4613-4624. (2023)
[8] Imaging intracellular metabolite and protein changes in live mammalian cells with bright fluorescent RNA-based genetically encoded sensors.
Fang, M., Li, H., Xie, X., Wang, H., Jiang, Y., Li, T., Zhang, B., Jiang, X., Cao, Y., Zhang, R., Zhang, D., Zhao, Y., Zhu, L., Chen, X., Yang, Y.
Biosensors & Bioelectronics, 235 , 115411. (2023)
[9] Genetically encoded RNA-based sensors with Pepper fluorogenic aptamer.
Chen, Z., Chen, W., Reheman, Z., Jiang, H., Wu, J., Li, X.
Nucleic Acids Research, 51(16) , 8322-8336. (2023)
[10] A universal orthogonal imaging platform for living-cell RNA detection using fluorogenic RNA aptamers.
Yin, P., Ge, M., Xie, S., Zhang, L., Kuang, S., Nie, Z.
Chemical Science, 14(48) , 14131-14139. (2023)
[11] An RNA Methylation-Sensitive AIEgen-Aptamer reporting system for quantitatively evaluating m6A methylase and demethylase activities.
Ying, X., Huang, C., Li, T., Li, T., Gao, M., Wang, F., Cao, J., Liu, J.
ACS chemical biology, 19(1) , 162-172. (2024)
[12] Optimization of RNA Pepper sensors for the detection of arbitrary RNA targets.
Tang, A. A., Afasizheva, A., Cano, C. T., Plath, K., Black, D., Franco, E.
ACS synthetic biology, 13(2) , 498-508. (2024)