Pepper aptamer

Mar 26, 2024 • Jiali Wang, Bo Fu

Timeline

Description

In 2019, Chen, X. et al. selected for aptamers bound with HBC using a systematic evolution of ligands by exponential enrichment (SELEX) approach, and identified one aptamer, D11. They termed this RNA aptamer “Pepper” and its complex with HBC “Pepper530”, indicative of its emission maximum. In 2021, Huang, K. et al. determined the structures of complexes of Pepper aptamer bound with its cognate HBC or HBC-like fluorophores at high resolution by X-ray crystallography. In 2022, to investigate how Pepper RNA folds to create a binding site for HBC, Rees, H. C. et al. used antibody-assisted crystallography to determine the structures of Pepper bound to HBC530 and HBC599 to 2.3 and 2.7 Å resolutions, respectively. The overall structure reported by Rees, H. C. et al. and Huang et al. is highly similar, despite different space groups and lattice packing interactions. The structures analyzed by Rees, H. C. et al. support the functional relevance of the observed 3D architecture^[1,2,3].

SELEX

In vitro selection experiments involve the construction of a single-stranded (ssDNA) library containing two 26-base random stretches separated by a 12-base fixed sequence and flanked from 5’ and 3’ ends with constant regions for PCR amplification and in vitro transcription. The ssDNA library was then used in a PCR reaction to amplify the library gently and to create double-stranded DNA (dsDNA) templates for RNA in vitro synthesis. Phenol chloroform extraction and ethanol precipitation of RNA transcripts was used to purify the transcript. Binding RNA to HBC through affinity chromatography. After eight rounds, the complementary DNA was cloned into pGEM-T Easy vector (Promega) and sequenced^[1].
Detailed information are accessible on SELEX page.

Structure

2D representation

Here we use ribodraw to complete the figure, through the 3D structure information^[2].

5'-GGCGCACUGGCGCUGCGCCUUCGGGCGCCAAUCGUAGCGUGUCGGCGCC-3'

3D visualisation

Huang K, Chen X, Li C, et al. determined the structures of complexes of Pepper aptamer bound with its cognate HBC or HBC-like fluorophores at high resolution by X-ray crystallography. The structure of the Pepper-HBC complex was refined at a high resolution of 1.64 Å. The PDB ID of this structure is 7EOH^[2].
Additional available structures that have been solved and detailed information are accessible on Structures page.
Rees HC, Gogacz W, Li NS, et al. determined the structures of complexes of Pepper aptamer bound with its cognate HBC530 or HBC599 at high resolution by X-ray crystallography. The structure of Pepper binding to HBC530 and HBC599 is the same, but the binding ligands are different. The structure of the Pepper-HBC599 complex was refined at a high resolution of 2.7 Å. The PDB ID of this structure is 7U0Y^[3].
Additional available structures that have been solved and detailed information are accessible on Structures page.

Binding pocket

Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 7EOH by NMR. (4-((2-hydroxyethyl)(methyl)amino)-benzylidene)-cyanophenyl-acetonitrile (HBC) (shown in sticks) is labeled in yellow. Right: The hydrogen bonds of binding sites of the aptamer bound with HBC.


Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 7U0Y by NMR. 4-[(Z)-1-cyano-2-{6-[(2-hydroxyethyl)(methyl)amino]-1-benzothiophen-2-yl}ethenyl]benzonitrile (HBC599) (shown in sticks) is labeled in yellow. Right: The hydrogen bonds of binding sites of the aptamer bound with HBC599.

Ligand information

SELEX ligand

To determine the Kd (dissociation rate constant) of Pepper-HBC complex, Pepper RNA labeled with biotin at the 3' end was synthesized and coupled to sepharose beads coated by streptavidin (GE Healthcare). Two-photon fluorescence images were taken immediately after the beads were transferred from (4-((2-hydroxyethyl)(methyl)amino)-benzylidene)-cyanophenylacetonitrile (HBC) containing buffer to HBC-free buffer. The quantitative fluorescence data were fitted to the formula of exponential decay (y = y0 + ae−bx)^[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	Molecular Formula	MW	CAS	Solubility	MedChemExpress ID
145712177	C19H17N3O	303.4g/mol	156840-13-0	125 mg/mL (in DMSO)	HBC

Similar compound

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	Named	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.
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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)