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TMR-DN aptamer
Timeline
The NMR resonance assignment of an RNA aptamer binding to the fluorescent ligand tetramethylrhodamine TMR was present[2]
The atomic resolution structure of an RNA-aptamer binding to the fluorescent xanthene dye tetramethylrhodamine was present[4]
RhoBAST bound to tetramethylrhodamine-dinitroaniline was isolated from random RNA nucleic acid libraries by in vitro selection[5]
A fluorogenic self-quenched dimer based on rhodamine whose cognate aptamer is o-Coral was rationally designed[6]
By embedding biRhoBAST and biSiRA into RNA scaffolds, Fluorescent light-up aptamers with higher fluorogenicity and more remarkable photostability were obtained[7]
The co-crystal structure of RhoBAST in complex with tetramethylrhodamine-dinitroaniline was determined to elucidate the molecular basis for ligand binding and fluorescence activation[8]
Description
In 2021, G. Ulrich Nienhaus and Andres Jaschke et al. isolated aptamer with rapid binding and dissociation kinetics for rhodamine dye through in vitro selection technology. In 2024, Xianyang Fang and Xing Li et al determined the co-crystal structure of RhoBAST in complex with tetramethylrhodamine-dinitroaniline by small-angle X-ray scattering. This RhoBAST-tetramethylrhodamine-dinitroaniline complex adopts an asymmetrical “A”-like architecture organized with a four-way[5,8].
SELEX
This work generated aptamers that bind TMR-DN using SELEX. An library consisting of a 15% doped 54-nucleotide SRB-2 sequence which containing ~3×1014 random library members was used and TMR-SS-Biotin was used as positive target. RhoBAST bound to TMR-DN was isolated from random RNA nucleic acid libraries by in vitro selection. The crystal structure of the RhoBAST-TMR-DN complex was reported in subsequent work[5,8].
Detailed information are accessible on SELEX page.
Structure
2D representation
The RhoBAST RNA aptamer adopts an asymmetric, "A"-shaped architecture, organised through a four-way junction rather than the previously predicted three-way configuration. This junction comprises three A-form helices—designated P1, P2, and P4—and a short pseudo-helix, P3, interconnected by three loops[8].
5'-GAACCUCCGCCCAUUGCACUCCGGGCGGUGAAGGAGAGGCGCAAGGUUAACCGCCUCAGGUUCC-3'
3D visualisation
In 2024, Xianyang Fang and Xing Li et al. determined the crystal structure of the RhoBAST aptamer bound to the fluorophore-quencher conjugate TMR-DN. The aptamer adopts an asymmetric "A"-shaped architecture featuring a four-way junction. The fluorophore-binding core is formed by the four-nucleotide capping loop L4 (G46–G47–U48–U49) and an underlying A32•A50•A45 base triple, creating a semi-open rectangular pocket. G47's orientation is stabilised by a hydrogen bond between its N7 and the O2' of G46, and by two hydrogen bonds involving U49's O4 and N3 with G47's N3 and N2. Notably, the DN quencher remains partially stacked over the phenyl ring of TMR, indicating an incompletely unquenched state. The PDB ID of the structure is 8JY0 (2.75 Å)[8].
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: 8JY0 by small-angle X-ray scattering. TMR-DN (shown in sticks) is labeled in magenta. Right: The hydrogen bonds of binding sites of the aptamer bound with TMR-DN or other nucleotides surround small molecules.
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Ligand information
SELEX ligand
The equilibrium dissociation constant of the aptamer: TMR-DN complex was determined by measuring fluorescence intensity as a function of RNA concentration at 25℃, with the fluorophore-quencher conjugate TMR-DN maintained at a fixed concentration of 1 nM. Fluorescence readings were recorded after each RNA addition, and the resulting data were fitted to a one-site binding model to calculate the Kd. This approach ensures that the measured fluorescence changes are directly attributable to complex formation under equilibrium conditions[5].
Structure ligand
TMR-DN, comprising 5-carboxytetramethylrhodamine conjugated to a dinitroaniline quencher, functions via contact-mediated fluorescence quenching. This fluorophore–quencher conjugate is non-toxic to cells and permeable to cell membranes, making it suitable for live-cell imaging applications.-----From Lumiprobe
PubChem CID: a unique identifier for substances in the PubChem database.
CAS number: a global registry number for chemical substances.
ChEBI ID: a unique identifier assigned to each molecular entity in the Chemical Entities of Biological Interest database.
| Name | PubChem CID | Molecular Formula | Molecular Weight | CAS | Solubility | CHEBI ID |
|---|---|---|---|---|---|---|
| TMR-DN | 9952143 | C24H22N203 | 386.4 g/mol | 120718-52-7 | NA | 52282 |
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Similar compound(s)
We screened the compounds with great similarity to TMR-DN 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 |
|---|---|---|---|---|
| ZINC5140632 | Tetramethylrhodamine | 120718-52-7 | 9952143 |  |
| ZINC4521863 | Rhodamine I | 115532-52-0 | 2762683 |  |
| ZINC4521864 | Rhodamine II | 115532-49-5 | 5009757 |  |
References
[1] Selecting RNA aptamers for synthetic biology: investigating magnesium dependence and predicting binding affinity.Carothers, J. M., Goler, J. A., Kapoor, Y., Lara, L., & Keasling, J. D.
Nucleic acids research, 38(8), 2736–2747. (2010)
[2] NMR resonance assignments for the tetramethylrhodamine binding RNA aptamer 3 in complex with the ligand 5-carboxy-tetramethylrhodamine.
Duchardt-Ferner, E., Juen, M., Kreutz, C., & Wöhnert, J.
Biomolecular NMR assignments, 11(1), 29–34. (2017)
[3] SiRA: A Silicon Rhodamine-Binding Aptamer for Live-Cell Super-Resolution RNA Imaging.
Wirth, R., Gao, P., Nienhaus, G. U., Sunbul, M., & Jäschke, A.
Journal of the American Chemical Society, 141(18), 7562–7571. (2019)
[4] Structure of an RNA aptamer in complex with the fluorophore tetramethylrhodamine.
Duchardt-Ferner, E., Juen, M., Bourgeois, B., Madl, T., Kreutz, C., Ohlenschläger, O., & Wöhnert, J.
Nucleic acids research, 48(2), 949–961. (2020)
[5] Super-resolution RNA imaging using a rhodamine-binding aptamer with fast exchange kinetics.
Sunbul, M., Lackner, J., Martin, A., Englert, D., Hacene, B., Grün, F., Nienhaus, K., Nienhaus, G. U., & Jäschke, A.
Nature biotechnology, 39(6), 686–690. (2021)
[6] Rational Design of Self-Quenched Rhodamine Dimers as Fluorogenic Aptamer Probes for Live-Cell RNA Imaging.
Fam, K. T., Pelletier, R., Bouhedda, F., Ryckelynck, M., Collot, M., & Klymchenko, A. S.
Analytical chemistry, 94(18), 6657–6664. (2022)
[7] Avidity-based bright and photostable light-up aptamers for single-molecule mRNA imaging.
Bühler, B., Schokolowski, J., Benderoth, A., Englert, D., Grün, F., Jäschke, A., & Sunbul, M.
Nature chemical biology, 19(4), 478–487. (2023)
[8] Structural mechanisms for binding and activation of a contact-quenched fluorophore by RhoBAST.
Zhang, Y., Xu, Z., Xiao, Y., Jiang, H., Zuo, X., Li, X., & Fang, X.
Nature communications, 15(1), 4206. (2024)
[9] Structural basis for ring-opening fluorescence by the RhoBAST RNA aptamer.
Siwik, S. H., Wierzba, A. J., Lennon, S. R., Olenginski, L. T., Palmer, A. E., & Batey, R. T.
bioRxiv : the preprint server for biology, 2024.12.30.630784. (2024)