Chili aptamer

Apr 22, 2024 • Bo Fu, Jiali Wang

Timeline

Description

In 2011, Samie R. Jaffrey et al. isolated Spinach RNA aptamer and obtained a 13-2 aptamer series that has affinity with DMHBI and can activate its fluorescence. In 2018, Claudia Höbartner et al. obtained an RNA aptamer with stronger affinity to DMHBI through rational design of 13-2-min, and named it Chili. In 2021, Claudia Höbartner et al. analyzed the structure of the Chili-DMHBI's derivatives complexs through crystallization, diffraction data collection and NMR spectroscopy^[1,2,3].

SELEX

This work generated aptamers that bind DMHBI using SELEX. A DNA library containing ~5×10¹³ random sequence members was used and DMHBI was conjugated to agarose beads as positive target. After 10 rounds of SELEX, an RNA aptamer that binds DFAME and activates its fluorescence was identified. These RNA mimics of GFP provide a useable approach for the genetic encoding of fluorescent RNAs^[1].

Detailed information are accessible on SELEX page.

Structure

2D representation

The Chili RNA folds into a single coaxial helical stack, consisting of two A-form duplexes, P1 and P2, which are separated by the central fluorophore-binding domain. Here we used ribodraw to complete the figure, through the 3D structure information^[2].

5'-GGCUAGCUGGAGGGGCGCCAGUUCGCUGGUGGUUGGGUGCGGUCGGCUAGCC-3'

3D visualisation

In 2021, Claudia Höbartner et al. analyzed the structure of the Chili-DMHBI's derivatives complexs through crystallization, diffraction data collection and NMR spectroscopy. The Chili RNA adopts a single coaxial helical stack (~70 Å in length), comprising two A-form duplexes (P1 and P2) flanking the central fluorophore-binding domain. The basal stem P1 forms an 8 bp duplex (nucleotides 1-8 and 45-52), while the apical stem P2 (5 bp, nt 17-21 and 26-30) is capped by the UUCG tetraloop L2 (nt 22–25). The FBD spans nucleotides 9-16 and 31-44, forming the ligand-binding pocket. Structural data obtained by X-ray crystallography were deposited in the Protein Data Bank and are available with the following accession codes: 7OAW (2.95 Å), 7OAX (2.24 Å), 7OA3 (2.8 Å), 7OAV (2.99 Å)^[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: 7OAW by NMR and X-ray crystallography. DMHBI+ (shown in sticks) is labeled in magenta. Right: The hydrogen bonds of binding sites of the aptamer bound with DMHBI+ or other nucleotides surround small molecules.

Ligand information

SELEX ligand

The Kd was determined by and measuring the increase in fluorescence as a function of increasing dye concentration in the presence of a fixed concentration of RNA aptamer^[2].

Structure ligand

DMHBI (4-[(4-hydroxy-3,5-dimethoxyphenyl)methylidene]-1,2-dimethyl-4,5-dihydro-1H-imidazol-5-one) is predominantly found in the phenolic form, the brighter DMFBI mimics EGFP and is anionic under standard conditions. The DMFBI-RNA complex has been called Spinach, it is the first fluorescent RNA tag that is both selective and non-toxic. It only fluoresces when bound to RNA, is resistant to photobleaching and has been used to follow RNA tagged molecules as they move through cells.-----From Green Fluorescent Protein

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
DMHBI	68787434	C14H16N2O4	276.29 g/mol	1241390-25-9	NA	NA

Similar compound(s)

We screened the compounds with great similarity to DMHBI 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
ZINC2051913110	DFHBI	NA	70808995
ZINC1857790839	DFHBI 1T	NA	101889712
ZINC2048528770	DFHBI 2T	NA	129080921
ZINC145340396	NA	NA	46898418
ZINC4918310	NA	NA	NA
ZINC34107441	NA	NA	6527199
ZINC8091545	NA	NA	19374977
ZINC409030759	NA	NA	126037191
ZINC408815341	NA	NA	125823074

References

[1] RNA mimics of green fluorescent protein.
Paige, J. S., Wu, K. Y., & Jaffrey, S. R.
Science (New York, N.Y.), 333(6042), 642–646. (2011)
[2] A multicolor large stokes shift fluorogen-activating RNA aptamer with cationic chromophores.
Steinmetzger, C., Palanisamy, N., Gore, K. R., & Höbartner, C.
Chemistry (Weinheim an der Bergstrasse, Germany), 25(8), 1931–1935. (2019)
[3] Structure-fluorescence activation relationships of a large Stokes shift fluorogenic RNA aptamer.
Steinmetzger, C., Bessi, I., Lenz, A. K., & Höbartner, C.
Nucleic acids research, 47(22), 11538–11550. (2019)
[4] Large Stokes shift fluorescence activation in an RNA aptamer by intermolecular proton transfer to guanine.
Mieczkowski, M., Steinmetzger, C., Bessi, I., Lenz, A. K., Schmiedel, A., Holzapfel, M., Lambert, C., Pena, V., & Höbartner, C.
Nature communications, 12(1), 3549. (2021)
[5] Large Stokes shift fluorescent RNAs for dual-emission fluorescence and bioluminescence imaging in live cells.
Trachman, R. J., 3rd, Autour, A., Jeng, S. C. Y., Abdolahzadeh, A., Andreoni, A., Cojocaru, R., Garipov, R., Dolgosheina, E. V., Knutson, Jiang, L., Xie, X., Su, N., Zhang, D., Chen, X., Xu, X., Zhang, B., Huang, K., Yu, J., Fang, M., Bao, B., Zuo, F., Yang, L., Zhang, R., Li, H., Huang, X., Chen, Z., Zeng, Q., Liu, R., Lin, Q., … Yang, Y.
Nature methods, 20(10), 1563–1572. (2023)