Spinach RNA aptamer, Aptamer 24-2-min(Spinach)

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Timeline

2011

The first fluorogenic RNA aptamer ‘Spinach’ which was capable of turning on the fluorescence were isolated upon the binding of small-molecule fluorophores[1]

2013

Spinach RNA was engineered as a genetically-encoded tag to the cellular metabolites and second messenger cyclic di-AMP (cdiA)[2]

2014

Piccirilli, J. A. et al. used antibody-assisted crystallography to determine the structures of Spinach RNA both with and without bound fluorophore at resolutions of 2.2 Å and 2.4 Å respectively[3]

2014

Ferré-D'Amaré, A. R. et al. solved the cocrystal structure of Spinach RNA bound to its cognate exogenous chromophore[4]

2015

Jaffrey, S. R. et al. showed that Spinach RNA can be swapped into various riboswitches, allowing metabolite binding to induce Spinach fluorescence directly[5]

2018

Piccirilli, J. A. et al. replaced the P2 stem loop of Spinach RNA with the pentaloop and crystallized the aptamer as a complex with Fab BL3–6[6]

Description

In 2011, Jaffrey, S. R. et al. isolated the first fluorogenic RNA aptamer ‘Spinach’ upon the binding of small-molecule fluorophores, which was capable of turning on the fluorescence. In 2018, Piccirilli, J. A. et al. replaced the P2 stem loop of Spinach RNA with the pentaloop and crystallized the aptamer as a complex with Fab BL3–6. Fab BL3-6S97N is an affinity-matured variant of the Fab BL3-6 antibody fragment, featuring a key S97N mutation in CDR L3. This mutation introduces asparagine, forming additional hydrogen bonds with RNA nucleobase A40, enhancing binding affinity by ~20-fold compared to the parent Fab. The crystal structure of Fab BL3-6S97N bound to the GAAACAC RNA motif shows N97’s carboxamide group within hydrogen-bonding distance of A40, strengthening interactions[1,6].



SELEX

In 2011, Jaffrey, S. R. et al. identified RNA sequences that bind and activate the fluorescence of GFP fluorophores beginning with 3,5-dimethoxy-4-hydroxybenzylidene imidazolinone (DMHBI). And then they performed SELEX with a library containing ~5 × 1013 RNA molecules and selected RNAs for their ability to bind DMHBI-agarose. After five rounds of selection, the pool of RNAs weakly activated DMHBI fluorescence, with further increases in fluorescence up to round 10[1].

Detailed information are accessible on SELEX page.



Structure

2D representation

In 2011, the first fluorogenic RNA aptamer ‘Spinach’ which was capable of turning on the fluorescence were isolated upon the binding of small-molecule fluorophores. Here we utilized RiboDraw to complete the figure, based the 3D structure information[1].

5'-GACGCGACCGAAAUGGUGAAGGACGGGUCCAGUGCGAAACACGCACUGUUGAGUAGAGUGUGAGCUCCGUAACUGGUCGCGUC-3'

drawing

3D visualisation

In 2018, Piccirilli, J.A. et al. determined the crystal structure of Spinach RNA aptamer in complex with Fab BL3-6S97N, at 1.64 Å resolution. The PDB ID of this structure is 6B14[6].

Additional available structures that have been solved and detailed information are accessible on Structures page.

(Clicking the "Settings/Controls info" to turn Spin off)      

drawing PDBe Molstar




Binding pocket

Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 6B14. Fab BL3-6S97N (shown in vacuumm electrostatics), blue is positive charge, red is negative charge. Right: The hydrogen bonds of binding sites of the aptamer bound with Fab BL3-6S97N.

drawing drawing


Ligand information

SELEX ligand

Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. Piccirilli, J. A, et al. mainly studied the crystal structure of the complex of Spinach RNA with Fab BL3-6S97N. According to the article, the affinity of Spinach RNA with Fab BL3-6S97N complex is 25 ± 6 nM. In addition, the crystal structure of the Spinach RNA complex with DFHBI was investigated with an affinity of 300 ± 68 nM. The binding constants of selected RNA clones and related mutants were determined by nitrocellulose filter binding assay as reported previously[6].

Name Sequence Ligand Affinity
Spinach aptamer 5'-GACGCGACCGAAAUGGUGAAGGACGGGUCCAGUGCGAAACACGCACUGUUGAGUAGAGUGUGAGCUCCGUAACUGGUCGCGUC-3' Fab BL3-6S97N 25 ± 6 nM
Spinach aptamer 5'-GACGCGACCGAAAUGGUGAAGGACGGGUCCAGUGCGAAACACGCACUGUUGAGUAGAGUGUGAGCUCCGUAACUGGUCGCGUC-3' DFHBI 300 ± 68 nM

Structure ligand

Fab (Antigen-binding fragment), also called antigen-binding fragment, is a region in the antibody structure that can bind to antigen. It consists of a complete light chain (variable region and constant region) and a partial heavy chain structure (variable region and a constant region fragment). The light chain and heavy chain are connected by a disulfide bond, which is small in size and has a molecular weight of 47-48 kDa.-----From article

UniProt ID: uniquely identifies protein sequences in the UniProt database, a resource for protein information.

Pfam: a widely recognised database of protein families and domains.

GenBank: maintained by NCBI(National Center for Biotechnology Information), is a database of nucleotide sequences from various organisms, vital for genetic and molecular biology research.

Mass: an intrinsic property of a body.

Uniprot ID Pfam Mass Protein sequence PDB ID GenBank
UniRef90_UPI001C68730C NA Heavy chain-24.74 kDa
Light chain-23.37 kDa
GGPYLQ ...... Heavy chain-EVQLVESGGGLVQPGGSLRLSCAASGFYISYSSIHWVRQAPGKGLEWVASISPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARQGYRRRSGRGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
Light chain-SDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSYSFPSTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
6B3K NA
drawing

Similar compound

We used the Dail server website to compare the structural similarities of ligand proteins, and selected the previous information with high similarity for presentation.

Dail server website: a network service for comparing protein structures in 3D. Dali compares them against those in the Protein Data Bank (PDB).

Z-score: a standard score that is converted from an original score. The list of neighbours is sorted by Z-score. Similarities with a Z-score lower than 2 are spurious.

RMSD: (Root Mean Square Deviation) is used to measure the degree to which atoms deviate from the alignment position.

PDB: PDB ID+ chain name.

PDB Z-score RMSD Description
6B14-H 34.1 0 Original chain
6RUL-A 22.5 1.8 GFP-lama-f98 a gfp enhancer nanobody with cpdhfr
8IQS-H 22.4 4 M11 vl-sarah
4HJJ-H 22 1.4 Interleukin-18
7JZ1-H 21.1 5.3 Mgc34 heavy chain
6XUX-A 20.3 1.3 Nanobody,glucosidase ygjk,glucosidase ygjk,nanobo
7CEA-A 20.2 4 Huts-4 vh(s112c)-sarah
7RGA-E 19.5 1.4 Nano clostridial antibody mimetic protein 3 vhh
4A6Y-A 19.3 3 Antibody bbe6.12h3 light chain
7KHF-A 18.8 14.9 Mdb1 fab heavy chain


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] RNA-based fluorescent biosensors for live cell imaging of second messengers cyclic di-GMP and cyclic AMP-GMP.
Kellenberger, C. A., Wilson, S. C., Sales-Lee, J., & Hammond, M. C.
Journal of the American Chemical Society, 135(13), 4906–4909. (2013)
[3] A G-quadruplex-containing RNA activates fluorescence in a GFP-like fluorophore.
Huang, H., Suslov, N. B., Li, N. S., Shelke, S. A., Evans, M. E., Koldobskaya, Y., Rice, P. A., & Piccirilli, J. A.
Nature chemical biology, 10(8), 686–691. (2014)
[4] Structural basis for activity of highly efficient RNA mimics of green fluorescent protein.
Warner, K. D., Chen, M. C., Song, W., Strack, R. L., Thorn, A., Jaffrey, S. R., & Ferré-D'Amaré, A. R.
Nature structural & molecular biology, 21(8), 658–663. (2014)
[5] Imaging metabolite dynamics in living cells using a Spinach-based riboswitch.
You, M., Litke, J. L., & Jaffrey, S. R.
Proceedings of the National Academy of Sciences of the United States of America, 112(21), E2756–E2765. (2015)
[6] Affinity maturation of a portable Fab-RNA module for chaperone-assisted RNA crystallography.
Koirala, D., Shelke, S. A., Dupont, M., Ruiz, S., DasGupta, S., Bailey, L. J., Benner, S. A., & Piccirilli, J. A.
Nucleic acids research, 46(5), 2624–2635. (2018)