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Spinach RNA aptamer, Aptamer 24-2-min(Spinach)
Timeline
Antibody fragments (Fab, Fv) that recognize native protein conformations have been shown to facilitate crystallization of other membrane proteins by increasing the polar surface area for protein-protein contacts and by restricting the flexibility of mobile domains[2]
The human β2-adrenergic G-protein-coupled receptor was crystallized as a complex with the antigen-binding fragment (Fab) derived from a monoclonal antibody[3]
With a higher molecular weight (50 kDa) than the U1A protein (11 kDa), Fab chaperones provide more surface area for crystal contacts, and their β-rich architecture is predisposed to making effective crystal contacts[4]
used antibody-assisted crystallography to determine the structures of Spinach both with and without bound fluorophore at 2.2-Å and 2.4-Å resolution[7]
Show that Pb(2+) induces formation of Spinach's G-quadruplex and activates fluorescence with high selectivity and sensitivity[8]
Split spinach aptamer (SSA) probes for fluorescent analysis of nucleic acids were designed and tested[9]
Description
In 1986, Satow Y et al. used X-ray for the first time to resolve the Phosphocholine binding immunoglobulin Fab. In 2014, Hao Huang and Nikolai B used antibody-assisted crystallography to determine the structures of Spinach (24-2 min aptamer). In 2018, Koirala D et al. studied the Crystal structure of Spinach RNA aptamer in complex with Fab BL3-6S97N[1,7,10].SELEX
Paige, J. S. 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. In 2014, Hao Huang and Nikolai B crystallized the minimal form of aptamer 24-2-min and they analyzed the complex structure of this aptamer[5].
Detailed information are accessible on SELEX page.
Structure
2D representation
An independent selection using a 40-nt randomized library unconstrained by secondary structure revealed a GNGACCC consensus sequence for Fab BL3–6 binding[5].
5'-GACGCGACCGAAAUGGUGAAGGACGGGUCCAGUGCGAAACACGCACUGUUGAGUAGAGUGUGAGCUCCGUAACUGGUCGCGUC-3'
Here we use ribodraw to complete the figure, through the 3D structure information[5].
5'-GGACGCGACCGAAAUGGUGAAGGACGGGUCCAGUGCGAAACACGCACUGUUGAGUAGAGUGUGAGCUCCGUAACUGGUCGCGUC-3'
3D visualisation
DasGupta, S., Shelke, S.A., Piccirilli, J.A. et al.sovled the crystal structure, at 1.64 Å resolution. The PDB ID of this structure is 6B14[5].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: 6B14 at 1.64 Å resolution. 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.
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Ligand information
SELEX ligand
DasGupta, S., Shelke, S.A., Piccirilli, J.A. et al. The binding constant of the aptamer was determined by nitrocellulose filtration experiment[5].| Name | Sequence | Ligand | Affinity |
|---|---|---|---|
| Spinash RNA | 5'-GACGCGACCGAAAUGGUGAAGGACGGGUCCAGUGCGAAACACGCACUGUUGAGUAGAGUGUGAGCUCCGUAACUGGUCGCGUC-3' | Fab BL3-6S97N | 45 ± 11 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.
| Uniprot ID | Pfam | MW | Amino acids sequences | PDB ID | GenBank |
|---|---|---|---|---|---|
| NA | NA | 47.19 KDa | heavy chain-EVQLVESGGGLVQPGGSLRLSCAASGFYISYSSIHWVRQAPGKGLEWVASISPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARQGYRRRSGRGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC light chain-SDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSYSFPSTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC |
6B3K | NA |
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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. The Dali server is a network service for comparing protein structures in 3D. Dali compares them against those in the Protein Data Bank (PDB).
| PDB | Z-score | RMSD | Description |
|---|---|---|---|
| 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 |
| 7L15-A | 18.5 | 6.3 | T cell receptor mu chain |
References
[1] Crystallisation of membrane proteins mediated by antibody fragments.Carola Hunte 1, Hartmut Michel.
Current opinion in structural biology, 12(4), 503–508 (2002)
[2] A monoclonal antibody for G protein-coupled receptor crystallography.
Day PW, Rasmussen SG, Parnot C, Fung JJ, Masood A, Kobilka TS, Yao XJ, Choi HJ, Weis WI, Rohrer DK, Kobilka BK
Nature methods, 4(11), 927–929 (2007)
[3] Engineering of recombinant crystallization chaperones.
Koide S.
Current opinion in structural biology, 19(4), 449–457 (2009)
[4] A portable RNA sequence whose recognition by a synthetic antibody facilitates structural determination.
Koldobskaya Y, Duguid EM, Shechner DM, Suslov NB, Ye J, Sidhu SS, Bartel DP, Koide S, Kossiakoff AA, Piccirilli JA
Nature structural & molecular biology, 18(1), 100–106 (2011)
[5] Affinity maturation of a portable Fab-RNA module for chaperone-assisted RNA crystallography.
Koirala D, Shelke SA, Dupont M, Ruiz S, DasGupta S, Bailey LJ, Benner SA, Piccirilli JA
Nucleic acids research, 46(5), 2624–2635 (2018)
[6] Phosphocholine binding immunoglobulin Fab McPC603. An X-ray diffraction study at 2.7 A.
Satow Y, Cohen GH, Padlan EA, Davies DR
Journal of molecular biology, 190(4), 593–604 (1986)