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B52-aptamer
Timeline
Isolated B52-binding RNAs by selection and amplification from a pool of random RNA sequences[1]
Demonstrate that Drosophila B52 protein can be specifically inhibited in vitro and in vivo by a multivalent RNA aptamer[2]
Deletion and substitution mutations defined the B52-binding site on these RNAs as a hairpin loop structure covering about 20 nucleotides[3]
Description
In 1997, Shi, H. et al. isolated B52-binding RNAs through selection and amplification from a pool of random RNA sequences, using the full-length B52 protein as the target. These RNAs contained a conserved consensus motif forming the core of a secondary structural element predicted by energy minimization. Deletion and substitution mutations defined the B52-binding site on these RNAs as a hairpin loop structure encompassing approximately 20 nucleotides. Subsequently they demonstrated inhibitory aptamer RNA binds B52 avidly and inhibits B52-stimulated pre-mRNA splicing. In 1999, Xu, D. & Shi, H. demonstrated that the performance of this 'aptabody' rivals that of a monoclonal antibody against B52[1].SELEX
They used, with some modification, an in vitro selection and amplification scheme (SELEX). And adapted the template-primer system to produce RNA molecules had a stretch of 40 random bases sandwiched between 59 and 39 constant regions for primer annealing. The 59 constant sequence included a promoter for T7 RNA polymerase. Synthetic oligodeoxynucleotide template (20 mg of a 108-mer) was amplified by PCR to make the original pool. This pool contained 3.4 * 1013 different sequences. The pool of random RNA was carried through nine rounds of selection and amplification. RNA-protein complexes were selected by binding to nitrocellulose filters. Observed a significant increase of the pool’s affinity for B52 as the selection progressed. Fractions of two final pools were cloned and sequenced[1].
Detailed information are accessible on SELEX page.
Structure
The 2D structure of the figures is based on the article by ribodraw tool to draw.5'-GGGAGAAUUCAACUGCCAUCUAGGCUGGUCAACCAGGCGACCGCCACCCGCGCGCGCAAUACCUAGUACUACAAGCUUCUGGACUCGGU-3'
Ligand information
SELEX ligand
SR proteins are a conserved family of proteins involved in RNA splicing. SR proteins are named because they contain a protein domain with long repeats of serine and arginine amino acid residues, whose standard abbreviations are "S" and "R" respectively. SR proteins are ~200-600 amino acids in length and composed of two domains, the RNA recognition motif (RRM) region and the RS domain. B52 is also called SRp55, splicing factor, arginine/serine-rich 6 is a protein that in humans is encoded by the SFRS6 gene.-----From Wiki
| Name | Uniprot ID | Pfam | MW | Amino acids sequences | PDB | Gene ID |
|---|---|---|---|---|---|---|
| SR Protein B52 (SRp55) | Q13247 | PF00076 | 39.6 KDa | MPRVYIGRLSYNVREKDIQRFFSGYGRLLEVDLKNGYGFVEFEDSRDADDAVYELNGKELCGERVIVEHARGPRRDRDGYSYGSRSGGGGYSSRRTSGRDKYGPPVRTEYRLIVENLSSRCSWQDLKDFMRQAGEVTYADAHKERTNEGVIEFRSYSDMKRALDKLDGTEINGRNIRLIEDKPRTSHRRSYSGSRSRSRSRRRSRSRSRRSSRSRSRSISKSRSRSRSRSKGRSRSRSKGRKSRSKSKSKPKSDRGSHSHSRSRSKDEYEKSRSRSRSRSPKENGKGDIKSKSRSRSQSRSNSPLPVPPSKARSVSPPPKRATSRSRSRSRSKSRSRSRSSSRD | 2M7S | 6431 |
Some isolated sequences bind to the affinity of the protein.
| Name | Sequence | Ligand | Affinity |
|---|---|---|---|
| BSS#8 | 5'-GGGAGAAUUCAACUGCCAUCUAGGCUGGUCAACCAGGCGACCGCCACCCGCGCGCGCAAUACCUAGUACUACAAGCUUCUGGACUCGGU-3' | B52 protein RRMs domain | 20 nM |
| BBS #4,14,15 | 5'-GGGAGAAUUCAACUGCCAUCUAGGCAGGGUAACGAUCAACCUGGCGACAGCUGCCCUGCCGUCCAAGUACUACAAGCUUCUGGACUCGGU-3' | B52 protein RRMs domain | 50 nM |
Similar compound
We used the Dail server website to compare the structural similarities of ligand proteins, and chose the top 10 in terms of 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). Z-score is 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) value is used to measure the degree to which atoms deviate from the alignment position.
| PDB | Z-socre | RMSD | Description |
|---|---|---|---|
| 2O3D-A | 10.7 | 2.1 | Splicing factor, arginine/serine-rich 1 |
| 4PKD-B | 10.5 | 6.7 | U1 snrna stem-loops 1 and 2 (55-mer) |
| 5D77-A | 10.5 | 1.6 | RNA-binding protein mip6 |
| 6N7P-F | 10.2 | 1.6 | U1 small nuclear ribonucleoprotein 70 kda homolog |
| 1HL6-A | 10.2 | 2.3 | Cg8781 protein |
| 6ELD-A | 9.6 | 1.9 | Nucleolysin tia-1 isoform p40,u1 small nuclear |
| 2MZQ-A | 9.5 | 2.4 | Single-strand telomeric dna-binding protein gbp2 |
| 3VF0-B | 9.5 | 2.0 | Vinculin |
| 8HNI-F | 9.5 | 1.7 | Heterogeneous nuclear ribonucleoproteins a2/b1 |
| 4WIJ-B | 9.5 | 3.0 | Splicing factor, proline- and glutamine-rich |
References
[1] A specific RNA hairpin loop structure binds the RNA recognition motifs of the Drosophila SR protein B52.Shi, H., Hoffman, B. E., & Lis, J. T.
Molecular and cellular biology, 17(5), 2649–2657. (1997)
[2] RNA aptamers as effective protein antagonists in a multicellular organism.
Shi, H., Hoffman, B. E., & Lis, J. T.
Proceedings of the National Academy of Sciences of the United States of America, 96(18), 10033–10038. (1999)
[3] Specific SR protein-dependent splicing substrates identified through genomic SELEX.
Kim, S., Shi, H., Lee, D. K., & Lis, J. T.
Nucleic acids research, 31(7), 1955–1961. (2003)
[4] Composite RNA aptamers as functional mimics of proteins.
Xu, D., & Shi, H.
Nucleic acids research, 37(9), e71. (2009)