Home
Database
Research
About us
NF-kappaB aptamer
Timeline
L J Maher 3rd isolated a small RNA aptamer that binds with nanomolar affinity to human transcription factor NF-kappa B in vitro selection[1]
Described here the 2.45-A resolution x-ray crystal structure of the p50 RHR/RNA aptamer complex[2]
Yeast genetic selections were used to optimize the RNA aptamer for binding to NF-kappaB in the eukaryotic nucleus, RNA aptamers with dramatically improved in vivo activity[3]
The first example of a post-modification of an NF-kappaB RNA aptamer by 4'-thioribonucleoside units[4]
The use of aptamers and siRNA together can be the most effective way to achieve maximal knock-down of protein activity[5]
The high affinity of the NF-kappaB transcription factor for this RNA aptamer may largely be due to the structural pre-organization of the RNA that results in its ability to mimic DNA[6]
In vitro selection of anti-NF-kappaB p65 RNA aptamers using parallel in vitro selections with either a fully randomized RNA library[7]
Description
In 1999, L J Maher 3rd isolated an aptamer that binds NF-kappaB p50, and its crystal structure was cracked in 2003. The results of competition experiments demonstrate that binding of the RNA aptamer blocks the ability of NF-kappa B to bind duplex DNA. Expression of this aptamer structure within heterologous nuclear RNA transcripts may provide a new strategy to inhibit NF-kappa B function in vivo. In 2008, He selected the RNA aptamer of anti-NF-kappaB p65. And reported the characterization of these aptamers with respect to NF-kappaB target specificity, affinity, minimal sequence requirements, secondary structure, and competition with DNA kappaB sites[1,2,7].SELEX
In 1999, L J Maher 3rd identified RNA aptamers capable of tightly binding p502 using cycles of in vitro affinity selection and amplification. The initial RNA pool represented ∼1014 different sequences consisting of a core of 60 random nucleotides flanked. Fourteen cycles of selection and amplification were performed,a dominant RNA was detected as 21 of 26 cloned and sequenced cDNAs[1].
Detailed information are accessible on SELEX page.
Structure
2D representation
Here we used ribodraw to complete the figure, through the 3D structure information[2].
5'-CAUACUUGAAACUGUAAGGUUGGCGUAUG-3'
3D visualisation
L. J., 3rd, & Ghosh, G. et al. sovled the 2.45-Å resolution x-ray crystal structure of the p50 RHR/RNA aptamer complex. The structural analysis indicates that two RNA molecules bind autonomously to the N-terminal Ig-like domains of p50. The RNA secondary structure comprises a stem and a stem-loop motif, with an internal loop adopting a kinked helical conformation due to the stacking interactions between three guanine residues within the loop. The PDB ID of this structure is 1OOA[2].Additional available structures that have been solved and detailed information are accessible on Structures page.
(Clicking the "Settings/Controls info" to turn Spin off)
|
|
Binding pocket
Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 1OOA at 2.45 Å resolution. NF-kB(p50) protein (shown in vacuumm electrostatics), blue is positive charge, red is negative charge. Right: The hydrogen bonds of binding sites of the aptamer bound with NF-kB(p50) protein.
|
|
Ligand information
SELEX ligand
Nitrocellulose filter binding experiments were employed to estimate the equilibrium dissociation constants of different RNA aptamer/NF-κB complexes. Binding reactions containing variable concentrations of NF-κB protein were assembled in binding buffer with a known concentration (typically 0.85 nM) of end-labeled RNA aptamer. Binding reactions were incubated for 10 min at room temperature and then filtered over 0.45 µm nitrocellulose filters ( Millipore catalog no. HAWP02500). Filters were washed with binding buffer, and radioactivity was determined by scintillation counting. p50dd: p50 dimerization domain. p65dd: p65 dimerization domain[2].| Name | Sequence | Ligand | Affinity |
|---|---|---|---|
| α-p50 RNA aptamer | CAUACUUGAAACUGUAAGGUUGGCGUAUG | NF-κB (p50)2 | 5.4±2.2 nM |
| α-p50 RNA aptamer | CAUACUUGAAACUGUAAGGUUGGCGUAUG | p50/p65 | 22±7 nM |
| α-p50 RNA aptamer | CAUACUUGAAACUGUAAGGUUGGCGUAUG | (p65)2 | 3500±2300 nM |
| α-p50 RNA aptamer | CAUACUUGAAACUGUAAGGUUGGCGUAUG | p50dd/p65 | 187±155 nM |
| α-p50 RNA aptamer | CAUACUUGAAACUGUAAGGUUGGCGUAUG | p50/p65dd | 10±4 nM |
| α-p50 RNA aptamer | CAUACUUGAAACUGUAAGGUUGGCGUAUG | p50dd/p65dd | No binding |
Structure ligand
This entry represents the N-terminal sub-domain of the Rel homology domain (RHD) of NF-kappaB subunit precursor p105, which can undergo cotranslational processing by the 26S proteasome to produce a 50kDa protein (p50). p50 is a DNA binding subunit of the NF-kappaB protein complex.-----From Pfam
| Name | Uniprot ID | Pfam | MW | Amino acids sequences | PDB ID | GenBank |
|---|---|---|---|---|---|---|
| NF-kappaB p50 protein | P25799 | IPR030503 | 36.61 kDa | GGPYLQILEQPKQRGFRFRYVCEGPSHGGLPGASSEKNKKSYPQVKICNYVGPAKVIVQLVTNGKNIHLHAHSLVGKHCEDGVCTVTAGPKDMVVGFANLGILHVTKKKVFETLEARMTEACIRGYNPGLLVHSDLAYLQAEGGGDRQLTDREKEIIRQAAVQQTKEMDLSVVRLMFTAFLPDSTGSFTRRLEPVVSDAIYDSKAPNASNLKIVRMDRTAGCVTGGEEIYLLCDKVQKDDIQIRFYEEEENGGVWEGFGDFSPTDVHRQFAIVFKTPKYKDVNITKPASVFVQLRRKSDLETSEPKPFLYYPEIKDKEEVQRKRQK | 1OOA | 10090 |
 |
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-score | RMSD | Description |
|---|---|---|---|
| 1OY3-C | 20.2 | 1.0 | Transcription factor p65 |
| 2O61-A | 20 | 6.1 | 36-mer |
| 8OW4-B | 16 | 2.7 | Nuclear factor of activated t-cells,cytoplasmic |
| 2CXK-A | 10.7 | 1.9 | Calmodulin binding transcription activator 1 |
| 5L56-A | 10.5 | 25.0 | Plexin-a1 |
| 6VXK-B | 10.4 | 22.2 | Semaphorin-like protein 139 |
| 3HRP-A | 10.2 | 2.6 | Uncharacterized protein |
| 3MLP-A | 10 | 10.3 | Transcription factor coe1 |
| 6STX-C | 9.9 | 8.3 | Kelch domain-containing protein |
| 7MO7-B | 9.6 | 11.3 | Hepatocyte growth factor |
References
[1] Selection and characterization of an RNA decoy for transcription factor NF-kappa B.Lebruska, L. L., & Maher, L. J., 3rd.
Biochemistry, 38(10), 3168–3174. (1999)
[2] Crystal structure of NF-kappaB (p50)2 complexed to a high-affinity RNA aptamer.
Huang, D. B., Vu, D., Cassiday, L. A., Zimmerman, J. M., Maher, L. J., 3rd, & Ghosh, G.
Proceedings of the National Academy of Sciences of the United States of America, 100(16), 9268–9273. (2003)
[3] Yeast genetic selections to optimize RNA decoys for transcription factor NF-kappa B.
Cassiday, L. A., & Maher, L. J., 3rd.
Proceedings of the National Academy of Sciences of the United States of America, 100(7), 3930–3935. (2003)
[4] Synthesis and physical and physiological properties of 4'-thioRNA: application to post-modification of RNA aptamer toward NF-kappaB.
Hoshika, S., Minakawa, N., & Matsuda, A.
Nucleic acids research, 32(13), 3815–3825. (2004)
[5] Co-expression of anti-NFkappaB RNA aptamers and siRNAs leads to maximal suppression of NFkappaB activity in mammalian cells.
Chan, R., Gilbert, M., Thompson, K. M., Marsh, H. N., Epstein, D. M., & Pendergrast, P. S.
Nucleic acids research, 34(5), e36. (2006)
[6] DNA mimicry by a high-affinity anti-NF-kappaB RNA aptamer.
Reiter, N. J., Maher, L. J., 3rd, & Butcher, S. E.
Nucleic acids research, 36(4), 1227–1236. (2008)
[7] Selection and characterization of anti-NF-kappaB p65 RNA aptamers.
Wurster, S. E., & Maher, L. J., 3rd.
RNA (New York, N.Y.), 14(6), 1037–1047. (2008)
[8] Characterization of anti-NF-kappaB RNA aptamer-binding specificity in vitro and in the yeast three-hybrid system.
Wurster, S. E., Bida, J. P., Her, Y. F., & Maher, L. J., 3rd.
Nucleic acids research, 37(18), 6214–6224. (2009)