NF-kappaB P50 aptamer

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Timeline

1999

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]

2003

Described here the 2.45-Å resolution x-ray crystal structure of the p50 RHR/RNA aptamer complex[2]

2003

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]

2004

The first example of a post-modification of an NF-kappaB RNA aptamer by 4'-thioribonucleoside units[4]

2006

The use of aptamers and siRNA together can be the most effective way to achieve maximal knock-down of protein activity[5]

2008

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]

2008

In vitro selection of anti-NF-kappaB p65 RNA aptamers using parallel in vitro selections with either a fully randomized RNA library[7]

2009

Described the further characterization of anti-NF-kappaB p50 and p65 RNA aptamers in vitro and in vivo[8]

Description

In 1999, L.J.Maher 3rd isolated an aptamer that binds to NF-kappaB p50, and its crystal structure was determined in 2003. The results of competition experiments demonstrated that the binding of the RNA aptamer blocked the ability of NF-kappa B to bind to duplex DNA. Expression of this aptamer structure within heterologous nuclear RNA transcripts may provide a novel strategy to inhibit NF-kappa B function in vivo. In 2008, He selected an RNA aptamer against NF-kappaB p65 and reported the characterisation 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 comprised approximately 1014 different sequences, consisting of a core of 60 random nucleotides flanked by conserved sequences. Fourteen cycles of selection and amplification were performed, and a dominant RNA was detected as 21 of 26 cloned and sequenced cDNAs[1].

Detailed information are accessible on SELEX page.



Structure

2D representation

The α-p50 RNA aptamer, which is an enhanced variant of the initial 31-nt aptamer, was selected in yeast. This selection was carried out within a yeast three-hybrid selection system to enhance its interactions with (p50)2. It has been demonstrated that this optimized RNA aptamer can impede the binding of (p50)2 to DNA in vivo in a yeast one-hybrid system. Here we used RiboDraw to complete the figure, through the 3D structure information[2]. The α-p50 RNA aptamer was named by L.J.Maher 3rd et al. in the article[1].

5'-CAUACUUGAAACUGUAAGGUUGGCGUAUG-3'

drawing

3D visualisation

L. J. Maher 3rd and G. Ghosh et al. resolved the 2.45 Å resolution X-ray crystal structure of the p50 RHR/RNA aptamer complex. The structural analysis indicated that two RNA molecules bound autonomously to the N-terminal Ig-like domains of p50. The RNA secondary structure comprised 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)      

drawing PDBe Molstar




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.

drawing drawing


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 5'-CAUACUUGAAACUGUAAGGUUGGCGUAUG-3' NF-κB (p50)2 5.4±2.2 nM
α-p50 RNA aptamer 5'-CAUACUUGAAACUGUAAGGUUGGCGUAUG-3' p50/p65 22±7 nM
α-p50 RNA aptamer 5'-CAUACUUGAAACUGUAAGGUUGGCGUAUG-3' (p65)2 3500±2300 nM
α-p50 RNA aptamer 5'-CAUACUUGAAACUGUAAGGUUGGCGUAUG-3' p50dd/p65 187±155 nM
α-p50 RNA aptamer 5'-CAUACUUGAAACUGUAAGGUUGGCGUAUG-3' p50/p65dd 10±4 nM
α-p50 RNA aptamer 5'-CAUACUUGAAACUGUAAGGUUGGCGUAUG-3' 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

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.

Name Uniprot ID Pfam Mass Protein sequence PDB ID GenBank
NF-kappaB p50 protein (39-363) P25799 IPR030503 36.61 kDa
GGPYLQ ...... GGPYLQILEQPKQRGFRFRYVCEGPSHGGLPGASSEKNKKSYPQVKICNYVGPAKVIVQLVTNGKNIHLHAHSLVGKHCEDGVCTVTAGPKDMVVGFANLGILHVTKKKVFETLEARMTEACIRGYNPGLLVHSDLAYLQAEGGGDRQLTDREKEIIRQAAVQQTKEMDLSVVRLMFTAFLPDSTGSFTRRLEPVVSDAIYDSKAPNASNLKIVRMDRTAGCVTGGEEIYLLCDKVQKDDIQIRFYEEEENGGVWEGFGDFSPTDVHRQFAIVFKTPKYKDVNITKPASVFVQLRRKSDLETSEPKPFLYYPEIKDKEEVQRKRQK
1OOA 10090
drawing

Similar compound(s)

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.

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 (Å) Description
1OOA-A 44.3 0 Original chain
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


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)