Tat aptamer

Apr 20, 2024 • Zhizhong Lu, Ying Ao

Timeline

Description

In 2000, Yamamoto, R.et al. utilized an in vitro selection approach to isolate a novel aptamer, Tat RNA aptamer, consisting of a 37-mer RNA oligomer, exhibiting high-affinity binding to the Tat protein of HIV-1. In this study, they explored various characteristics of the RNA aptamer. The RNA aptamer maintained significant binding affinity to Tat even under conditions of a substantial excess of HIV TAR, indicating its potential utility as a molecular recognition element in biosensors. In 2003, Matsugami, A., Kobayashi, S.et al determined the structure of the aptamer complexed with argininamide, the simplest analog of Tat, has been determined by NMR. Unique structural features responsible for the high affinity have been found: the formation of two adjacent U:A:U base triples, the resultant widening of the major groove, the formation of hydrogen bonds between a G base and argininamide, and the stabilization of the binding through stacking interaction of a guanidinium group with bases^[1,3].

SELEX

In 2000, Yamamoto, R.et al. Initially, binding buffer containing 5.0 mM RNA and 0.5 mM Tat protein facilitated the first cycle of selection. Successive cycles involved manipulation of RNA pool concentrations and competition assays with nonspecific RNA (E. coli tRNA) and specific competitor RNA (TAR RNA). Additionally, during cycles 7 to 11, competition with another specific pool of competitor RNAs (12-18N pool) was introduced. The final two cycles included significant reduction in Tat protein concentration. The binding buffer comprised 50 mM Tris-HCl (pH 7.8) and 50 mM KCl. Pre-filtering through a nitrocellulose acetate filter was performed to eliminate RNAs selectively bound to the filter. After each cycle, Tat-RNA complexes were collected on a filter and eluted using sodium acetate, EDTA, and urea. Reverse transcription and PCR amplification were conducted post elution. Mutagenic PCR was employed during cycles 9 to 11. Ligation of PCR products into the pCRII vector followed the 11th cycle. Individual clones were sequenced for analysis^[1].
Detailed information are accessible on SELEX page.

Structure

2D representation

Here we use ribodraw to complete the figure, through the 3D structure information^[3].

5'-GGGAGCUUGAUCCCGGAAACGGUCGAUCGCUCCC-3'

3D visualisation

Matsugami, A., Kobayashi, S. et al. determined the Aptamer-Argininamide Complex structure by NMR. Unique structural features responsible for the high affinity have been found: the formation of two adjacent U:A:U base triples, the resultant widening of the major groove, the formation of hydrogen bonds between a G base and argininamide, and the stabilization of the binding through stacking interaction of a guanidinium group with bases. Structural characterization of the aptamer complexed with the RG peptide has also been carried out. Simultaneous interactions of the aptamer with two arginine residues of the RG peptide at two binding sites are strongly suggested. A combination of structural studies on the aptamer-argininamide and aptamer-RG peptide complexes provides a comprehensive explanation of the extremely high affinity of the aptamer to Tat. The PDB ID of the NMR structure is 1NBK^[3].
Additional available structures that have been solved and detailed information are accessible on Structures page.

Binding pocket

Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 1NBK. Argininamide (shown in sticks) is labeled in yellow. Right: The hydrogen bonds of binding sites of the aptamer bound with the argininamide. HIV-1 Tat peptides is a cell penetrating peptide rich in arginine.

Ligand information

SELEX ligand

The affinity was determined by Gel-shift binding assay. Yamamoto, R.et al performed gel-shift assays at four concentrations of RNA with varying concentrations of CQ(0.1-64 nM).The TAR-1-CQ and aptamer-CQ complexes were resolved on nondenaturing gels and estimated the amount of each complexformed. The equilibrium dissociation constants were calculated by nonlinear regression to fit the saturation radiolabelled ligand-binding isotherm. CQ and Tat-1 protein have similar binding affinity for TAR RNA, used CQ instead of Tat-1 in binding kinetics experiments mainly to avoidproblems with possible denaturation of Tat-1 protein during expression and purification. CQ: Tat-1-derived peptids (amino acid residues 37-72)^[1].

Structure ligand

The retroviral Tat protein binds to the Tar RNA. This activates transcriptional initiation and elongation from the LTR promoter. Binding is mediated by an arginine rich region.-----From Pfam

Name	Uniprot ID	Pfam	MW	Amino acids sequences	PDB ID	GenBank
HIV Tat Protein arginine rich region	P04608	PF00539	9.8 KDa	MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFITKALGISYGRKKRRQRRRAHQNSQTHQASLSKQPTSQPRGDPTGPKE	6CYT	155871

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
3MI9-C	9.7	0	Cell division protein kinase 9
3MIA-C	8.9	0.2	Cell division protein kinase 9
4OR5-C	8.6	0.4	Cyclin-dependent kinase 9
4OGR-D	8.6	0.5	Cyclin-dependent kinase 9
4OR5-H	8.6	0.3	Cyclin-dependent kinase 9
4OGR-H	8.6	0.8	Cyclin-dependent kinase 9
4OGR-M	8.5	0.5	Cyclin-dependent kinase 9
6CYT-D	8.4	0.5	Cyclin-dependent kinase 9
5L1Z-D	8.1	0.9	Cyclin-dependent kinase 9

References

[1] A novel RNA motif that binds efficiently and specifically to the Tat protein of HIV and inhibits the trans‐activation by Tat of transcription in vitro and in vivo.
Yamamoto, R., Katahira, M., Nishikawa, S., Baba, T., Taira, K., & Kumar, P.K.
Genes to Cells, 5(5), 371-388 (2000)
[2] Molecular beacon aptamer fluoresces in the presence of Tat protein of HIV-1.
Yamamoto, R., Baba, T., & Kumar, P. K.
Genes to cells, 5(5), 389–396. (2000)
[3] Structural basis of the highly efficient trapping of the HIV Tat protein by an RNA aptamer.
Matsugami, A., Kobayashi, S., Ouhashi, K., Uesugi, S., Yamamoto, R., Taira, K., Nishikawa, S., Kumar, P. K., & Katahira, M.
Structure (London, England : 1993), 11(5), 533–545. (2003)
[4] Aptamer-based biosensors for the detection of HIV-1 Tat protein.
Tombelli, S., Minunni, M., Luzi, E., & Mascini, M.
Bioelectrochemistry (Amsterdam, Netherlands), 67(2), 135–141. (2005)
[5] Effects of diamond-FET-based RNA aptamer sensing for detection of real sample of HIV-1 Tat protein.
Rahim Ruslinda, A., Tanabe, K., Ibori, S., Wang, X., & Kawarada, H.
Biosensors & bioelectronics, 40(1), 277–282. (2013)
[6] Spectrophotometric ellipsometry based Tat-protein RNA-aptasensor for HIV-1 diagnosis.
Caglayan, M. O., & Üstündağ, Z.
Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy, 227, 117748. (2020)
[7] Complex Formation of an RNA Aptamer with a Part of HIV-1 Tat through Induction of Base Triples in Living Human Cells Proven by In-Cell NMR.
Eladl, O., Yamaoki, Y., Kondo, K., Nagata, T., & Katahira, M.
International journal of molecular sciences, 24(10), 9069. (2023)