HTLV-1 Rex aptamer

Apr 17, 2024 • Ziyu Guo, Zhizhong Lu

Timeline

Description

In 1999, Baskerville, S., and Zapp, M used RNA selection techniques to screen RNA molecules that can bind tightly to REX-fusion proteins, namely anti-Rex aptamers, from a pool of randomly sequenced RNA with conformational constraints. The study suggests that anti-Rex aptamers may serve as RNA decoys for Rex proteins and have potential antiviral applications. In 1999, Jiang, F., Gorin, A. determined the solution structure of binding site of Rex peptide RNA aptamer complex by using combined NMR molecular dynamics method^[1,2].

SELEX

In 1999, Baskerville, S., and Zapp, M used a similarly compact pool (79.9) as a starting point for in vitro selection experiments. The 79.9 pool contained two random sequence tracts of 12 to 18 residues pinioned between constant sequence stems. Since the 79.9 pool could have contained only around 1011 possible species and since 10¹³ molecules were introduced into the first round of selection, all possible sequences and stem-like structures were likely included within the pool. Complexes between Rex and members of the RNA pool were isolated by coimmobilization on nitrocellulose filters. The captured RNAs were eluted and amplified by reverse transcription, PCR, and in vitro transcription. tRNA was included in the binding reaction as a nonspecific competitor, and its concentration was increased through the course of selection. Stem IID of the XRE (containing the XBE) was introduced as a specific competitor in the third round, and its concentration was also progressively increased. The selected population was assayed for the ability to bind Rex after four and eight rounds of selection and amplification. In the presence of nonspecific (tRNA) and specific (XBE) competitors, only 1.3% of the native RNA population could bind to Rex, while 7% of the population from the fourth round of selection could bind Rex, as could 13% from the eighth round of selection. The RNA populations from rounds 0, 4, and 8 were also assayed in direct competition with the wild-type binding element^[2].
Detailed information are accessible on SELEX page.

Structure

2D representation

Here we use ribodraw to complete the figure, through the 3D structure information. 33-mer RNA aptamer is modified by aptamer mutation in the SELEX article, but the core region has not changed，It may be an extension of 8-20 aptamer^[2].

5'-GGGCGCCGGUACGCAAGUACGACGGUACGCUCC-3'

3D visualisation

Jiang, F., Gorin, A. determined the solution of binding site of Rex peptide RNA aptamer complex by using combined NMR molecular dynamics method. The complex contains a pair of two-base bulges on opposite strands separated by three base pairs (2 G•C and 1 A•U). This stem-loop IID sequence has features that are distinct from the RNA aptamer, with the latter containing a pair of two-base bulges on the same strand separated by three base pairs (all G•C). The stem-loop IID RxRE site, which apparently binds two Rex ARM peptides, gave very poor quality imino proton nuclear magnetic resonance (NMR) spectra following addition of either one or two equivalents of bound peptide, making this system unsuitable for further study. The PDB ID of this structure is 1EXY^[2].
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: 1EXY. HTLV-1 arginine-rich Rex peptide (shown in vacuumm electrostatics), blue is positive charge, red is negative charge. Right: The hydrogen bonds of binding sites of the aptamer bound with HTLV-1 arginine-rich Rex peptide.

Ligand information

SELEX ligand

In vitro selection of anti-Rex aptamers. Anti-Rex aptamers were isolated from the 79.9 pool by iterative selection for binding followed by amplification of bound species. In each round of selection, tRNA and Rex protein (see Table 1 for amounts) were incubated for 10 min at ambient temperature in 30 pl of 1x binding buffer (50 mM Tris-HCI (pH 8.0), 50 mM KCI). The RNA pool in 20 μl of 1× binding buffer was heated to 90C for 2 min, was cooled to ambient temperature over 10 min to equilibrate conformers, and in some rounds was passed over HAWP 25 modified cellulose filters (Millipore, New Bedford, Mass.) to remove filter-binding sequences. The solutions containing the Rex protein and the RNA pool were mixed; In some rounds a specific competitor RNA, the XRE, was also thermally equilibrated and added to the mixture (see Table 1). The final reaction mix (60 μl) was incubated at ambient temperature for 60 min. The amounts and final concentrations of tRNA, Rex, pool RNA, and XRE were varied during the course of selection as detailed in Table 1. RNA-protein com-l plexes were separated from free RNA by vacuum filtration (5 mm Hg) over HAWP 25 modified cellulose filters (Millipore). Following application of the RNA-protein mixtures, filters were washed twice with 500 μl of 1x binding buffer. The final two rounds of selection included a dilution step following the binding reaction; the dilution step would have favored the retention of RNA. Protein complexes with low off-rates. RNAs that were coretained with Rex were eluted from the filters with 400 μl of 2× PK buffer (0.2 M Tris-Cl [pH 7.6], 2.5 mM EDTA, 0.3 M NaCl, 2% SDS) for 30 min at 75°C. The eluate was extracted with phenol-chloroform and precipitated with ethanol, and the pellet was resuspended in 25 μl of water^[1].

Structure ligand

Human T-cell lymphotropic virus type 1 or human T-lymphotropic virus (HTLV-1), also called the adult T-cell lymphoma virus type 1, is a retrovirus of the human T-lymphotropic virus (HTLV) family. As with human immunodeficiency virus type 1 (HIV-1), HTLV-1 is a complex retrovirus, with a genome that encodes regulatory proteins that modulate the expression of the viral proteins.-----From WiKi

Name	Uniprot ID	Pfam	MW	Amino acids sequences	PDB ID	GenBank
HTLV-1	P0C206	P0C206	20.56 kDa	MPKTRRRPRRSQRKRP	1EXY	AF033817

Similar compound

We used the RCSB PDB website's similar structure search to find the top 10 structures similar to HIV-1 REV PROTEIN (RESIDUES 34-50), and calculated TM-socre values and RMSD values using the TM-align website.

PDB	TM-score	RMSD	Description
1EXY-B	1	0	Solution structure of HTLV-1 peptide bound to its rna aptamer target
7SVR-A	0.26	2.35	The complex of dephosphorylated human cystic fibrosis transmembrane conductance regulator (CFTR) and Lumacaftor (VX-809)
4QMG-B	0.22	3.35	The Structure of MTDH-SND1 Complex Reveals Novel Cancer-Promoting Interactions
2RNW-A	0.21	1.96	The Structural Basis for Site-Specific Lysine-Acetylated Histone Recognition by the Bromodomains of the Human Transcriptional Co-Activators PCAf and CBP
6IAW-D	0.27	1.53	Structure of head fiber and inner core protein gp22 of native bacteriophage P68
2MZ6	0.16	2.22	NMR structure of Protegrin-3 (PG3) in the presence of DPC micelles
7TT1-F	0.16	2.66	BamABCDE bound to substrate EspP class 4
4XAI-B	0.24	2.31	Crystal Structure of red flour beetle NR2E1/TLX

References

[1] Anti-Rex aptamers as mimics of the Rex-binding element.
Baskerville, S., Zapp, M., & Ellington, A. D.
Journal of virology, 73(6), 4962–4971. (1999)
[2] Anchoring an extended HTLV-1 Rex peptide within an RNA major groove containing junctional base triples.
Jiang, F., Gorin, A., Hu, W., Majumdar, A., Baskerville, S., Xu, W., Ellington, A., & Patel, D. J.
Structure (London, England : 1993), 7(12), 1461–1472. (1999)
[3] Fluorescence-based methods for evaluating the RNA affinity and specificity of HIV-1 Rev-RRE inhibitors.
Luedtke, N. W., & Tor, Y.
Biopolymers, 70(1), 103–119. (2003)
[4] Amino acid requirement for the high affinity binding of a selected arginine-rich peptide with the HIV Rev-response element RNA.
Sugaya, M., Nishino, N., Katoh, A., & Harada, K.
Journal of peptide science : an official publication of the European Peptide Society, 14(8), 924–935.  (2008)
[5] Bioavailable inhibitors of HIV-1 RNA biogenesis identified through a Rev-based screen.
Prado, S., Beltrán, M., Coiras, M., Bedoya, L. M., Alcamí, J., & Gallego, J.
Biochemical pharmacology, 107, 14–28. (2016)