NMR structure of a kissing complex formed between the TAR RNA element of HIV-1 and a LNA-modified aptamer

Abstract

The trans-activating responsive (TAR) RNA element located in the 5' untranslated region of the HIV-1 genome is a 57-nt imperfect stem-loop essential for the viral replication. TAR regulates transcription by interacting with both viral and cellular proteins. RNA hairpin aptamers specific for TAR were previously identified by in vitro selection [Ducongé,F. and Toulmé,J.J. (1999) In vitro selection identifies key determinants for loop-loop interactions: RNA aptamers selective for the TAR RNA element of HIV-1. RNA, 5, 1605-1614]. These aptamers display a 5'-GUCCCAGA-3' consensus apical loop, partially complementary to the TAR one, leading to the formation of a TAR-aptamer kissing complex. The conserved GA combination (underlined in the consensus sequence) has been shown to be crucial for the formation of a highly stable complex. To improve the nuclease resistance of the aptamer and to increase its affinity for TAR, locked nucleic acid (LNA) nucleotides were introduced in the aptamer apical loop. LNA are nucleic acids analogues that contain a 2'-O,4'-C methylene linkage and that raise the thermostablity of duplexes. We solved the NMR solution structure of the TAR-LNA-modified aptamer kissing complex. Structural analysis revealed the formation of a non-canonical G*A pair leading to increased stacking at the stem-loop junction. Our data also showed that the introduction of LNA residues provides an enhanced stability while maintaining a normal Watson-Crick base pairing with a loop-loop conformation close to an A-type.

Figure 1.

Comparison of TAR/R06(GA) and TAR/LR06(GA) complexes. (A) Secondary structure of TAR/R06(GA) (left) and TAR/LR06(GA) (right) complexes based on exchangeable and non-exchangeable proton NMR data. Two Gs were introduced at the 5′-end of R06(GA) to improve the yield of the transcription. LNAs residues are in red (asterisk indicates aptamer residues). (B) View of a C ribonucleotide residue (top) and of a C-LNA residue (bottom). (C) Imino proton region of 1D spectra of TAR/R06(GA) (top) and TAR/LR06(GA) (bottom).

Figure 2.

Assignment of proton, nitrogen and carbon resonances of TAR/LR06(GA)complex. (A) Imino proton region of the 1D spectrum (top) and the ¹H/¹⁵N HSQC spectrum (bottom). (B) H6/8-H1′ region of a NOESY spectrum recorded at 15°C in 100% D₂O with a mixing time of 200 ms. Dashed lines represent sequential assignment of H6/8-H1′. (C) ¹H-¹³C HSQC spectrum showing the aromatic H8-C8, H6-C6 and H2-C2 correlations of TAR ¹³C-¹⁵N labelled complexed with LR06(GA).

Figure 3.

Overall structure of TAR/LR06(GA) complex. (A) Superposition of 10 converged NMR structures. Bases are shown in dark blue and the backbone in salmon. (B) View of the average structure of TAR/LR06(GA) complex. LNAs residues are coloured in yellow, G5*•A12* and U6*•A11 in salmon. (C) The TAR stem region has been superimposed for the 10 converged structures (left). The central helix has been superimposed for the 10 converged structures (middle). The LR06(GA) stem region has been superimposed for the 10 converged structures (right).

Figure 4.

Description of the loop–loop Interaction of TAR/LR06(GA) and comparison to LNA/RNA and RNA/RNA complexes. (A) View of the loop–loop interaction of TAR/LR06(GA) with LNA residues, C9* and A10*, coloured in yellow. View of the minor groove water accessible surface of (B) TAR/LR06(GA) loop–loop helix, (C) an A-type helix, (D) TAR/TAR*(UA) (1KIS), (E) LNA:RNA hybrid (1HOQ) and (F) SL1 HIV-1 dimer loop–loop helix (2F4X). The surfaces are generated using a water probe of 1.4 Å radius on MOLMOL software. PDB ID are indicated in brackets.

Figure 5.

Comparison of G11*•C6 base pair in TAR/R06(GA) and TAR/LR06(GA) complexes. Imino region of NOESY spectra recorded at 4°C in H2O/D2O 90%/10% of TAR/R06(GA) (top) and TAR/LR06(GA) (bottom).

Figure 6.

Structure at the stem-loop junctions. (A) View of the G5*•A12* base pair in TAR/LR06(GA) complex. (B) View of A11 and U6* at stem-loop junction in TAR/LR06(GA) complex. (C) View of turns typically observed at the stem-loop junctions. TAR is represented in blue and the aptamer in salmon. Turns between C5 and C6 and between G5* and U6* in TAR/LR06(GA) complex (left). Turns between C5 and C6 and between U5* and U6* in TAR/TAR*(UA) complex (right) (47).