DNA aptamers selected against the HIV-1 trans-activation-responsive RNA element form RNA-DNA kissing complexes

In vitro selection was performed in a DNA library, made of oligonucleotides with a 30-nucleotide random sequence, to identify ligands of the human immunodeficiency virus type-1 trans-activation-responsive (TAR) RNA element. Aptamers, extracted after 15 rounds of selection-amplification, either from a classical library of sequences or from virtual combinatorial libraries, displayed an imperfect stem-loop structure and presented a consensus motif 5'ACTCCCAT in the apical loop. The six central bases of the consensus were complementary to the TAR apical region, giving rise to the formation of RNA-DNA kissing complexes, without disrupting the secondary structure of TAR. The RNA-DNA kissing complex was a poor substrate for Escherichia coli RNase H, likely due to steric and conformational constraints of the DNA/RNA heteroduplex. 2'-O-Methyl derivatives of a selected aptamer were binders of lower efficiency than the parent aptamer in contrast to regular sense/antisense hybrids, indicating that the RNA/DNA loop-loop region adopted a non-canonical heteroduplex structure. These results, which allowed the identification of a new type of complex, DNA-RNA kissing complex, demonstrate the interest of in vitro selection for identifying non-antisense oligonucleotide ligands of RNA structures that are of potential value for artificially modulating gene expression.     

Aptamers targeted to an RNA hairpin show improved specificity compared to that of complementary oligonucleotides

Aptamers interacting with RNA hairpins through loop-loop (so-called kissing) interactions have been described as an alternative to antisense oligomers for the recognition of RNA hairpins. R06, an RNA aptamer, was previously shown to form a kissing complex with the TAR (trans-activating responsive) hairpin of HIV-1 RNA (Ducongé and Toulmé (1999) RNA 5, 1605). We derived a chimeric locked nucleic acid (LNA)/DNA aptamer from R06 that retains the binding properties of the originally selected R06 aptamer. We demonstrated that this LNA/DNA aptamer competes with a peptide of the retroviral protein Tat for binding to TAR, even though the binding sites of the two ligands do not overlap each other. This suggests that upon binding, the aptamer TAR adopts a conformation that is no longer appropriate for Tat association. In contrast, a LNA/DNA antisense oligomer, which exhibits the same binding constant and displays the same base-pairing potential as the chimeric aptamer, does not compete with Tat. Moreover, we showed that the LNA/DNA aptamer is a more specific TAR binder than the LNA/DNA antisense sequence. These results demonstrate the benefit of reading the three-dimensional shape of an RNA target rather than its primary sequence for the design of highly specific oligonucleotides.     

Dominant role of the 5' TAR bulge in dimerization of HIV-1 genomic RNA, but no evidence of TAR-TAR kissing during in vivo virus assembly

The 5' untranslated region of HIV-1 genomic RNA (gRNA) contains two stem-loop structures that appear to be equally important for gRNA dimerization: the 57-nucleotide 5' TAR, at the very 5' end, and the 35-nucleotide SL1 (nucleotides 243-277). SL1 is well-known for containing the dimerization initiation site (DIS) in its apical loop. The DIS is a six-nucleotide palindrome. Here, we investigated the mechanism of TAR-directed gRNA dimerization. We found that the trinucleotide bulge (UCU24) of the 5' TAR has dominant impacts on both formation of HIV-1 RNA dimers and maturation of the formed dimers. The ΔUCU trinucleotide deletion strongly inhibited the first process and blocked the other, thus impairing gRNA dimerization as severely as deletion of the entire 5' TAR, and more severely than deletion of the DIS, inactivation of the viral protease, or most severe mutations in the nucleocapsid protein. The apical loop of TAR contains a 10-nucleotide palindrome that has been postulated to stimulate gRNA dimerization by a TAR-TAR kissing mechanism analogous to the one used by SL1 to stimulate dimerization. Using mutations that strongly destabilize formation of the TAR palindrome duplex, as well as compensatory mutations that restore duplex formation to a wild-type-like level, we found no evidence of TAR-TAR kissing, even though mutations nullifying the kissing potential of the TAR palindrome could impair dimerization by a mechanism other than hindering of SL1. However, nullifying the kissing potential of TAR had much less severe effects than ΔUCU. By not uncovering a dimerization mechanism intrinsic to TAR, our data suggest that TAR mutations exert their effect 3' of TAR, yet not on SL1, because TAR and SL1 mutations have synergistic effects on gRNA dimerization.     

Bimodal loop-loop interactions increase the affinity of RNA aptamers for HIV-1 RNA structures

Multiple loop-loop interactions between adjacent RNA hairpins regulate gene expression in different organisms. To demonstrate that such natural interactions could be mimicked for generating RNA ligands that are able to recognize simultaneously at least two structured RNA targets, a double kissing complex model was designed. The target consisted of two HIV-1 transactivating responsive (TAR) RNA variants, BRU and MAL, connected by a non-nucleotidic linker. The double ligand was generated by combining the corresponding hairpin aptamers, R06BRU and R06MAL, identified previously by in vitro selection [Ducongé, F., and Toulmé, J. J (1999) RNA 5, 1605-1614]. The resulting interaction was analyzed by thermal denaturation monitored by UV spectroscopy, electrophoretic mobility shift assays (EMSAs), and surface plasmon resonance (SPR) experiments. The bimodal complex was characterized by a binding equilibrium constant increased by at least 1 order of magnitude compared to that of the complexes between the individual parent hairpins. This resulted from a slower dissociation rate. We then made use of such a strategy for targeting two structured functional motifs of the folded 5' untranslated region (5'UTR) of HIV-1. Two bivalent RNA ligands were designed that targeted simultaneously the TAR and dimerization initiation site (DIS) hairpins or the TAR and poly(A) ones. The results show that these ligands also displayed enhanced affinity for their target compared to the individual molecules. The work reported here suggests that bimodal structured RNA ligands might provide a way of increasing the affinity of aptamers for folded RNA targets.     

LNA/DNA chimeric oligomers mimic RNA aptamers targeted to the TAR RNA element of HIV-1

One of the major limitations of the use of phosphodiester oligonucleotides in cells is their rapid degradation by nucleases. To date, several chemical modifications have been employed to overcome this issue but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this work conformationally restricted nucleotides, locked nucleic acid (LNA), were investigated to design nuclease resistant aptamers targeted against the HIV-1 TAR RNA. LNA/DNA chimeras were synthesized from a shortened version of the hairpin RNA aptamer identified by in vitro selection against TAR. The results indicate that these modifications confer good protection towards nuclease digestion. Electrophoretic mobility shift assays, thermal denaturation monitored by UV-spectroscopy and surface plasmon resonance experiments identified LNA/DNA TAR ligands that bind to TAR with a dissociation constant in the low nanomolar range as the parent RNA aptamer. The crucial G, A residues that close the aptamer loop remain a key structural determinant for stable LNA/DNA chimera–TAR complexes. This work provides evidence that LNA modifications alternated with DNA can generate stable structured RNA mimics for interacting with folded RNA targets.     

RNA Dimerization Promotes PKR Dimerization and Activation

The double-stranded RNA (dsRNA)-activated protein kinase [protein kinase R (PKR)] plays a major role in the innate immune response in humans. PKR binds dsRNA non-sequence specifically and requires a minimum of 15-bp dsRNA for one protein to bind and 30-bp dsRNA to induce protein dimerization and activation by autophosphorylation. PKR phosphorylates eukaryotic initiation factor 2α, a translation initiation factor, resulting in the inhibition of protein synthesis. We investigated the mechanism of PKR activation by an RNA hairpin with a number of base pairs intermediate between these 15- to 30-bp limits: human immunodeficiency virus type 1 transactivation-responsive region (TAR) RNA, a 23-bp hairpin with three bulges that is known to dimerize. TAR monomers and dimers were isolated from native gels and assayed for RNA and protein dimerization to test whether RNA dimerization affects PKR dimerization and activation. To modulate the extent of dimerization, we included TAR mutants with different secondary features. Native gel mixing experiments and analytical ultracentrifugation indicate that TAR monomers bind one PKR monomer and that TAR dimers bind two or three PKRs, demonstrating that RNA dimerization drives the binding of multiple PKR molecules. Consistent with functional dimerization of PKR, TAR dimers activated PKR while TAR monomers did not, and RNA dimers with fewer asymmetrical secondary-structure defects, as determined by enzymatic structure mapping, were more potent activators. Thus, the secondary-structure defects in the TAR RNA stem function as antideterminants to PKR binding and activation. Our studies support that dimerization of a 15- to 30-bp hairpin RNA, which effectively doubles its length, is a key step in driving activation of PKR and provide a model for how RNA folding can be related to human disease.     

The in vitro loose dimer structure and rearrangements of the HIV-2 leader RNA

RNA dimerization is an essential step in the retroviral life cycle. Dimerization and encapsidation signals, closely linked in HIV-2, are located in the leader RNA region. The SL1 motif and nucleocapsid protein are considered important for both processes. In this study, we show the structure of the HIV-2 leader RNA (+1-560) captured as a loose dimer. Potential structural rearrangements within the leader RNA were studied. In the loose dimer form, the HIV-2 leader RNA strand exists in vitro as a single global fold. Two kissing loop interfaces within the loose dimer were identified: SL1/SL1 and TAR/TAR. Evidence for these findings is provided by RNA probing using SHAPE, chemical reagents, enzymes, non-denaturing PAGE mobility assays, antisense oligonucleotides hybridization and analysis of an RNA mutant. Both TAR and SL1 as isolated domains are bound by recombinant NCp8 protein with high affinity, contrary to the hairpins downstream of SL1. Foot-printing of the SL1/NCp8 complex indicates that the major binding site maps to the SL1 upper stem. Taken together, these data suggest a model in which TAR hairpin III, the segment of SL1 proximal to the loop and the PAL palindromic sequence play specific roles in the initiation of dimerization.     

In vitro selection of DNA aptamers against the HIV-1 TAR RNA hairpin

In vitro selection was performed to identify DNA aptamers against the TAR RNA stem-loop structure of HIV-1. A counterselection step allowed the elimination of kissing complex-forming aptamers previously selected (Boiziau et al. J. Biol. Chem. 1999; 274:12730). This led to the emergence of oligonucleotides, most of which contained two consensus sequences, one targeted to the stem 3'-strand (5'-CCCTAGTTA) and the other complementary to the TAR apical loop (5'-CTCCC). The best aptamer could be shortened to a 19-mer oligonucleotide, characterized by a dissociation constant of 50 nM. A 16-mer oligonucleotide complementary to the TAR stem 3'-strand could also be derived from the identified aptamers, with an equal affinity (Kd = 50 nM). Experiments performed to elucidate the interaction between TAR and the aptamers (UV melting measures, enzymatic and chemical footprints) demonstrated that the TAR stem 5'-strand was not simply displaced as a result of the complex formation but unexpectedly remained associated on contact with the antisense oligonucleotide. We suggest that a multistranded structure could be formed.     

Electron paramagnetic resonance dynamic signatures of TAR RNA-small molecule complexes provide insight into RNA structure and recognition

Electron paramagnetic resonance (EPR) spectroscopy was utilized to investigate the correlation between RNA structure and RNA internal dynamics in complexes of HIV-1 TAR RNA with small molecules. TAR RNAs containing single nitroxide spin-labels in the 2'-position of U23, U25, U38, or U40 were incubated with compounds known to inhibit TAR-Tat complex formation. The combined changes in nucleotide mobility at all four sites, as monitored by their EPR spectral width, yield a dynamic signature for each compound. The multicyclic dyes Hoechst 33258, DAPI, and berenil bind to TAR RNA in a similar manner and gave nearly identical signatures. Different signatures were obtained for the acridine derivative CGP 40336A and the aminoglycoside antibiotic neomycin, which bind to different regions of the RNA. The dynamic signature for guanidinoneomycin was remarkably similar to that obtained for argininamide and is evidence for guanidinoneomycin binding to the same site as arginine 52 of the Tat protein, rather than to the neomycin binding site. The data presented here show that the dynamic signatures provide strong insights into RNA structure and recognition and demonstrate the value of EPR spectroscopy for the investigation of small molecule binding to RNA.     

Systematic screening of LNA/2'-O-methyl chimeric derivatives of a TAR RNA aptamer

We synthesized and evaluated by surface plasmon resonance 64 LNA/2'-O-methyl sequences corresponding to all possible combinations of such residues in a kissing aptamer loop complementary to the 6-nt loop of the TAR element of HIV-1. Three combinations of LNA/2'-O-methyl nucleoside analogues where one or two LNA units are located on the 3' side of the aptamer loop display an affinity for TAR below 1nM, i.e. one order of magnitude higher than the parent RNA aptamer. One of these combinations inhibits the TAR-dependent luciferase expression in a cell assay.     

Discovery of selective bioactive small molecules by targeting an RNA dynamic ensemble

Current approaches used to identify protein-binding small molecules are not suited for identifying small molecules that can bind emerging RNA drug targets. By docking small molecules onto an RNA dynamic ensemble constructed by combining NMR spectroscopy and computational molecular dynamics, we virtually screened small molecules that target the entire structure landscape of the transactivation response element (TAR) from HIV type 1 (HIV-1). We quantitatively predict binding energies for small molecules that bind different RNA conformations and report the de novo discovery of six compounds that bind TAR with high affinity and inhibit its interaction with a Tat peptide in vitro (K(i) values of 710 nM-169 μM). One compound binds HIV-1 TAR with marked selectivity and inhibits Tat-mediated activation of the HIV-1 long terminal repeat by 81% in T-cell lines and HIV replication in an HIV-1 indicator cell line (IC(50) ～23.1 μM).