Circular dichroism studies suggest that TAR RNA changes conformation upon specific binding of arginine or guanidine.

Short basic peptides from the HIV Tat protein bind specifically to a bulge region in TAR RNA, with a single arginine residue providing the only sequence-specific contact. The free amino acid arginine also binds specifically to TAR. Previous circular dichroism (CD) experiments suggested that peptide binding induces a conformational change in TAR. Here we confirm this observation using single arginine-containing peptides and show that arginine or guanidine binding also induces a conformational change in TAR. A peptide containing a single arginine within a stretch of histidines (CYHHHRHHHHHA) shows pH-dependent binding and a corresponding change in TAR conformation, as detected by a decrease in the CD signal at 265 nm. Arginine and guanidine, which bind to TAR with apparent Kd's of approximately 1.5 mM, induce similar CD changes. In contrast, lysine, which does not bind specifically to TAR, has no effect. Mutants of TAR that abolish specific binding (a U-->C substitution in the three-nucleotide bulge, a deletion of the bulge, or an A-U to U-A base pair change above the bulge) show no change in the CD signal upon binding of peptides, arginine, or guanidine. The results suggest that binding of a single guanidinium group to a specific site in TAR induces a change in RNA conformation.     

Identification of a novel HIV-1 TAR RNA bulge binding protein.

The Tat protein binds to TAR RNA to stimulate the expression of the human immunodeficiency virus type 1 (HIV-1) genome. Tat is an 86 amino acid protein that contains a short region of basic residues (aa49-aa57) that are required for RNA binding and TAR is a 59 nucleotide stem-loop with a tripyrimidine bulge in the upper stem. TAR is located at the 5' end of all viral RNAs. In vitro, Tat specifically interacts with TAR by recognising the sequence of the bulge and upper stem, with no requirement for the loop. However, in vivo the loop sequence is critical for activation, implying a requirement for accessory cellular TAR RNA binding factors. A number of TAR binding cellular factors have been identified in cell extracts and various models for the function of these factors have been suggested, including roles as coactivators and inhibitors. We have now identified a novel 38 kD cellular factor that has little general, single-stranded or double-stranded RNA binding activity, but that specifically recognises the bulge and upper stem region of TAR. The protein, referred to as BBP (bulge binding protein), is conserved in mammalian and amphibian cells and in Schizosaccharomyces pombe but is not found in Saccharomyces cerevisiae. BBP is an effective competitive inhibitor of Tat binding to TAR in vitro. Our data suggest that the bulge-stem recognition motif in TAR is used to mediate cellular factor/RNA interactions and indicates that Tat action might be inhibited by such competing reactions in vivo.     

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.     

Polyamide amino acids trimers as TAR RNA ligands and anti-HIV agents

Based on a split-and-mix strategy, a library of trimeric Polyamide Amino Acids (PAA) incorporating four different amino acids (Lys, Ala, Arg, and Phe) has been prepared. Screening of the batches for HIV TAR RNA binding in a fluorescent assay allowed the identification of several components that interact with TAR RNA at a micromolar concentration, with a good TAR versus tRNA specificity. Some of these compounds compete efficiently with the association of TAR and Tat protein. In cell cultures, these compounds display a moderate antiviral activity, associated nevertheless with some toxicity. Overall, these results confirm that this new family can be a basis for the design of novel RNA targeting drugs.     

Polyamidoamine dendrimers inhibit binding of Tat peptide to TAR RNA

The binding of polyamidoamine (PAMAM) dendrimer or Tat peptide to trans-acting responsive element (TAR) RNA has been studied using microgravimetric quartz crystal microbalance (QCM). Experimental results showed that PAMAM dendrimer could form complexes with TAR RNA. Especially, PAMAM dendrimer could disrupt the interaction of Tat peptide with TAR RNA, which is essential for HIV-1 virus replication, suggesting that QCM is a powerful tool for studying the binding processes of Tat peptide-TAR RNA and drug-TAR RNA and has great significance for the design of new drugs. An equation to measure the binding ability between TAR RNA and other species has been proposed.     

Essential structural requirements for specific recognition of HIV TAR RNA by peptide mimetics of Tat protein

The pharmacological disruption of the interaction between the HIV Tat protein and its cognate transactivation response RNA (TAR) would generate novel anti-viral drugs with a low susceptibility to drug resistance, but efforts to discover ligands with sufficient potency to warrant pharmaceutical development have been unsuccessful. We have previously described a family of structurally constrained β-hairpin peptides that potently inhibits viral growth in HIV-infected cells. The nuclear magnetic resonance (NMR) structure of an inhibitory complex revealed that the peptide makes intimate contacts with the 3-nt bulge and the upper helix of the RNA hairpin, but that a single residue contacts the apical loop where recruitment of the essential cellular co-factor cyclin T₁ occurs. Attempting to extend the peptide to form more interactions with the RNA loop, we examined a library of longer peptides and achieved >6-fold improvement in affinity. The structure of TAR bound to one of the extended peptides reveals that the peptide slides down the major groove of the RNA, relative to our design, in order to maintain critical interactions with TAR. These conserved contacts involve three amino acid side chains and identify critical interaction points required for potent and specific binding to TAR RNA. They constitute a template of essential interactions required for inhibition of this RNA.     

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).     

The design, synthesis, and biological evaluation of novel substituted purines as HIV-1 Tat-TAR inhibitors

A series of novel substituted purines containing a side chain with a terminal amino or guanidyl group were designed and synthesized as HIV-1 Tat-TAR inhibitors. All the compounds could effectively block the TAR transactivation in human 293T cells with the CAT expression percentage ranging from 34.4% to 65.7% and showed high antiviral effects with low cytotoxicities in inhibiting the formation of SIV-induced syncytium in CEM174 cells. Molecular modeling studies by Auto-dock process suggest that the compounds bind to TAR RNA in two different modes.     

Evidence for a base triple in the free HIV-1 TAR RNA

We propose the existence of a novel base triple in the HIV-1 TAR hairpin. This triple is supported by covariation of loop residue 31 with residue 22, which is part of an unusual base pair with U40 below the 3-nucleotide bulge. A set of mutants was constructed to test the involvement of bases A22, U31, and U40 in a triple interaction. RNA structure probing, trans-activation assays, and structure modeling are consistent with the existence of this base triple in a bent conformation of the free TAR element. However, disruption of the base triple does not affect binding of a Tat-derived peptide. We therefore compared the structure of free and Tat-bound TAR RNA by footprinting and site-specific cross-linking analyses. These studies indicate that the Tat arginine-rich motif, in addition to its known binding site at the bulge, is in close contact with U31 in the TAR loop. Because binding of Tat to TAR is known to coincide with the formation of a base triple with residues U23, A27, and U38, we hypothesize that Tat binding and the associated straightening of TAR triggers the disruption of the (A22-U40)U31 triple.     

A comparative binding study of modified bovine immunodeficiency virus TAR RNA against its TAT peptide

Besides generating novel binding peptides or small molecules to their RNA target, successful design of chemically modified RNA constructs capable of tighter binding with their binding peptides is also of significant importance. Herein, the synthesis and binding studies of a series of both wt and mutant bovine immunodeficiency virus (BIV) TAR RNA constructs against its Tat peptide are reported. Understanding the requirements that enable RNA construct binding properties, especially at the hairpin loop or internal bulge, would afford potential therapeutic approaches to control the BIV life cycle.     

Inhibition of the HIV-1 rev-RRE complex formation by unfused aromatic cations

RNA viruses cause a wide range of human diseases. Development of new agents to target such viruses is an active area of research. Towards this goal, a series of diphenylfuran cations as potential inhibitors of the Rev-RRE complex have been designed and synthesized. Analysis of the interaction of the diphenylfurans with RRE and TAR RNA model systems by gel shift assays indicates that they exhibit both sequence and structure-dependent binding modes. Our results show a strong interaction between the diphenylfuran ring system and RRE bases, while the TAR interactions are much weaker with the compounds that are the best inhibitors of Rev-RRE.     

Concerted motions in HIV-1 TAR RNA may allow access to bound state conformations: RNA dynamics from NMR residual dipolar couplings.

Ground-state dynamics in RNA is a critical precursor for structural adaptation observed ubiquitously in protein-RNA recognition. A tertiary conformational analysis of the stem-loop structural element in the transactivation response element (TAR) from human immunodeficiency virus type 1 (HIV-I) RNA is presented using recently introduced NMR methods that rely on the measurement of residual dipolar couplings (RDC) in partially oriented systems. Order matrix analysis of RDC data provides evidence for inter-helical motions that are of amplitude 46(+/-4) degrees, of random directional character, and that are executed about an average conformation with an inter-helical angle between 44 degrees and 54 degrees. The generated ensemble of TAR conformations have different organizations of functional groups responsible for interaction with the trans-activator protein Tat, including conformations similar to the previously characterized bound-state conformation. These results demonstrate the utility of RDC-NMR for simultaneously characterizing RNA tertiary dynamics and average conformation, and indicate an avenue for TAR complex formation involving tertiary structure capture.     

A Small-Molecule Probe Induces a Conformation in HIV TAR RNA Capable of Binding Drug-Like Fragments

The HIV-1 transactivation response (TAR) element-Tat interaction is a potentially valuable target for treating HIV infection, but efforts to develop TAR-binding antiviral drugs have not yet yielded a successful candidate for clinical development. In this work, we describe a novel approach toward screening fragments against RNA that uses a chemical probe to target the Tat-binding region of TAR. This probe fulfills two critical roles in the screen: by locking the RNA into a conformation capable of binding other fragments, it simultaneously allows the identification of proximal binding fragments by ligand-based NMR. Using this approach, we have discovered six novel TAR-binding fragments, three of which were docked relative to the probe-RNA structure using experimental NMR restraints. The consistent orientations of functional groups in our data-driven docked structures and common electrostatic properties across all fragment leads reveal a surprising level of selectivity by our fragment-sized screening hits. These models further suggest linking strategies for the development of higher-affinity lead compounds for the inhibition of the TAR-Tat interaction.     

A high-throughput screening utilizing intramolecular fluorescence resonance energy transfer for the discovery of the molecules that bind HIV-1 TAR RNA specifically

A 16-residue peptide, including the Tat(49-57) sequence was labeled with a fluorescein and a tetramethylrhodamine at its N- and C-terminus, respectively. This double dye-labeled peptide was prepared as a tracer for high-throughput screening utilizing intramolecular fluorescence resonance energy transfer (FRET). The binding of the competitor molecules for HIV-1 TAR RNA were monitored and dissociation constants of those molecule were determined by using this tracer. This novel screening system might be useful to discover the drug for HIV-1 TAR RNA.