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.     

Specific modulation of the anti-DNA autoantibody-nucleic acids interaction by the high affinity RNA aptamer

Anti-DNA autoantibodies are one of the frequently found autoantibodies in systemic lupus erythematosus patient sera. RNA aptamers for the monoclonal G6-9 anti-DNA autoantibody were selected from a random pool of RNA library. Binding affinity of the best aptamer is around 2nM, which is at least 100-fold higher than that of cognate DNA antigen to the autoantibody. Aptamer binds specifically to the G6-9 autoantibody but not to other similar autoantibodies. Minimal binding motif of the aptamer was mapped, providing a hint for a natural epitope of the autoantibody. DNA binding to the G6-9 autoantibody is shown to be efficiently inhibited by the aptamer. Such binding property of the RNA aptamer could be used not only as a modulator for the pathogenic anti-DNA autoantibody, but also as a useful biochemical reagent for elucidating a fine specificity of the autoantibody-nucleic acid interaction.     

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.     

Molecular recognition of amino acids by RNA aptamers: the evolution into an L-tyrosine binder of a dopamine-binding RNA motif

We report the evolution of an RNA aptamer to change its binding specificity. RNA aptamers that bind the free amino acid tyrosine were in vitro selected from a degenerate pool derived from a previously selected dopamine aptamer. Three independent sequences bind tyrosine in solution, the winner of the selection binding with a dissociation constant of 35 microM. Competitive affinity chromatography with tyrosine-related ligands indicated that the selected aptamers are highly L-stereo selective and also recognize L-tryptophan and L-dopa with similar affinity. The binding site was localized by sequence comparison, analysis of minimal boundaries, and structural probing upon ligand binding. Tyrosine-binding sites are characterized by the presence of both tyrosine (UAU and UAC) and termination (UAG and UAA) triplets.     

DNA aptamers as potential anti-HIV agents

Guanine (G)-rich DNA sequences can adopt stable G-quadruplex structures by G-tetrad hydrogen-bonding and hydrophobic stacking. Recently, it has been shown that a DNA sequence forms an aptamer (termed 93del) and adopts a novel dimeric quadruplex folding topology in K+ solution. This aptamer exhibits anti-HIV1 integrase activity in the nanomolar range in vitro. A docking-based model of the 93del-integrase complex positions the DNA aptamer within a channel of the tetrameric integrase. This mutual fitting blocks several catalytic amino acid residues that are essential for integrase function, and accounts for the anti-HIV1 activity of the 93del aptamer.     

Crystal structure of an RNA aptamer bound to thrombin

Aptamers, an emerging class of therapeutics, are DNA or RNA molecules that are selected to bind molecular targets that range from small organic compounds to large proteins. All of the determined structures of aptamers in complex with small molecule targets show that aptamers cage such ligands. In structures of aptamers in complex with proteins that naturally bind nucleic acid, the aptamers occupy the nucleic acid binding site and often mimic the natural interactions. Here we present a crystal structure of an RNA aptamer bound to human thrombin, a protein that does not naturally bind nucleic acid, at 1.9 A resolution. The aptamer, which adheres to thrombin at the binding site for heparin, presents an extended molecular surface that is complementary to the protein. Protein recognition involves the stacking of single-stranded adenine bases at the core of the tertiary fold with arginine side chains. These results exemplify how RNA aptamers can fold into intricate conformations that allow them to interact closely with extended surfaces on non-RNA binding proteins.     

A simple and direct electrochemical detection of interferon-gamma using its RNA and DNA aptamers

Tuberculosis is the most frequent cause of infection-related death worldwide. We constructed a simple and direct electrochemical sensor to detect interferon (IFN)-gamma, a selective marker for tuberculosis pleurisy, using its RNA and DNA aptamers. IFN-gamma was detected by its 5'-thiol-modified aptamer probe immobilized on the gold electrode. Interaction between IFN-gamma and the aptamer was recorded using electrochemical impedance spectroscopy and quartz crystal microbalance (QCM) with high sensitivity. The RNA-aptamer-based sensor showed a low detection limit of 100 fM, and the DNA-aptamer-based sensor detected IFN-gamma to 1 pM in sodium phosphate buffer. With QCM analysis, the aptamer immobilized on the electrode and IFN-gamma bound to the aptamer probe was quantified. This QCM result shows that IFN-gamma exists in multimeric forms to interact with the aptamers, and the RNA aptamer prefers the high multimeric state of IFN-gamma. Such a preference may describe the low detection limit of the RNA aptamer shown by impedance analysis. In addition, IFN-gamma was detected to 10 pM by the DNA aptamer in fetal bovine serum, a mimicked biological system, which has similar components to pleural fluid.     

NMR resonance assignments for the tetramethylrhodamine binding RNA aptamer 3 in complex with the ligand 5-carboxy-tetramethylrhodamine

RNA aptamers are used in a wide range of biotechnological or biomedical applications. In many cases the high resolution structures of these aptamers in their ligand-complexes have revealed fundamental aspects of RNA folding and RNA small molecule interactions. Fluorescent RNA-ligand complexes in particular find applications as optical sensors or as endogenous fluorescent tags for RNA tracking in vivo. Structures of RNA aptamers and aptamer ligand complexes constitute the starting point for rational function directed optimization approaches. Here, we present the NMR resonance assignment of an RNA aptamer binding to the fluorescent ligand tetramethylrhodamine (TMR) in complex with the ligand 5-carboxy-tetramethylrhodamine (5-TAMRA) as a starting point for a high-resolution structure determination using NMR spectroscopy in solution.     

A simple fluorescent biosensor for theophylline based on its RNA aptamer

Theophylline is a potent bronchodilator with a narrow therapeutic index. A simple fluorescent biosensor that detects clinically relevant theophylline concentrations has been developed using the well-characterized theophylline binding RNA aptamer. Hybridization of the RNA aptamer to a fluorescently labeled DNA strand (FL-DNA) yields a fluorescent RNA:DNA hybrid that is sensitive to theophylline. The biosensor retains the remarkable selectivity of the RNA aptamer for theophylline over caffeine and is sensitive to 0-2 muM theophylline, well below the clinically relevant concentration (5-20 mg/L or approximately 10-50 muM). Adding a dabcyl quenching dye to the 3'-terminus of the fluorescently labeled DNA strand yielded a dual-labeled DNA strand (FL-DNA-Q) and increased the dynamic range of this simple biosensor from 1.5-fold to 4-fold.     

Evolution of aptamers with a new specificity and new secondary structures from an ATP aptamer

Small changes in target specificity can sometimes be achieved, without changing aptamer structure, through mutation of a few bases. Larger changes in target geometry or chemistry may require more radical changes in an aptamer. In the latter case, it is unknown whether structural and functional solutions can still be found in the region of sequence space close to the original aptamer. To investigate these questions, we designed an in vitro selection experiment aimed at evolving specificity of an ATP aptamer. The ATP aptamer makes contacts with both the nucleobase and the sugar. We used an affinity matrix in which GTP was immobilized through the sugar, thus requiring extensive changes in or loss of sugar contact, as well as changes in recognition of the nucleobase. After just five rounds of selection, the pool was dominated by new aptamers falling into three major classes, each with secondary structures distinct from that of the ATP aptamer. The average sequence identity between the original aptamer and new aptamers is 76%. Most of the mutations appear to play roles either in disrupting the original secondary structure or in forming the new secondary structure or the new recognition loops. Our results show that there are novel structures that recognize a significantly different ligand in the region of sequence space close to the ATP aptamer. These examples of the emergence of novel functions and structures from an RNA molecule with a defined specificity and fold provide a new perspective on the evolutionary flexibility and adaptability of RNA.