Molecular recognition of cAMP by an RNA aptamer

Two classes of RNA aptamers that bind the second messenger adenosine 3',5'-cyclic monophosphate (cAMP; 1) were isolated from a random-sequence pool using in vitro selection. Class I and class II aptamers are formed by 33- and 31-nucleotide RNAs, respectively, and each is comprised of similar stem-loop and single-stranded structural elements. Class II aptamers, which dominate the final selected RNA population, require divalent cations for complex formation and display a dissociation constant (K(D)) for cAMP of approximately 10 microM. A representative class II aptamer exhibits substantial discrimination against 5'- and 3'-phosphorylated nucleosides such as ATP, 5'-AMP, and 3'-AMP. However, components of cAMP such as adenine and adenosine also are bound, indicating that the adenine moiety is the primary positive determinant of ligand binding. Specificity of cAMP binding appears to be established by hydrogen bonding interactions with the adenine base as well as by steric interactions with groups on the ribose moiety. In addition, the aptamer recognizes 8,5'-O-cycloadenosine (2) but not N(3), 5'-cycloadenosine (3), indicating that this RNA might selectively recognize the anti conformation of the N-glycosidic bond of cAMP.     

An aptamer based competition assay for protein detection using CNT activated gold-interdigitated capacitor arrays

An aptamer can specifically bind to its target molecule, or hybridize with its complementary strand. A target bound aptamer complex has difficulty to hybridize with its complementary strand. It is possible to determine the concentration of target based on affinity separation system for the protein detection. Here, we exploited this property using C-reactive protein (CRP) specific RNA aptamers as probes that were immobilized by physical adsorption on carbon nanotubes (CNTs) activated gold interdigitated electrodes of capacitors. The selective binding ability of RNA aptamer with its target molecule was determined by change in capacitance after allowing competitive binding with CRP and complementary RNA (cRNA) strands in pure form and co-mixtures (CRP:cRNA=0:1, 1:0, 1:1, 1:2 and 2:1). The sensor showed significant capacitance change with pure forms of CRP/cRNA while responses reduced considerably in presence of CRP:cRNA in co-mixtures (1:1 and 1:2) because of the binding competition. At a critical CRP:cRNA ratio of 2:1, the capacitance response was dramatically lost because of the dissociation of adsorbed aptamers from the sensor surface to bind when excess CRP. Binding assays showed that the immobilized aptamers had strong affinity for cRNA (K(d)=1.98 μM) and CRP molecules (K(d)=2.4 μM) in pure forms, but low affinity for CRP:cRNA ratio of 2:1 (K(d)=8.58 μM). The dynamic detection range for CRP was determined to be 1-8 μM (0.58-4.6 μg/capacitor). The approach described in this study is a sensitive label-free method to detect proteins based on affinity separation of target molecules that can potentially be used for probing molecular interactions.     

A sol-gel-integrated protein array system for affinity analysis of aptamer-target protein interaction

A sol-gel microarray system was developed for a protein interaction assay with high activity. Comparing to 2-dimensional microarray surfaces, sol-gel can offer a more dynamic and broad range for proteins. In the present study, this sol-gel-integrated protein array was used in binding affinity analysis for aptamers. Six RNA aptamers and their target protein, yeast TBP (TATA-binding protein), were used to evaluate this method. A TBP-containing sol-gel mixture was spotted using a dispensing workstation under high-humidity conditions and each Cy-3-labeled aptamer was incubated. The dissociation constants (K(d)) were calculated by plotting the fluorescent intensity of the bound aptamers as a function of the TBP concentrations. The K(d) value of the control aptamer was found to be 8?nM, which agrees well with the values obtained using the conventional method, electric mobility shift assay. The sol-gel-based binding affinity measurements fit well with conventional binding affinity measurements, suggesting their possible use as an alternative to the conventional method. In addition, aptamer affinity measurements by the sol-gel-integrated protein chip make it possible to develop a simple high-throughput affinity method for screening high-affinity aptamers.     

The affinity ratio--its pivotal role in gold nanoparticle-based competitive colorimetric aptasensor

We present an important role of the ratio of affinities in unmodified gold nanoparticles-based colorimetric aptasensor reactions. An affinity ratio, representing the competitive interactions among aptamers, targets, and unmodified gold nanoparticles (umAuNPs), was found to be an important factor for the sensitivity (the performance), where the affinity ratio is the affinity of the aptamer to targets divided by the affinity to umAuNPs (K(dAuNP)/K(dTarget)). In this study, the five different aptamers having different affinity ratios to both umAuNPs and targets are used, and the degree of color change is well correlated with its affinity ratio. This result is verified by using a tetracycline binding aptamer (TBA) showing different affinities to its three derivatives, tetracycline, oxytetracycline and doxycycline. Based on this model, the sensitivity of umAuNPs based colorimetric detection for ibuprofen can be enhanced simply through reducing the ibuprofen binding aptamer's affinity to umAuNP by using bis (p-sulfonatophenyl) phenylphosphine as an AuNP-capping ligand, instead of using the citrate. As a result, a clear color change is observed even at a 20-fold less amount of ibuprofen. This study presents that the performance (detection sensitivity) of umAuNPs-based colorimetric aptasensors could be improved by simply adjusting the affinity ratio of the aptamers to targets and umAuNPs, without knowing the conformational changes of aptamers upon the target binding or needing any modification of aptamer sequences.     

Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR

RNA aptamers are in vitro-selected binding domains that recognize their respective ligand with high affinity and specificity. They are characterized by complex three-dimensional conformations providing preformed binding pockets that undergo conformational changes upon ligand binding. Small molecule-binding aptamers have been exploited as synthetic riboswitches for conditional gene expression in various organisms. In the present study, double electron-electron resonance (DEER) spectroscopy combined with site-directed spin labeling was used to elucidate the conformational transition of a tetracycline aptamer upon ligand binding. Different sites were selected for post-synthetic introduction of either the (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate by reaction with a 4-thiouridine modified RNA or of 4-isocyanato-2,6-tetramethylpiperidyl-N-oxid spin label by reaction with 2'-aminouridine modified RNA. The results of the DEER experiments indicate the presence of a thermodynamic equilibrium between two aptamer conformations in the free state and capture of one conformation upon tetracycline binding.     

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.     

Entropy and Mg2+ control ligand affinity and specificity in the malachite green binding RNA aptamer

The binding of small molecule targets by RNA aptamers provides an excellent model to study the versatility of RNA function. The malachite green aptamer binds and recognizes its ligand via stacking and electrostatic interactions. The binding of the aptamer to its original selection target and three related molecules was determined by isothermal titration calorimetry, equilibrium dialysis, and fluorescence titration. The results reveal that the entropy of complex formation plays a large role in determining binding affinity and ligand specificity. These data combined with previous structural studies show that metal ions are required to stabilize the complexes with non-native ligands whereas the complex with the original selection target is stable at low salt and in the absence of divalent metal ions.     

Is a closing "GA pair" a rule for stable loop-loop RNA complexes?

RNA hairpin aptamers specific for the trans-activation-responsive (TAR) RNA element of human immunodeficiency virus type 1 were identified by in vitro selection (Ducongé, F., and Toulmé, J. J. (1999) RNA 5, 1605-1614). The high affinity sequences selected at physiological magnesium concentration (3 mm) were shown to form a loop-loop complex with the targeted TAR RNA. The stability of this complex depends on the aptamer loop closing "GA pair" as characterized by preliminary electrophoretic mobility shift assays. Thermal denaturation monitored by UV-absorption spectroscopy and binding kinetics determined by surface plasmon resonance show that the GA pair is crucial for the formation of the TAR-RNA aptamer complex. Both thermal denaturation and surface plasmon resonance experiments show that any other "pairs" leads to complexes whose stability decreases in the order AG > GG > GU > AA > GC > UA > CA, CU. The binding kinetics indicate that stability is controlled by the off-rate rather than by the on-rate. Comparison with the complex formed with the TAR* hairpin, a rationally designed TAR RNA ligand (Chang, K. Y., and Tinoco, I. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8705-8709), demonstrates that the GA pair is a key determinant which accounts for the 50-fold increased stability of the TAR-aptamer complex (K(d) = 2.0 nm) over the TAR-TAR* one (K(d) = 92. 5 nm) at physiological concentration of magnesium. Replacement of the wild-type GC pair next to the loop of RNA I' by a GA pair stabilizes the RNA I'-RNA II' loop-loop complex derived from the one involved in the control of the ColE1 plasmid replication. Thus, the GA pair might be the preferred one for stable loop-loop interactions.     

In vitro selection of RNA molecules that inhibit the activity of ricin A-chain

The cytotoxin ricin disables translation by depurinating a conserved site in eukaryotic rRNA. In vitro selection has been used to generate RNA ligands (aptamers) specific for the catalytic ricin A-chain (RTA). The anti-RTA aptamers bear no resemblance to the normal RTA substrate, the sarcin-ricin loop (SRL), and were not depurinated by RTA. An initial 80-nucleotide RNA ligand was minimized to a 31-nucleotide aptamer that contained all sequences and structures necessary for interacting with RTA. This minimal RNA formed high affinity complexes with RTA (K(d) = 7.3 nM) which could compete directly with the SRL for binding to RTA. The aptamer inhibited RTA depurination of the SRL and could partially protect translation from RTA inhibition. The IC(50) of the aptamer for RTA in an in vitro translation assay is 100 nM, roughly 3 orders of magnitude lower than a small molecule inhibitor of ricin, pteroic acid, and 2 orders of magnitude lower than the best known RNA inhibitor. The novel anti-RTA aptamers may find application as diagnostic reagents for a potential biological warfare agent and hold promise as scaffolds for the development of strong ricin inhibitors.     

NMR resonance assignments for the class II GTP binding RNA aptamer in complex with GTP

The structures of RNA-aptamer-ligand complexes solved in the last two decades were instrumental in realizing the amazing potential of RNA for forming complex tertiary structures and for molecular recognition of small molecules. For GTP as ligand the sequences and secondary structures for multiple families of aptamers were reported which differ widely in their structural complexity, ligand affinity and ligand functional groups involved in RNA-binding. However, for only one of these families the structure of the GTP-RNA complex was solved. In order to gain further insights into the variability of ligand recognition modes we are currently determining the structure of another GTP-aptamer—the so-called class II aptamer—bound to GTP using NMR-spectroscopy in solution. As a prerequisite for a full structure determination, we report here ¹H, ¹³C, ¹⁵N and partial ³¹P-NMR resonance assignments for the class II GTP-aptamer bound to GTP.     

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.     

Aptamers that bind to the human complement component receptor hC5aR1 interfere with hC5aR1 interaction to its hC5a ligand

The complement system plays an important role in inflammation and immunity. In this system, a potent inflammatory ligand is C5a, which initiates its effects by activating its core receptor C5aR1. Thus, compounds that interfere with the C5a–C5aR1 interaction could alleviate some inflammatory conditions. Consequently, several ligands that bind to either C5a or C5aR1 have previously been isolated and evaluated. In the present study, two RNA aptamers, aptamer 1 and aptamer 9, that specifically bind to hC5aR1 with much higher affinity than antibodies were isolated. These two aptamers were tested for their ability to interfere with the cognate ligand of hC5aR1, C5a, using a chemotaxis assay. Both aptamer 1 and 9 interfered with the C5a interaction, suggesting that the aptamers recognized the extracellular domain of hC5aR1 responsible for hC5a ligand binding. Considering the higher affinity of aptamers to the hC5aR1 and their interference with hC5a ligand binding, further study is warranted to explore not only their applications in the diagnosis of inflammatory diseases but also their usefulness in modulating hC5a and hC5aR1 interactions.     

Sephadex-binding RNA ligands: rapid affinity purification of RNA from complex RNA mixtures

Sephadex-binding RNA ligands (aptamers) were obtained through in vitro selection. They could be classified into two groups based on their consensus sequences and the aptamers from both groups showed strong binding to Sephadex G-100. One of the highest affinity aptamers, D8, was chosen for further characterization. Aptamer D8 bound to dextran B512, the soluble base material of Sephadex, but not to isomaltose, isomaltotriose and isomaltotetraose, suggesting that its optimal binding site might consist of more than four glucose residues linked via alpha-1,6 linkages. The aptamer was very specific to the Sephadex matrix and did not bind appreciably to other supporting matrices, such as Sepharose, Sephacryl, cellulose or pustulan. Using Sephadex G-100, the aptamer could be purified from a complex mixture of cellular RNA, giving an enrichment of at least 60 000-fold, compared with a non-specific control RNA. These RNA aptamers can be used as affinity tags for RNAs or RNA subunits of ribonucleoproteins to allow rapid purification from complex mixtures of RNA using only Sephadex.     

Integration of multiple computer modeling software programs for characterization of a brain natriuretic peptide sandwich DNA aptamer complex

Several molecular modeling programs including Pep‐Fold 3, Vienna RNA, RNA Composer, Avogadro, PatchDock, RasMol, and VMD were used to define the three‐dimensional and basic binding characteristics of an extant sandwich DNA aptamer assay complex for human brain natriuretic peptide (BNP). In particular, the theoretical question of demonstrating likely binding of 72 base capture and reporter aptamers to at least two separate “epitopes” or binding sites on the small 32‐amino acid BNP target was addressed, and the data support the existence of separate aptamer binding sites on BNP. The binding model was based on first docking BNP to the capture aptamer based on shape complementarity with PatchDock, followed by docking the capture aptamer‐BNP complex with the reporter aptamer in PatchDock. Although, shape complementarity clearly dominated this binding model and aptamers are known to be somewhat flexible, the model demonstrates hydrogen bond stabilization within each of the two different aptamers and between the aptamers and the BNP target, thus suggesting a strong binding and high affinity sandwich assay that matches the author's former published assay results (Bruno et al., Microchem. J. 2014;115:32‐38) with subpicogram per milliliter sensitivity and good specificity. Other aspects such as capture and reporter aptamer interactions in the absence of BNP are illustrated and suggest means for potentially improving the existing assay by truncating the capture and reporter aptamers where they overlap to further decrease background signal levels.     

Characterization and application of a novel RNA aptamer against the mouse prion protein

In order to isolate RNA aptamers against the mouse prion protein (mPrP), we carried out in vitro selection from RNA pools containing a 30-nucleotide randomized region. Aptamer 60-3 was found to have a high affinity for mPrP (K(d) = 5.6 +/- 1.5 nM), and 2'-fluoro-pyrimidine modifications for RNase resistance did not abolish its binding activity (K(d) = 22 +/- 4 nM). Following 5' biotinylation, aptamer 60-3 specifically detected PrP in mouse brain homogenate in a Northwestern blotting assay. To determine the mPrP-aptamer binding region, we performed protein-deletion-mutant analysis and competition-binding analysis using heparin. The results showed that aptamer 60-3 appears to have binding sites located between amino acids 23-108.     

Monitoring complex formation in the blood-coagulation cascade using aptamer-coated SAW sensors

Specific binding of the anticoagulants heparin and antithrombin III to the blood clotting cascade factor human thrombin was recorded as a function of time with a Love-wave biosensor array consisting of five sensor elements. Two of the sensor elements were used as references. Three sensor elements were coated with RNA or DNA aptamers for specific binding of human thrombin. The affinity between the aptamers and thrombin, measured using the biosensor, was within the same range as the value of K(D) measured by filter binding experiments. Consecutive binding of the thrombin inhibitors heparin, antithrombin III or the heparin-antithrombin III complex to the immobilized thrombin molecules, and binding of a ternary complex of heparin, anithrombin III, and thrombin to aptamers was evaluated. The experiments showed attenuation of binding to thrombin due to heparin-antithrombin III complex formation. Binding of heparin activated the formation of the inhibitory complex of antithrombin III with thrombin about 2.7-fold. Binding of the DNA aptamer to exosite II appeared to inhibit heparin binding to exosite I.     

The structural basis for molecular recognition by the vitamin B 12 RNA aptamer

Previous solution structures of ligand-binding RNA aptamers have shown that molecular recognition is achieved by the folding of an initially unstructured RNA around its cognate ligand, coupling the processes of RNA folding and binding. The 3 A crystal structure of the cyanocobalamin (vitamin B12) aptamer reported here suggests a different approach to molecular recognition in which elements of RNA secondary structure combine to create a solvent-accessible docking surface for a large, complex ligand. Central to this structure is a locally folding RNA triplex, stabilized by a novel three-stranded zipper. Perpendicular stacking of a duplex on this triplex creates a cleft that functions as the vitamin B12 binding site. Complementary packing of hydrophobic surfaces, direct hydrogen bonding and dipolar interactions between the ligand and the RNA appear to contribute to binding. The nature of the interactions that stabilize complex formation and the possible uncoupling of folding and binding for this RNA suggest a strong mechanistic similarity to typical protein-ligand complexes.     

An adaptable pentaloop defines a robust neomycin-B RNA aptamer with conditional ligand-bound structures

Aptamers can be highly specific for their targets, which implies precise molecular recognition between aptamer and target. However, as small polymers, their structures are more subject to environmental conditions than the more constrained longer RNAs such as those that constitute the ribosome. To understand the balance between structural and environmental factors in establishing ligand specificity of aptamers, we examined the RNA aptamer (NEO1A) previously reported as specific for neomycin-B. We show that NEO1A can recognize other aminoglycosides with similar affinities as for neomycin-B and its aminoglycoside specificity is strongly influenced by ionic strength and buffer composition. NMR and 2-aminopurine (2AP) fluorescence studies of the aptamer identified a flexible pentaloop and a stable binding pocket. Consistent with a well-structured binding pocket, docking analysis results correlated with experimental measures of the binding energy for most ligands. Steady state fluorescence studies of 2AP-substituted aptamers confirmed that A16 moves to a more solvent accessible position upon ligand binding while A14 moves to a less solvent accessible position, which is most likely a base stack. Analysis of binding affinities of NEO1A sequence variants showed that the base in position 16 interacts differently with each ligand and the interaction is a function of the buffer constituents. Our results show that the pentaloop provides NEO1A with the ability to adapt to external influences on its structure, with the critical base at position 16 adjusting to incorporate each ligand into a stable pocket by hydrophobic interactions and/or hydrogen bonds depending on the ligand and the ionic environment.     

Ligand binding by the tandem glycine riboswitch depends on aptamer dimerization but not double ligand occupancy

The glycine riboswitch predominantly exists as a tandem structure, with two adjacent, homologous ligand-binding domains (aptamers), followed by a single expression platform. The recent identification of a leader helix, the inclusion of which eliminates cooperativity between the aptamers, has reopened the debate over the purpose of the tandem structure of the glycine riboswitch. An equilibrium dialysis-based assay was combined with binding-site mutations to monitor glycine binding in each ligand-binding site independently to understand the role of each aptamer in glycine binding and riboswitch tertiary interactions. A series of mutations disrupting the dimer interface was used to probe how dimerization impacts ligand binding by the tandem glycine riboswitch. While the wild-type tandem riboswitch binds two glycine equivalents, one for each aptamer, both individual aptamers are capable of binding glycine when the other aptamer is unoccupied. Intriguingly, glycine binding by aptamer-1 is more sensitive to dimerization than glycine binding by aptamer-2 in the context of the tandem riboswitch. However, monomeric aptamer-2 shows dramatically weakened glycine-binding affinity. In addition, dimerization of the two aptamers in trans is dependent on glycine binding in at least one aptamer. We propose a revised model for tandem riboswitch function that is consistent with these results, wherein ligand binding in aptamer-1 is linked to aptamer dimerization and stabilizes the P1 stem of aptamer-2, which controls the expression platform.     

Characterization of a fluorophore binding RNA aptamer by fluorescence correlation spectroscopy and small angle X-ray scattering

Using fluorescence correlation spectroscopy (FCS), we have established an in vitro assay to study RNA dynamics by analyzing fluorophore binding RNA aptamers at the single molecule level. The RNA aptamer SRB2m, a minimized variant of the initially selected aptamer SRB-2, has a high affinity to the disulfonated triphenylmethane dye sulforhodamine B. A mobility shift of sulforhodamine B after binding to SRB2m was measured. In contrast, patent blue V (PBV) is visible only if complexed with SRB2m due to increased molecular brightness and minimal background. With small angle X-ray scattering (SAXS), the three-dimensional structure of the RNA aptamer was characterized at low resolution to analyze the effect of fluorophore binding. The aptamer and sulforhodamine B-aptamer complex was found to be predominantly dimeric in solution. Interaction of PBV with SRB2m led to a dissociation of SRB2m dimers into monomers. Radii of gyration and hydrodynamic radii, gained from dynamic light scattering, FCS, and fluorescence cross-correlation experiments, led to comparable conclusions. Our study demonstrates how RNA-aptamer fluorophore complexes can be simultaneously structurally and photophysically characterized by FCS. Furthermore, fluorophore binding RNA aptamers provide a tool for visualizing single RNA molecules.