RNA architecture dictates the conformations of a bound peptide.

BACKGROUND: The biological function of several viral and bacteriophage proteins, and their arginine-rich subdomains, involves RNA-mediated interactions. It has been shown recently that bound peptides adopt either beta-hairpin or alpha-helical conformations in viral and phage peptide-RNA complexes. We have compared the structures of the arginine-rich peptide domain of HIV-1 Rev bound to two RNA aptamers to determine whether RNA architecture can dictate the conformations of a bound peptide. RESULTS: The core-binding segment of the HIV-1 Rev peptide class II RNA aptamer complex spans the two-base bulge and hairpin loop of the bound RNA and the carboxy-terminal segment of the bound peptide. The bound peptide is anchored in place by backbone and sidechain intermolecular hydrogen bonding and van der Waals stacking interactions. One of the bulge bases participates in U*(A*U) base triple formation, whereas the other is looped out and flaps over the bound peptide in the complex. The seven-residue hairpin loop is closed by a sheared G*A mismatch pair with several pyrimidines looped out of the hairpin fold. CONCLUSIONS: Our structural studies establish that RNA architecture dictates whether the same HIV-1 Rev peptide folds into an extended or alpha-helical conformation on complex formation. Arginine-rich peptides can therefore adapt distinct secondary folds to complement the tertiary folds of their RNA targets. This contrasts with protein-RNA complexes in which elements of RNA secondary structure adapt to fit within the tertiary folds of their protein targets.     

Controlled intracellular localization and enhanced antisense effect of oligonucleotides by chemical conjugation

Oligonucleotides can be covalently linked to peptides composed of any sequence of amino acids by solid phase fragment condensation. The peptides incorporated into the conjugates include nuclear localizing signals (NLS), nuclear export signals (NES), membrane fusion domain of some viral proteins and some designed peptides with amphipathic character. Evaluation of biological properties of DNA-peptide conjugates indicated that (a) the conjugates could bind to target RNA and dsDNA with increased affinity, (b) the conjugates were more resistant to cellular nuclease degradation, (c) the conjugate-RNA hybrids could activate RNase H as effectively as native oligonucleotides, (d) the conjugates with fusion peptides showed largely enhanced cellular uptake, (e) the conjugates with NLS could be predominantly delivered into the cell nucleus, (f) the conjugates with NES could be localized in the cytoplasm. As a result, antisense oligonucleotides conjugated with NLS could inhibit human telomerase in human leukemia cells much more strongly than phosphorothioate oligonucleotides.     

Structural basis of DNA folding and recognition in an AMP-DNA aptamer complex: distinct architectures but common recognition motifs for DNA and RNA aptamers complexed to AMP

Background: Structural studies by nuclear magnetic resonance (NMR) of RNA and DNA aptamer complexes identified through in vitro selection and amplification have provided a wealth of information on RNA and DNA tertiary structure and molecular recognition in solution. The RNA and DNA aptamers that target ATP (and AMP)' with micromolar affinity exhibit distinct binding site sequences and secondary structures. We report below on the tertiary structure of the AMP-DNA aptamer complex in solution and compare it with the previously reported tertiary structure of the AMP-RNA aptamer complex in solution.Results: The solution structure of the AMP-DNA aptamer complex shows, surprisingly, that two AMP molecules are intercalated at adjacent sites within a rectangular widened minor groove. Complex formation involves adaptive binding where the asymmetric internal bubble of the free DNA aptamer zippers up through formation of a continuous six-base mismatch segment which includes a pair of adjacent three-base platforms. The AMP molecules pair through their Watson-Crick edges with the minor groove edges of guanine residues. These recognition G·A mismatches are flanked by sheared G·A and reversed Hoogsteen G·G mismatch pairs.Conclusions: The AMP-DNA aptamer and AMP-RNA aptamer complexes have distinct tertiary structures and binding stoichiometries. Nevertheless, both complexes have similar structural features and recognition alignments in their binding pockets. Specifically, AMP targets both DNA and RNA aptamers by intercalating between purine bases and through identical G·A mismatch formation. The recognition G·A mismatch stacks with a reversed Hoogsteen G·G mismatch in one direction and with an adenine base in the other direction in both complexes. It is striking that DNA and RNA aptamers selected independently from libraries of 10¹⁴ molecules in each case utilize identical mismatch alignments for molecular recognition with micromolar affinity within binding-site pockets containing common structural elements.     

Nonequilibrium capillary electrophoresis of equilibrium mixtures: a universal tool for development of aptamers

Aptamers are DNA (or RNA) ligands selected from large libraries of random DNA sequences and capable of binding different classes of targets with high affinity and selectivity. Both the chances for the aptamer to be selected and the quality of the selected aptamer are largely dependent on the method of selection. Here we introduce selection of aptamers by nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). The new method has a number of advantages over conventional approaches. First, NECEEM-based selection has exceptionally high efficiency, which allows aptamer development with fewer rounds of selection. Second, NECEEM can be equally used for selecting aptamers and finding their binding parameters. Finally, due to its comprehensive kinetic capabilities, the new method can potentially facilitate selection of aptamers with predefined K(d), k(off), and k(on) of the aptamer-target interaction. In this proof-of-principle work, we describe the theoretical bases of the method and demonstrate its application to a one-step selection of DNA aptamers with nanomolar affinity for protein farnesyltransferase.     

Tuning the specificity of a synthetic receptor using a selected nucleic acid receptor

Because of their relative simplicity, synthetic receptors often lack the selectivity observed for biopolymer receptors, such as aptamers. However, aptamer recognition of ligands is limited by the chemistries inherent in the four canonical nucleotides. Here, we report the design and selection of a ternary complex in which the specificity of a bis-boronic acid synthetic host (1) that binds to various carboxylic acids is tuned by a surrounding aptamer. Although, the synthetic receptor alone has higher selectivity for citrate over DL-tartrate, the formation of the aptamer:receptor complex reversed the organic host selectivity to preferentially bind tartrate. The RNA conformation changed upon the introduction of the synthetic host, consistent with an induced-fit mechanism for binding.     

Streptavidin binding bifunctional aptamers and their interaction with low molecular weight ligands

This paper describes the measurement of the binding affinities of two bifunctional RNA aptamers to their respective ligands. The aptamers comprise either a theophylline or malachite green binding sequence fused to a streptavidin binding sequence. These bifunctional aptamers are shown to bind simultaneously to both the small ligand and to streptavidin whether in free solution or on gold surfaces. Binding isotherms for both interactions were measured by different physiochemical techniques: surface plasmon resonance, fluorescence spectroscopy and dynamic light scattering. Both qualitatively and quantitatively there is little difference in binding affinities between the bifunctional aptamers and their monofunctional components. The respective K_d values for streptavidin binding in the monofunctional aptamer and in the theophylline bifunctional aptamer were 12 nM and 65 nM, respectively whilst the K_d values for theophylline binding in the monofunctional aptamer and the streptavidin bifunctional aptamer were 300 nM and 120 nM. These results are consistent with treating each aptamer sequence as a module that can be combined with others without significant loss of function. This allows for the use of streptavidin based immobilization strategies without either the cost of biotinylated dNTPs or the variable yields associated with the chemical biotinylation of RNA.     

Probing the structure of DNA aptamers with a classic heterocycle

DNA aptamers are synthetic, single-stranded DNA oligonucleotides selected by SELEX methods for their binding with specific ligands. Here we present ethidium binding results for three related DNA aptamers (PDB code: 1OLD, 1DB6, and 2ARG)that bind L-argininamide (L-Arm). The ligand bound form of each aptamer's structure has been reported and each are found to be composed primarily of two domains consisting of a stem helical region and a loop domain that forms a binding pocket for the cognate ligand. Previous thermodynamic experiments demonstrated that the DNA aptamer 1OLD undergoes a large conformational ordering upon binding to L-Arm. Here we extend those linkage binding studies by examining the binding of the heterocyclic intercalator ethidium to each of the three aptamers by fluorescence and absorption spectrophotometric titrations. Our results reveal that ethidium binds to each aptamer with DeltaG degree's in the range of -8.7 to -9.4 kcal/mol. The stoichiometry of binding is 2:1 for each aptamer and is quantitatively diminished in the presence of L-Arm as is the overall fluorescence intensity of ethidium. Together, these results demonstrate that a portion of the bound ethidium is excluded from the aptamer in the presence of a saturating amount of L-Arm. These results demonstrate the utility of ethidium and related compounds for the probing of non-conventional DNA structures and reveal an interesting fundamental thermodynamic linkage in DNA aptamers. Results are discussed in the context of the thermodynamic stability and structure of each of the aptamers examined.     

Supramolecular Fluorescence Resonance Energy Transfer in Nucleobase‐Modified Fluorogenic RNA Aptamers

RNA aptamers form compact tertiary structures and bind their ligands in specific binding sites. Fluorescence‐based strategies reveal information on structure and dynamics of RNA aptamers. Herein, we report the incorporation of the universal emissive nucleobase analog 4‐cyanoindole into the fluorogenic RNA aptamer Chili, and its application as a donor for supramolecular FRET to the bound ligands DMHBI⁺ or DMHBO⁺. The photophysical properties of the new nucleobase–ligand‐FRET pair revealed structural restraints for the overall RNA aptamer organization and identified nucleotide positions suitable for FRET‐based readout of ligand binding. This strategy is generally suitable for binding‐site mapping and may also be applied for responsive aptamer devices.     

Supramolecular Fluorescence Resonance Energy Transfer in Nucleobase-Modified Fluorogenic RNA Aptamers

RNA aptamers form compact tertiary structures and bind their ligands in specific binding sites. Fluorescence-based strategies reveal information on structure and dynamics of RNA aptamers. Herein, we report the incorporation of the universal emissive nucleobase analog 4-cyanoindole into the fluorogenic RNA aptamer Chili, and its application as a donor for supramolecular FRET to the bound ligands DMHBI⁺ or DMHBO⁺. The photophysical properties of the new nucleobase–ligand-FRET pair revealed structural restraints for the overall RNA aptamer organization and identified nucleotide positions suitable for FRET-based readout of ligand binding. This strategy is generally suitable for binding-site mapping and may also be applied for responsive aptamer devices.     

Investigation of the interaction between a bivalent aptamer and thrombin by AFM

Aptamers are a new class of molecular probes for protein recognition, detection, and inhibition. Multivalent aptamer-protein binding through aptamer assembly has been currently developed as an effective way to achieve higher protein affinity and selectivity. In this study, the specific interaction between bivalent aptamer Bi-8S and thrombin has been measured directly and quantitatively by atomic force microscopy to investigate the unbinding dynamics and dissociation energy landscape of the multivalent interaction. Bivalent aptamer Bi-8S contains thrombin's two aptamers, 15apt and 27apt, which are linked by eight spacer phosphoramidites. The results revealed the sequential dissociation of the two aptamers. Moreover, the dynamic force spectroscopy data revealed that the 27apt's binding to the thrombin remains largely unaffected by the eight-spacer phosphoramidites within Bi-8S. In contrast, the eight-spacer phosphoramidites stabilized the 15apt-thrombin binding.     

Kinetic capillary electrophoresis-based affinity screening of aptamer clones

DNA aptamers are single stranded DNA (ssDNA) molecules artificially selected from random-sequence DNA libraries for their specific binding to a certain target. DNA aptamers have a number of advantages over antibodies and promise to replace them in both diagnostic and therapeutic applications. The development of DNA aptamers involves three major stages: library enrichment, obtaining individual DNA clones, and the affinity screening of the clones. The purpose of the screening is to obtain the nucleotide sequences of aptamers and the binding parameters of their interaction with the target. Highly efficient approaches have been recently developed for the first two stages, while the third stage remained the rate-limiting one. Here, we introduce a new method for affinity screening of individual DNA aptamer clones. The proposed method amalgamates: (i) aptamer amplification by asymmetric PCR (PCR with a primer ratio different from unity), (ii) analysis of aptamer-target interaction, combining in-capillary mixing of reactants by transverse diffusion of laminar flow profiles (TDLFP) and affinity analysis using kinetic capillary electrophoresis (KCE), and (iii) sequencing of only aptamers with satisfying binding parameters. For the first time we showed that aptamer clones can be directly used in TDLFP/KCE-based affinity analysis without an additional purification step after asymmetric PCR amplification. We also demonstrated that mathematical modeling of TDLFP-based mixing allows for the determination of K_d values for the in-capillary reaction of an aptamer and a target and that the obtained K_d values can be used for the accurate affinity ranking of aptamers. The proposed method does not require the knowledge of aptamer sequences before screening, avoids lengthy (3-5 h) purification steps of aptamer clones, and minimizes reagent consumption to nanoliters.     

Nuclear annexin II negatively regulates growth of LNCaP cells and substitution of ser 11 and 25 to glu prevents nucleo-cytoplasmic shuttling of annexin II

Background  

Annexin II heavy chain (also called p36, calpactin I) is lost in prostate cancers and in a majority of prostate intraepithelial neoplasia (PIN). Loss of annexin II heavy chain appears to be specific for prostate cancer since overexpression of annexin II is observed in a majority of human cancers, including pancreatic cancer, breast cancer and brain tumors. Annexin II exists as a heterotetramer in complex with a protein ligand p11 (S100A10), and as a monomer. Diverse cellular functions are proposed for the two forms of annexin II. The monomer is involved in DNA synthesis. A leucine-rich nuclear export signal (NES) in the N-terminus of annexin II regulates its nuclear export by the CRM1-mediated nuclear export pathway. Mutation of the NES sequence results in nuclear retention of annexin II.     

Aptamers selected for higher-affinity binding are not more specific for the target ligand

Previous study of eleven different in vitro-selected RNA aptamers that bind guanosine triphosphate (GTP) with K(d)s ranging from 8 microM to 9 nM showed that more information is required to specify the structures of the higher-affinity aptamers. We are interested in understanding how the more complex aptamers achieve higher affinities for the ligand. In vitro selection produces structural solutions to a functional problem that are are as simple as possible in terms of the information content needed to define them. It has long been assumed that the simplest way to improve the affinity of an aptamer is to increase the shape and functional group complementarity of the RNA binding pocket for the ligand. This argument underlies the hypothesis that selection for higher-affinity aptamers automatically leads to structures that bind more specifically to the target molecule. Here, we examined the binding specificities of the eleven GTP aptamers by carrying out competition binding studies with sixteen different chemical analogues of GTP. The aptamers have distinct patterns of specificity, implying that each RNA is a structurally unique solution to the problem of GTP binding. However, these experiments failed to provide evidence that higher-affinity aptamers bind more specifically to GTP. We suggest that the simplest way to improve aptamer K(d)s may be to increase the stability of the RNA tertiary structure with additional intramolecular RNA-RNA interactions; increasingly specific ligand binding may emerge only in response to direct selection for specificity.     

RNA aptamers that bind flavin and nicotinamide redox cofactors

RNA molecules that specifically bind riboflavin (Rb) and beta-nicotinamide mononucleotide (NMN) have been isolated by in vitro selection. A simple structural motif containing intramolecular G-quartets was found to bind tightly to oxidized riboflavin (Kd = 1-5 micromolar). DNA versions of the consensus sequence also bind, but with weaker affinity. DMS protection experiments show that the quartet structure of these aptamers is stabilized by interaction with the flavin. As a measure of their redox specificity, the aptamers do not show significant differential binding between oxidized and reduced forms of a 5-deazariboflavin derivative that is a close structural analog of riboflavin. In contrast to the lack of redox specificity of the riboflavin aptamers, RNAs selected for binding to the nicotinamide portion of NAD discriminate between NAD and NADH in solution by over an order of magnitude. A mutagenized pool based on one of the NMN aptamer sequences was used to reselect for NMN binding. Comparison of the reselected sequences led to the identification of the binding region of the aptamer. A complex secondary structure containing two interacting stem-loops is proposed for the minimal NMN-binding RNA. The same mutagenized pool was used to select for increased discrimination between NMN and NMNH. From these reselected sequences, a mutation within the binding region was identified that increases specificity for NMN. These experiments show that RNA can bind these cofactors with low micromolar affinity and, in the case of nicotinamide cofactors, can discriminate between the two redox states. These cofactor binding motifs may provide a framework for generating new ribozymes that catalyze redox reactions similar to those found in basic metabolic pathways.     

Aptamer-based biosensors for the detection of HIV-1 Tat protein

Two biosensors have been constructed using an RNA aptamer as biorecognition element. The aptamer, specific for HIV-1 Tat protein, has been immobilised on the gold surface of piezoelectric quartz crystals or surface plasmon resonance (SPR) chips to develop a quartz crystal microbalance (QCM)-based and an SPR-based biosensor, respectively. Both the biosensors were modified with the same immobilisation chemistry based on the binding of a biotinylated aptamer on a layer of streptavidin. The binding between the immobilised aptamer and its specific protein has been evaluated with the two biosensors in terms of sensitivity, reproducibility and selectivity. A protein very similar to Tat, Rev protein, has been used as negative control. The two biosensors both were very reproducible in the immobilisation and the binding steps. The selectivity was high in both cases.     

Modified capillary electrophoresis based measurement of the binding between DNA aptamers and an unknown concentration target

The recognition of targets such as biomacromolecules, viruses and cells by their aptamers is crucial in aptamer-based biosensor platforms and research into protein function. However, it is difficult to evaluate the binding constant of aptamers and their targets that are hard to purify and quantify, especially when the targets are undefined. Therefore, we aimed to develop a modified capillary electrophoresis based method to determine the dissociation constant of aptamers whose targets are hard to quantify. A protein target, human thrombin, and one of its aptamers were used to validate our modified method. We demonstrated that the result calculated by our method, only depending on the aptamer’s concentrations, was consistent with the classical method, which depended on the concentrations of both the aptamers and the targets. Furthermore, a series of DNA aptamers binding with avian influenza virus H9N2 were confirmed by a four-round selection of capillary electrophoresis–systematic evolution of ligands by exponential enrichment, and we identified the binding constant of these aptamers by directly using the whole virus as the target with the modified method. In conclusion, our modified method was validated to study the interaction between the aptamer and its target, and it may also advance the evaluation of other receptor–ligand interactions.     

Assembling OX40 aptamers on a molecular scaffold to create a receptor-activating aptamer

We show that a molecular scaffold can be utilized to convert a receptor binding aptamer into a receptor agonist. Many receptors (including tumor necrosis receptor family members) are activated when they are multimerized on the cell surface. Molecular scaffolds have been utilized to assemble multiple receptor binding peptide ligands to generate activators of such receptors. We demonstrate that an RNA aptamer that recognizes OX40, a member of the tumor necrosis factor receptor superfamily, can be converted into a receptor-activating aptamer by assembling two copies on an olignucleotide-based scaffold. The OX40 receptor-activating aptamer is able to induce nuclear localization of nuclear factor-kappaB, cytokine production, and cell proliferation, as well as enhance the potency of dendritic cell-based tumor vaccines when systemically delivered to mice.     

Selective Hemin Binding by a Non-G-quadruplex Aptamer with Higher Affinity and Better Peroxidase-like Activity

Previous aptamers for porphyrins and metalloporphyrins were all guanine-rich sequences that can fold in G-quadruplex structures. Due to stacking-based binding, these aptamers can hardly tell different porphyrins apart, and they can also bind other planar molecules, hindering their practical applications. In this work, we used the capture selection method to obtain aptamers for hemin and protoporphyrin IX (PPIX). The hemin aptamer (Hem1) features two highly conserved repeating binding loops, and it cannot form a G-quadruplex, which was supported by its Mg²⁺-dependent but K⁺-independent hemin binding and CD spectroscopy. Isothermal titration calorimetry revealed much higher enthalpy change for the new aptamer, and the best aptamer showed a K_d of 43 nM hemin. Hem1 can also enhance the peroxidase-like activity of hemin. This work demonstrates that aptamers have alternative ways to bind porphyrins allowing selective recognition of different porphyrins.