A colorimetric detection method of pesticide acetamiprid by fine-tuning aptamer length

This work investigates the effect of shortening aptamer sequences on the colorimetric detection of acetamiprid using aptamer-wrapped gold nanoparticles (AuNPs). Truncated 37-mer and 25-mer aptamers were generated by deleting excess flanking nucleotides from parental 49-mer acetamiprid-target aptamer. In comparing the responses of the three sequences, truncated aptamers did not improve the ability to discriminate against other tested pesticides. However, comparison between 49-mer and other shorter aptamers showed that shortening aptamer sequences through removing excess flanking nucleotides outsides of binding region improved colorimetric sensitivity for acetamiprid by 3.3 fold. Due to excess bases, the target-bound aptamer might still adhere to AuNPs, resulting in incomplete dissociation of aptamer from AuNPs and therefore the suppression of aggregation responses. This work provides further insight to the effects of aptamer structure on detection of the target, as well as a method by fine-tuning aptamer length for rapid detection of pesticide residues in environments or food.     

Engineering a structure switching mechanism into a steroid-binding aptamer and hydrodynamic analysis of the ligand binding mechanism

The steroid binding mechanism of a DNA aptamer was studied using isothermal titration calorimetry (ITC), NMR spectroscopy, quasi-elastic light scattering (QELS), and small-angle X-ray spectroscopy (SAXS). Binding affinity determination of a series of steroid-binding aptamers derived from a parent cocaine-binding aptamer demonstrates that substituting a GA base pair with a GC base pair governs the switch in binding specificity from cocaine to the steroid deoxycholic acid (DCA). Binding of DCA to all aptamers is an enthalpically driven process with an unfavorable binding entropy. We engineered into the steroid-binding aptamer a ligand-induced folding mechanism by shortening the terminal stem by two base pairs. NMR methods were used to demonstrate that there is a transition from a state where base pairs are formed in one stem of the free aptamer, to where three stems are formed in the DCA-bound aptamer. The ability to generate a ligand-induced folding mechanism into a DNA aptamer architecture based on the three-way junction of the cocaine-binding aptamer opens the door to obtaining a series of aptamers all with ligand-induced folding mechanisms but triggered by different ligands. Hydrodynamic data from diffusion NMR spectroscopy, QELS, and SAXS show that for the aptamer with the full-length terminal stem there is a small amount of structure compaction with DCA binding. For ligand binding by the short terminal stem aptamer, we propose a binding mechanism where secondary structure forms upon DCA binding starting from a free structure where the aptamer exists in a compact form.     

ValFold: Program for the aptamer truncation process

DNA or RNA aptamers have gained attention as the next generation antibody-like molecules for medical or diagnostic use. Conventional secondary structure prediction tools for nucleic acids play an important role to truncate or minimize sequence, or introduce limited chemical modifications without compromising or changing its binding affinity to targets in the design of improved aptamers selected by Systematic Evolution of Ligands by EXponential enrichment (SELEX). We describe a novel software package, ValFold, capable of predicting secondary structures with improved accuracy based on unique aptamer characteristics. ValFold predicts not only the canonical Watson-Crick pairs but also G-G pairs derived from G-quadruplex (known structure for many aptamers) using the stem candidate selection algorithm. AVAILABILITY: The database is available for free at http://code.google.com/p/valfold/     

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.     

Rational truncation of an RNA aptamer to prostate-specific membrane antigen using computational structural modeling

RNA aptamers represent an emerging class of pharmaceuticals with great potential for targeted cancer diagnostics and therapy. Several RNA aptamers that bind cancer cell-surface antigens with high affinity and specificity have been described. However, their clinical potential has yet to be realized. A significant obstacle to the clinical adoption of RNA aptamers is the high cost of manufacturing long RNA sequences through chemical synthesis. Therapeutic aptamers are often truncated postselection by using a trial-and-error process, which is time consuming and inefficient. Here, we used a "rational truncation" approach guided by RNA structural prediction and protein/RNA docking algorithms that enabled us to substantially truncateA9, an RNA aptamer to prostate-specific membrane antigen (PSMA),with great potential for targeted therapeutics. This truncated PSMA aptamer (A9L; 41mer) retains binding activity, functionality, and is amenable to large-scale chemical synthesis for future clinical applications. In addition, the modeled RNA tertiary structure and protein/RNA docking predictions revealed key nucleotides within the aptamer critical for binding to PSMA and inhibiting its enzymatic activity. Finally, this work highlights the utility of existing RNA structural prediction and protein docking techniques that may be generally applicable to developing RNA aptamers optimized for therapeutic use.     

Dissection of the functional structure of aptamer17, which specifically recognizes differentiated PC12 cells

A specific single-stranded DNA (ssDNA) aptamer (aptamer17) that specifically recognizes differentiated PC12 cells had been previously obtained after 6 rounds of whole cell-based subtractive systematic evolution of ligands by exponential enrichment selection from a random ssDNA library. To further investigate the relationship between the structure and function of this aptamer, 3 truncated ssDNA aptamers were designed according to the predicted secondary structure of aptamer17. Our results show that the stem-loop is the core structure of the aptamers required for specific binding to differentiated PC12 cells, specifically loops I and II. Aptamer17 and the truncated aptamers with this basic structure could bind specifically to differentiated PC12 cells and identify these cells from a mixture of differentiated and undifferentiated PC12 cells. Therefore, truncated forms of aptamer17 may be useful in the clinic to identify undifferentiated and differentiated PC12 cells from a mixture of cells.     

Structural characterization of an anti-gp120 RNA aptamer that neutralizes R5 strains of HIV-1

We recently described the isolation of 2'-fluoropyrimidine-substituted RNA aptamers that bind specifically to the surface glycoprotein (gp 120) of the R5 strain, HIV-1(Ba-L), as presented in a previous study. These aptamers potently neutralize HIV-1 infectivity in human peripheral blood mononuclear cells of both tissue culture lab-adapted strains and diverse R5 clinical isolates from multiple clades. Here, we report a detailed structural characterization of one such neutralizing aptamer, B40, using enzymatic and chemical probing methods. We identify the minimal region of the aptamer essential for binding gp120 and accordingly design a 77-nucleotide truncated aptamer, B40t77. We then quantitatively analyze the binding affinity and neutralization potency of the parental and truncated (minimal) aptamer, and show them to be comparable. Furthermore, using results from secondary structure analysis, RNA mutagenesis and BIAcore surface plasmon resonance (SPR) binding assays, we hypothesize that a folded RNA structure is required to present specific nucleotide sequences to allow gp120-recognition and binding. The information gained from this study may provide leads for development of novel anti-HIV-1 therapies and can be used to extend our understanding of the molecular interactions between the virus and its host cell.     

Conservative secondary structure motif of streptavidin-binding aptamers generated by different laboratories

Aptamers that are selected in vitro from random pools of DNA or RNA molecules by SELEX (Systematic evolution of ligands by exponential enrichment) technique have been extensively explored for analytical and biomedical applications. Although many aptamers with high affinity and specificity against specific ligands have been reported, there is still a lack of well characterized DNA aptamers. Here we report the selection of a group of aptamer candidates (85 mer) against streptavidin. Through comparing the predicted secondary structures of all the candidates, a conservative bulge-hairpin structure section (about 29 mer) was found, and then it was determined to be the binding motif to streptavidin. This binding motif was further discovered to also exist in streptavidin-binding aptamers (SBAs) selected by three other laboratories using different methods. The primary sequences of this secondary structure motif are very different, only several nucleotides in the loop and bulge area are critical for binding and other nucleotides are variable. The streptavidin binding of all the SBAs could be competed by biotin implying that they bind to the same site on streptavidin. These results suggest that the evolution of SBA is predominated by specific groups on streptavidin. The highly variable sequence composition of streptavidin-binding aptamer would make the design of aptameric sensor or device based on streptavidin more flexible and easy.     

Improvement of a streptavidin-binding aptamer by LNA- and α-l-LNA-substitutions

Forty modified versions of a streptavidin-binding aptamer each containing single or multiple LNA or α-l-LNA-substitutions were synthesized and their dissociation constants determined by surface plasmon resonance experiments. Both full-length and truncated versions of the aptamer were studied and compared with the unmodified DNA aptamers. A ∼two-fold improvement in binding affinity was achieved by incorporation of LNA nucleotides in the 3′-part of the stems of the streptavidin-binding aptamer whereas LNA- and α-l-LNA-substitutions in the terminal stem increased the serum stability.     

Solution structure of an informationally complex high-affinity RNA aptamer to GTP

Higher-affinity RNA aptamers to GTP are more informationally complex than lower-affinity aptamers. Analog binding studies have shown that the additional information needed to improve affinity does not specify more interactions with the ligand. In light of those observations, we would like to understand the structural characteristics that enable complex aptamers to bind their ligands with higher affinity. Here we present the solution structure of the 41-nt Class I GTP aptamer (K(d) = 75 nM) as determined by NMR. The backbone of the aptamer forms a reverse-S that shapes the binding pocket. The ligand nucleobase stacks between purine platforms and makes hydrogen bonds with the edge of another base. Interestingly, the local modes of interaction for the Class I aptamer and an RNA aptamer that binds ATP with a K(d) of 6 microM are very much alike. The aptamers exhibit nearly identical levels of binding specificity and fraction of ligand sequestered from the solvent (81%-85%). However, the GTP aptamer is more informationally complex (approximately 45 vs. 35 bits) and has a larger recognition bulge (15 vs. 12 nucleotides) with many more stabilizing base-base interactions. Because the aptamers have similar modes of ligand binding, we conclude that the stabilizing structural elements in the Class I aptamer are responsible for much of the difference in K(d). These results are consistent with the hypothesis that increasing the number of intra-RNA interactions, rather than adding specific contacts to the ligand, is the simplest way to improve binding affinity.     

Chemical basis of glycine riboswitch cooperativity

The glycine binding riboswitch forms a unique tandem aptamer structure that binds glycine cooperatively. We employed nucleotide analog interference mapping (NAIM) and mutagenesis to explore the chemical basis of glycine riboswitch cooperativity. Based on the interference pattern, at least two sites appear to facilitate cooperative tertiary interactions, namely, the minor groove of the P1 helix from aptamer 1 and the major groove of the P3a helix from both aptamers. Mutation of these residues altered both the cooperativity and binding affinity of the riboswitch. The data support a model in which the P1 helix of the first aptamer participates in a tertiary interaction important for cooperativity, while nucleotides in the P1 helix of the second aptamer interface with the expression platform. These data have direct analogy to well-characterized mutations in hemoglobin, which provides a framework for considering cooperativity in this RNA-based system.     

Solution structure of an ATP-binding RNA aptamer reveals a novel fold.

In vitro selection has been used to isolate several RNA aptamers that bind specifically to biological cofactors. A well-characterized example in the ATP-binding RNA aptamer family, which contains a conserved 11-base loop opposite a bulged G and flanked by regions of double-stranded RNA. The nucleotides in the consensus sequence provide a binding pocket for ATP (or AMP), which binds with a Kd in the micromolar range. Here we present the three-dimensional solution structure of a 36-nucleotide ATP-binding RNA aptamer complexed with AMP, determined from NMR-derived distance and dihedral angle restraints. The conserved loop and bulged G form a novel compact, folded structure around the AMP. The backbone tracing of the loop nucleotides can be described by a Greek zeta (zeta). Consecutive loop nucleotides G, A, A form a U-turn at the bottom of the zeta, and interact with the AMP to form a structure similar to a GNRA tetraloop, with AMP standing in for the final A. Two asymmetric G. G base pairs close the stems flanking the internal loop. Mutated aptamers support the existence of the tertiary interactions within the consensus nucleotides and with the AMP found in the calculated structures.     

Structure determination and binding kinetics of a DNA aptamer-argininamide complex

The structure of a DNA aptamer, which was selected for specific binding to arginine, was determined using NMR spectroscopy. The sequence forms a hairpin loop, with residues important for binding occurring in the loop region. Binding of argininamide induces formation of one Watson-Crick and two non-Watson-Crick base pairs, which facilitate generation of a binding pocket. The specificity for arginine seems to arise from contacts between the guanidino end of the arginine and phosphates, with atoms positioned by the shape of the pocket. Complex binding kinetics are observed suggesting that there is a slow interconversion of two forms of the DNA, which have different binding affinities. These data provide information on the process of adaptive recognition of a ligand by an aptamer.     

Anti-bovine prion protein RNA aptamer containing tandem GGA repeat interacts both with recombinant bovine prion protein and its �� isoform with high affinity

In order to obtain RNA aptamers against bovine prion protein (bPrP), we carried out in vitro selection from RNA pools containing a 55-nucleotide randomized region to target recombinant bPrP. Most of obtained aptamers contained conserved GGA tandem repeats (GGA)₄ and aptamer #1 (apt #1) showed a high affinity for both bPrP and its β isoform (bPrP-β). The sequence of apt #1 suggested that it would have a G-quadruplex structure, which was confirmed using CD spectra in titration with KCl. A mutagenic study of this conserved region, and competitive assays, showed that the conserved (GGA)₄ sequence is important for specific binding to bPrP and bPrP-β. Following 5′-biotinylation, aptamer #1 specifically detected PrP^c in bovine brain homogenate in a Northwestern blotting assay. Protein deletion mutant analysis showed that the bPrP aptamer binds within 25–131 of the bPrP sequence. Interestingly, the minimized aptamer #1 (17 nt) showed greater binding to bPrP and bPrP-β as compared to apt #1. This minimized form of aptamer #1 may therefore be useful in the detection of bPrP, diagnosis of prion disease, enrichment of bPr and ultimately in gaining a better understanding of prion diseases.Key words: RNA aptamer, prion protein, SELEX, GGA repeat, G-quadruplex     

Development of RNA aptamer that inhibits methyltransferase activity of dengue virus

Objectives

To develop an RNA aptamer specific for the methyltransferase (MTase) of dengue virus (DENV) which is essential for viral genome replication and translation acting directly on N-7 and 2′-O-methylation of the type-I cap structure of the viral RNA.

Results

We identified 2′-fluoro-modified RNA aptamers that can specifically bind DENV serotype 2 (DENV2) MTase using systematic evolution of ligands by exponential enrichment technology. We truncated the chosen aptamer into a 45-mer RNA sequence that can bind DENV2 MTase with K _d  ~ 28 nM and inhibit N-7 methylation activity of the protein. Moreover, the 45-mer truncated aptamer could not only bind with an K _d  ~ 15.6 nM but also inhibit methylation activity of DENV serotype 3 (DENV3) MTase. The 45-mer aptamer competitively impeded binding of both DENV2 and DENV3 genomic RNA to MTase of each serotype.

Conclusion

The selected 45-mer truncated RNA aptamer specifically and avidly bound DENV MTase and competitively inhibited its methylation activity, and thus could be useful for the development of anti-DENV agents.

     

Circular L-RNA aptamer promotes target recognition and controls gene activity

Rational design of aptamers to incorporate unnatural nucleotides and special chemical moieties can expand their functional complexity and diversity. Spiegelmer (L-RNA aptamer) is a unique class of aptamer that is composed of unnatural L-RNA nucleotides, and so far there are limited L-RNA aptamer candidates and applications being reported. Moreover, the target binding properties of current L-RNA aptamers require significant improvement. Here, using L-Apt.4-1c as an example, we develop a simple and robust strategy to generate the first circular L-RNA aptamer, cycL-Apt.4-1c, quantitatively, demonstrate substantial enhancement in binding affinity and selectivity toward its target, and notably report novel applications of circular L-RNA aptamer in controlling RNA–protein interaction, and gene activity including telomerase activity and gene expression. Our approach and findings will be applicable to any L-RNA aptamers and open up a new avenue for diverse applications.     

Stability and binding properties of a modified thrombin binding aptamer

Aptamer-based drugs represent an attractive approach in pharmacological therapy. The most studied aptamer, thrombin binding aptamer (TBA), folds into a well-defined quadruplex structure and binds to its target with good specificity and affinity. Modified aptamers with improved biophysical properties could constitute a new class of therapeutic aptamers. In this study we show that the modified thrombin binding aptamer (mTBA), ^3′GGT^5′-^5′TGGTGTGGTTGG^3′, which also folds into a quadruplex structure, is more stable than its unmodified counterpart and shows a higher thrombin affinity. The stability of the modified aptamer was investigated using differential scanning calorimetry, and the energetics of mTBA and TBA binding to thrombin was characterized by means of isothermal titration calorimetry (ITC). ITC data revealed that TBA/thrombin and mTBA/thrombin binding stoichiometry is 1:2 for both interactions. Structural models of the two complexes of thrombin with TBA and with mTBA were also obtained and subjected to molecular dynamics simulations in explicit water. Analysis of the models led to an improvement of the understanding of the aptamer-thrombin recognition at a molecular level.