Structural determinants of conformationally selective, prion-binding aptamers

We have recently described the isolation of 2'-fluoropyrimidine-substituted RNA aptamers that bind selectively to disease-associated beta-sheet-rich forms of the prion protein, PrP, from a number of mammalian species. These aptamers inhibit the accumulation of protease-resistant forms of PrP in a prion-seeded, in vitro conversion assay. Here we identify the minimal portions of two of these aptamers that retain binding specificity. We determine their secondary structures by a combination of modeling and solution probing. Finally, we identify an internal site for biotinylation of a minimized, synthetic aptamer and use the resultant reagent in the detection of abnormal forms of PrP in vitro.     

DNA aptamers that bind to PrP(C) and not PrP(Sc) show sequence and structure specificity

DNA aptamers were selected against recombinant human (rhu) cellular prion protein (PrP(C)) 23-231 by systematic evolution of ligands via a systematic evolution of ligands by exponential (SELEX) enrichment procedure using lateral flow chromatography. The SELEX procedure was performed with an aptamer library consisting of a randomized 40-nucleotide core flanked by 28-mer primer-binding sites that, theoretically, represented approximately 10(24) distinct nucleic acid species. Sixty nanograms of rhuPrP(C)23-231 immobilized in the center of a lateral flow device was used as the target molecule for SELEX. At the end of 6 iterations of SELEX, 13 distinct candidate aptamers were identified, of which, 3 aptamers represented 32%, 8%, and 5% of the sequences respectively. Eight aptamers, including the three most frequently occurring candidates, were selected for further evaluation. Selected aptamers bound to rhuPrP(C)23-231 at 10(-6) M to 10(-8) M concentrations. Two of the eight aptamers bound at higher concentrations to rhuPrP(C)90-231. Theoretical thermodynamic modeling of selected aptamer sequences identified several common motifs among the selected aptamers that could play a role in PrP binding. Binding affinity to rhuPrP(C)23-231 was both aptamer sequence and structure dependent. Further, selected aptamers bound to mammalian PrPs derived from brain of healthy sheep, calf, piglet, and deer, and to PrP(C) expressed in mouse neuroblastoma cells. None of the aptamers bound to proteinase K-digested scrapie-infected mouse neuroblastoma cells or untreated PrP-null cells, which further confirmed the PrP(C) specificity of the aptamers. In summary, we enriched and selected DNA aptamers that bind specifically to rhuPrP(C) and mammalian PrP(C) with varying affinities and can be applied to biological samples for PrP(C) enrichment and as diagnostic tools in double ligand assay systems.     

RNA aptamers specifically interact with the prion protein PrP.

We have isolated RNA aptamers which are directed against the recombinant Syrian golden hamster prion protein rPrP23-231 (rPrPc) fused to glutathione S-transferase (GST). The aptamers did not recognize the fusion partner GST or the fusion protein GST::rPrP90-231 (rPrP27-30), which lacks 67 amino acids from the PrP N terminus. The aptamer-interacting region of PrPc was mapped to the N-terminal amino acids 23 to 52. Sequence analyses suggest that the RNA aptamers may fold into G-quartet-containing structural elements. Replacement of the G residues in the G quartet scaffold with uridine residues destroyed binding to PrP completely, strongly suggesting that the G quartet motif is essential for PrP recognition. Individual RNA aptamers interact specifically with prion protein in brain homogenates from wild-type mice (C57BL/6), hamsters (Syrian golden), and cattle as shown by supershifts obtained in the presence of anti-PrP antibodies. No interaction was observed with brain homogenates from PrP knockout mice (prn-p(0/0)). Specificity of the aptamer-PrP interaction was further confirmed by binding assays with antisense aptamer RNA or a mutant aptamer in which the guanosine residues in the G tetrad scaffold were replaced by uridine residues. The aptamers did not recognize PrP27-30 in brain homogenates from scrapie-infected mice. RNA aptamers may provide a first milestone in the development of a diagnostic assay for the detection of transmissible spongiform encephalopathies.     

Screening of DNA Aptamer Against Mouse Prion Protein by Competitive Selection

Prion disease is a neurodegenerative disorder, in which the normal prion protein (PrP) changes structurally into an abnormal form and accumulates in the brain. There is a great demand for the development of a viable approach to diagnosis and therapy. Not only has the ligand against PrP been used for diagnosis, but it has also become a promising tool for therapy, as an antibody. Aptamers are a novel type of ligand composed of nucleic acids. DNA aptamers in particular have many advantages over antibodies. Therefore, we tried to isolate the DNA aptamer for mouse PrP. We developed a competitive selection method and tried to screen the DNA aptamer with it. In the fourth round of selection, several clones of the aptamer with an affinity to PrP were enriched, and clone 4–9 showed the highest affinity of all. The investigation by aptamer blotting and Western blotting showed that clone 4–9 was specifically able to recognize both α-PrP and β-PrP. Moreover, it was indicated that clone 4–9 could recognize the flexible region of the N-terminal domain of PrP. These characteristics suggest that clone 4–9 might be a useful tool in prion-disease diagnosis and research.Key Words: aptamer, DNA, SELEX, competitive selection, prion protein, β-PrP, detection     

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.     

Screening of DNA Aptamer Against Mouse Prion Protein by Competitive Selection

Prion disease is a neurodegenerative disorder, in which the normal prion protein (PrP) changes structurally into an abnormal form and accumulates in the brain. There is a great demand for the development of a viable approach to diagnosis and therapy. Not only has the ligand against PrP been used for diagnosis, but it has also become a promising tool for therapy, as an antibody. Aptamers are a novel type of ligand composed of nucleic acids. DNA aptamers in particular have many advantages over antibodies. Therefore, we tried to isolate the DNA aptamer for mouse PrP. We developed a competitive selection method and tried to screen the DNA aptamer with it. In the fourth round of selection, several clones of the aptamer with an affinity to PrP were enriched, and clone 4-9 showed the highest affinity of all. The investigation by aptamer blotting and Western blotting showed that clone 4-9 was specifically able to recognize both α-PrP and β-PrP. Moreover, it was indicated that clone 4-9 could recognize the flexible region of the N-terminal domain of PrP. These characteristics suggest that clone 4-9 might be a useful tool in prion-disease diagnosis and research.     

Detection of prion epitopes on PrP and PrP of transmissible spongiform encephalopathies using specific monoclonal antibodies to PrP

Amino acid residues 90-120 of the prion protein (PrP) are likely to be critical for the conversion of PrP(c) to PrP(sc) in the transmissible spongiform encephalopathies. We raised 10 monoclonal antibodies against the 90-120 amino acid region, mapped the epitope specificity of these anti-PrP antibodies, and investigated the expression of epitopes recognized by the antibodies in both PrP(c) and PrP(sc). Four out of five of the anti-PrP antibodies raised in a prion knockout mouse immunized with the linear peptide of PrP90-120 could detect PrP(sc) in 'native' and denatured forms and PrP(c) in normal cells, as well as recognize epitopes within PrP93-112 residues. In contrast, the other six anti-PrP reagents, including five raised from the two knockout mice immunized with conformationally modified PrP90-120 peptide, could detect PrP(c) and recognize epitopes within PrP93-107 residues. Four of these reagents could also detect denatured PrP(sc) on western blots but not PrP(sc) plaques in brain tissue. The results indicate that residues PrP93-102 are exposed in PrP(c) but are buried upon conversion to the PrP(sc) isoform. Furthermore, PrP103-107 residues are partially buried in PrP(sc) while only the PrP107-112 epitope remains exposed, suggesting that the region PrP93-112 undergoes conformational changes during its conversion to PrP(sc).     

Toward molecular dissection of PrPC-PrPSc interactions

Direct interaction between endogenous cellular prion protein (PrP(C)) and misfolded, disease-associated (PrP(Sc)) conformers is a key event in prion propagation, which precedes templated conversion of PrP(C) into nascent PrP(Sc) and prion infectivity. Although almost none of the molecular details of this pivotal process are understood, the persistence of individual prion strains suggests that assembly of the prion replicative complex is mechanistically precise. To systematically map defined regions of PrP(C) sequence that bind tightly to PrP(Sc), we have generated a comprehensive panel of over 45 motif-grafted antibodies containing overlapping peptide grafts collectively spanning PrP residues 19-231. Grafted antibody binding experiments, performed under stringent conditions, clearly identified only three distinct and independent high affinity PrP(Sc) recognition motifs. The first of these binding motifs lies at the very N-terminal region of the mature PrP molecule within PrP-(23-33); the second motif lies within PrP-(98-110); and the third is contained within PrP-(136-158). Mutational analyses of these PrP(Sc)-binding regions revealed that reactivity of the 23-33 and 98-110 segments are largely dependent upon the presence of multiple positively charged amino acid residues. These studies yield new insight into critical peptidic components composing one side of the prion replicative interface.     

In vitro selection of RNA aptamers that bind to colicin E3 and structurally resemble the decoding site of 16S ribosomal RNA

Colicin E3 is a ribonuclease that specifically cleaves at the site after A1493 of 16S rRNA in Escherichia coli ribosomes, thus inactivating translation. To analyze the interaction between colicin E3 and 16S rRNA, we used in vitro selection to isolate RNA ligands (aptamers) that bind to the C-terminal ribonuclease domain of colicin E3, from a degenerate RNA pool. Although the aptamers were not digested by colicin E3, they specifically bound to the protein (K(d) = 2-14 nM) and prevented the 16S rRNA cleavage by the C-terminal ribonuclease domain. Among these aptamers, aptamer F2-1 has a sequence similar to that of the region around the cleavage site from residue 1484 to 1506, including the decoding site, of E. coli 16S rRNA. The secondary structure of aptamer F2-1 was determined by the base pair covariation among the variants obtained by a second in vitro selection, using a doped RNA pool based on the aptamer F2-1 sequence. The sequence and structural similarities between the aptamers and 16S rRNA provide insights into the recognition of colicin E3 by this specific 16S rRNA region.     

Detection system based on the conformational change in an aptamer and its application to simple bound/free separation

Aptamers are good molecular recognition elements for biosensors. Especially, their conformational change, which is induced by the binding to the target molecule, enables the development of several types of useful detection systems. We applied this property to bound/free separation, which is a crucial process for highly sensitive detection. We designed aptamers which change their conformation upon binding to the target molecule and thereby expose a single-strand bearing the complementary sequence to the capture probe immobilized onto the support. We named the designed aptamers "capturable aptamers" and the capture probe "capture DNA". Three capturable aptamers were designed based on the PrP aptamer, which binds to prion protein. One of these capturable aptamers was demonstrated to recognize prion protein and change its conformation upon binding to it. A detection system using this designed capturable aptamer for prion protein was developed. Capturable aptamers and capture DNA allow us to perform simple bound/free separation with only one target ligand.     

High affinity binding between copper and full-length prion protein identified by two different techniques

The cellular prion protein is known to be a copper-binding protein. Despite the wide range of studies on the copper binding of PrP, there have been no studies to determine the affinity of the protein on both full-length prion protein and under physiological conditions. We have used two techniques, isothermal titration calorimetry and competitive metal capture analysis, to determine the affinity of copper for wild type mouse PrP and a series of mutants. High affinity copper binding by wild type PrP has been confirmed by the independent techniques indicating the presence of specific tight copper binding sites up to femtomolar affinity. Altogether, four high affinity binding sites of between femto- and nanomolar affinities are located within the octameric repeat region of the protein at physiological pH. A fifth copper binding site of lower affinity than those of the octameric repeat region has been detected in full-length protein. Binding to this site is modulated by the histidine at residue 111. Removal of the octameric repeats leads to the enhancement of affinity of this fifth site and a second binding site outside of the repeat region undetected in the wild type protein. High affinity copper binding allows PrP to compete effectively for copper in the extracellular milieu. The copper binding affinities of PrP have been compared with those of proteins of known function and are of magnitudes compatible with an extracellular copper buffer or an enzymatic function such as superoxide dismutase like activity.     

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.     

Preferential Cu2+ coordination by His96 and His111 induces beta-sheet formation in the unstructured amyloidogenic region of the prion protein

The prion protein (PrP) is a Cu(2+) binding cell surface glycoprotein that can misfold into a beta-sheet-rich conformation to cause prion diseases. The majority of copper binding studies have concentrated on the octarepeat region of PrP. However, using a range of spectroscopic techniques, we show that copper binds preferentially to an unstructured region of PrP between residues 90 and 115, outside of the octarepeat domain. Comparison of recombinant PrP with PrP-(91-115) indicates that this prion fragment is a good model for Cu(2+) binding to the full-length protein. In contrast to previous reports we show that Cu(2+) binds to this region of PrP with a nanomolar dissociation constant. NMR and EPR spectroscopy indicate a square-planar or square-pyramidal Cu(2+) coordination utilizing histidine residues. Studies with PrP analogues show that the high affinity site requires both His(96) and His(111) as Cu(2+) ligands, rather than a complex centered on His(96) as has been previously suggested. Our circular dichroism studies indicate a loss of irregular structure on copper coordination with an increase in beta-sheet conformation. It has been shown that this unstructured region, between residues 90 and 120, is vital for prion propagation and different strains of prion disease have been linked with copper binding. The role of Cu(2+) in prion misfolding and disease must now be re-evaluated in the light of these findings.     

Anti-bovine Prion protein RNA aptamer containing tandem GGA repeat interacts both with recombinant bovine prion protein and its β 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 PrPc 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 (17nt) 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 bPrP, and ultimately in gaining a better understanding of prion diseases.     

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     

Antibodies to the scrapie protein decorate prion rods

Scrapie is a degenerative, transmissible neurologic disease of sheep and goats which occurs in the absence of any detectable host immune response. Antibodies to the scrapie agent have been produced after immunization of rabbits with either scrapie prions or the prion protein, PrP 27-30, purified from infected hamster brain. Immunoreactivity of the antisera was assessed by dot and Western immunoblots with purified prions and PrP 27-30. Antibodies raised against infectious prions were more immunoreactive with native than denatured preparations, whereas those raised against PrP 27-30 were more reactive with denatured prion preparations. As determined by second antibody-colloidal gold, both antisera were found to decorate scrapie prion rods in purified preparations. Antibodies to cellular filamentous proteins failed to react with PrP 27-30 or the scrapie prion rods; conversely, antibodies to PrP 27-30 did not exhibit immunoreactivity with cellular filamentous proteins. The monospecificity of the rabbit antiserum raised against PrP 27-30 was established by its reactivity after affinity purification. The purified antibodies reacted with PrP 27-30 on Western blots and with the prion rods. Considerable evidence indicates that the scrapie rods are aggregates of infectious prions; the findings presented here provide an immunologic demonstration that PrP 27-30 is a structural component of the prion rods.     

Use of magnetic beads in selection and detection of biotoxin aptamers by electrochemiluminescence and enzymatic methods.

Systematic evolution of ligands by exponential enrichment (SELEX) was used to develop DNA ligands (aptamers) to cholera whole toxin and staphylococcal enterotoxin B (SEB). Affinity selection of aptamers was accomplished by conjugating the biotoxins to tosyl-activated magnetic beads. The use of magnetic beads reduces the volumes needed to perform aptamer selection, thus obviating alcohol precipitation and allowing direct PCR amplification from the bead surface. Following five rounds of SELEX, 5'-biotinylated aptamers were bound to streptavidin-coated magnetic beads and used for the detection of ruthenium trisbypyridine [Ru(bpy)3(2+)]-labeled cholera toxin and SEB by an electrochemiluminescence methodology. A comparison of control (double-stranded) aptamer binding was made with aptamers that were heat denatured at 96 degrees C (single-stranded) and allowed to cool (conform) in the presence of biotoxin-conjugated magnetic beads. Results suggest that control aptamers performed equally well when compared to heat-denatured DNA aptamers in the cholera toxin electrochemiluminescence assay and a colorimetric microplate assay employing peroxidase-labeled cholera toxin and 5'-amino terminated aptamers conjugated to N-oxysuccinimide-activated microtiter wells. Interestingly, however, in the SEB electrochemiluminescence assay, double-stranded aptamers exceeded the performance of single-stranded aptamers. The detection limits of all aptamer assays were in the low nanogram to low picogram ranges.     

Thioaptamer interactions with prion proteins: sequence-specific and non-specific binding sites

Binding of nucleic acids to the prion protein (PrP) created a conundrum that required distinguishing between non-specific interactions and biologically important polynucleotides. In the process of developing selective ligands for PrP, we found using a single-stranded DNA thioaptamer library that the binding of thioaptamers to PrP occurs on at least two different sites on the protein. Selection against recombinant (rec) PrP of Syrian hamster (SHa) sequence 90-231 folded into an alpha-helical-rich conformation identified a 12-base consensus sequence within a series of 20 thioaptamers, all of which consist of 40 bases. Each thioaptamer was comprised of both normal and thio-dA modified bases. One thioaptamer designated 97 bound to recSHaPrP with affinity of 0.58(+/-0.1) nM; lower affinities for bovine (Bo), and human (Hu) were found, establishing that binding is dependent on the primary structure of PrP. High affinity binding of thioaptamer 97 to PrP was found to be mediated through the dodecyl sequence GACACAAGCCGA within the consensus region with five critical backbone modifications 5' to each dA residue. A control oligonucleotide with an equivalent number of phosphorothioates to thioaptamer 97 and a scrambled consensus sequence could not distinguish among the three PrP sequences. Control oligonucleotides bearing non-selected sequences bound to PrP at a sequence-independent DNA-binding site. In contrast, the high-affinity binding of thioaptamer 97 to PrP depends on (1) backbone modifications, (2) oligonucleotide sequence, and (3) PrP sequence.     

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.     

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.