JBC Transcription and Nuclear Factor Monoclonals

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M310928200 on January 7, 2004

J. Biol. Chem., Vol. 279, Issue 13, 13102-13109, March 26, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
279/13/13102    most recent
M310928200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sayer, N. M.
Right arrow Articles by James, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sayer, N. M.
Right arrow Articles by James, W.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Structural Determinants of Conformationally Selective, Prion-binding Aptamers*

Natalie M. Sayer{ddagger}, Matthew Cubin, Alexandre Rhie, Marc Bullock, Abdessamad Tahiri-Alaoui§, and William James

From the Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom

Received for publication, October 3, 2003 , and in revised form, November 21, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transmissible spongiform encephalopathies (TSEs)1 involve spongiform degeneration and astrocyte gliosis in the central nervous system leading to dementia and death (1). They can have genetic, sporadic, or infectious etiologies, exemplified by fatal familial insomnia, sporadic Creutzfeldt-Jakob disease, and variant Creutzfeldt-Jakob disease in humans, respectively. They all involve the modification of prion precursor protein PrPc, which is constitutively expressed in mammalian cells, particularly neurons, as a GPI-anchored plasma membrane glycoprotein. During many TSE diseases, infectivity is found to be associated with amyloid deposits of PrP, in a form referred to as PrPSc (2), and lack of a gene for PrP renders animals insusceptible to prion diseases (3, 4). These and related observations support the prion theory of TSE etiology, in which self-templated conformational changes in PrP are necessary and sufficient for disease progression and transmission (5). The encipherment of the pathological properties of TSEs within the conformation of PrP is not fully understood, but whereas the membrane-proximal domain of PrPc is mainly {alpha}-helical in structure, PrPsc is dominated by {beta}-sheets (6). In contrast to PrPC, PrPsc is insoluble in nondenaturing conditions and is partially resistant to proteases (7).

Diagnostic tests for the in vivo identification of prions are not yet ideal. Disease cannot be confirmed until clinical signs appear or even until a post mortem of brain tissue shows spongiform changes and prion accumulation using immunohistochemical methods. Only recently has it been possible to isolate antibodies that are able to differentiate between PrPc and PrPsc (8), so current methods rely on a pretreatment with protease, which may obscure the presence of relevant, protease-sensitive conformations. The most promising of diagnostic tests have been using fluorescence, such as multi-spectral ultraviolet fluorescence looking at specific fluorescence of proteins when excited by UV radiation (9) and fluorescence correlation spectroscopy, which recognizes single fluorescent molecules in solution as they pass between the exciting laser beam and the objective of a confocal microscope (10). Another interesting approach by Safar et al. (11) is that of a conformation-dependent immunoassay; this uniquely quantifies PrP isoforms by the binding pattern of different antibodies to both denatured and native forms. There has been some evidence that PrPsc can be detected in the blood of infected rodents (12, 13), as well as in the urine of scrapie-infected hamsters (14). The amount of PrPsc outside the central nervous system is very small compared with that in the brain, so it might be necessary to amplify the small amount there using, for example, the cyclic PrPsc amplification process (15). For a review of these and other diagnostic assays see Ref. 16.

Given the limitations of antibodies, aptamers provide an alternative approach to the ligand-based detection of PrPsc. Aptamers are nucleic acids isolated in vitro for their ability to bind a molecule of interest by the SELEX procedure (17). This involves cycles of binding and amplification starting from about 1014 different nucleic acid molecules, which eventually results in the isolation of a few sequences that have good affinity for targets such as CD4 (18), streptavidin (19), and HIV-1 gp120 (20, 21). Protein binding by aptamers results mainly from hydrogen bonds and electrostatic forces, which means that the best targets are often basic, positively charged molecules. Aptamers can have a subnanomolar affinity and a high specificity (for a review see Ref. 22), Because of the number of different potential ligands that can be screened by the SELEX process, aptamers have often been raised against molecules when other approaches have failed. They have many advantages over antibodies in that they are nonimmunogenic, smaller, and usually of a better affinity and can be chemically synthesized. This, together with their contrasting physico-chemical properties to antibodies, makes aptamers potentially useful reagents for the detection of previously overlooked conformations of PrP. We have recently described the isolation of 2'-fluoropyrimidine-substituted RNA aptamers that were selected from randomized libraries using infectious prions as the target material (23). One of these aptamers, SAF-93 was shown to bind to two regions of PrP: (i) a conformationally insensitive, diffuse region lying between residues 23 and 110 that binds nonspecifically to nucleic acids and (ii) a site lying between residues 110 and 230. The latter site was occluded in the normal {alpha}-helix-rich conformation of the protein but exposed in the disease-associated {beta}-sheet-rich forms and was not bound by control nucleic acids. SAF-93 also inhibited the conversion of {alpha} helix-rich to {beta} sheet-rich forms in a prion-nucleated, in vitro conversion assay (23). SAF-93 is therefore an interesting ligand for PrP and may reveal conformations of PrP not yet identified. Before the properties of this and another aptamer, SAF-76 (also described in this paper), could be exploited, more information was required about the structural features essential for their binding to prions. In this paper, we present the results of experiments designed to facilitate the use of prion-binding aptamers as molecular reagents for the investigation of prion diseases. Firstly we determined the solution structure of the aptamers and mapped the binding motifs onto the determined structure. We then characterized the minimal structural features that were responsible for specific binding and designed a novel method for determining a residue within the essential structure of the best aptamer that could be modified without affecting its affinity for PrP. With this information, we went on to synthesize modified aptamers for a range of analytical and diagnostic applications.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Protein Preparation and Refolding—Full-length bovine PrP, corresponding to residues 23-230 (human numbering) was cloned, expressed, and purified as described before (23). Briefly, the protein was expressed in Escherichia coli and purified from inclusion bodies by nickel-nitrilotriacetic acid affinity chromatography and reversed phase high pressure liquid chromatography. The purified PrP was refolded into native {alpha}-helical conformation or {beta}-oligomeric isoform following the protocol described in Ref. 24. Finally, the monomeric {alpha}-helical rich form and {beta}-oligomer isoform of PrP were purified by high performance size exclusion chromatography (23). The protein structures were checked using CD, as previously described (23).

In Vitro Transcription—Transcription of 2'-fluoropyrimidine-containing RNA was carried out largely as described in Ref. 25. Briefly, transcription by T7 RNA polymerase (New England Biolabs) was incubated at 37 °C for at least 6 h in 40 mM Tris, pH 8.1, 6 mM magnesium chloride, 1 mM spermidine, 5 mM dithiothreitol, and 1 mM NTPs. The products were then treated for 30 min at 37 °C with 50 units of DNase 1 (Roche Applied Science) in 150 mM sodium acetate, pH 5.2, 10 mM magnesium chloride. The reaction was stopped by phenol extraction, and the transcript was recovered by ethanol precipitation.

32P End Labeling of RNA—For 5' labeling, the terminal 5' phosphate was removed using bacterial alkaline phosphatase (New England Bio-labs) and replaced with [32P]phosphate from [{gamma}-32P]ATP using T4 polynucleotide kinase (Roche Applied Science). Labeling at the 3' end was achieved by ligating [32P]pCp to the 3' end of the aptamer using T4 RNA ligase (New England Biolabs). Ligation was carried out for 2 h at 37 °C. The labeled RNAs were electrophoresed on 12% polyacrylamide, 8 M urea gels, visualized by autoradiography, and recovered by passive elution from gel slices.

Identification of the Core Binding Sequence Using Boundary Experiments—A library of nested deletion fragments was generated from either 3' or 5' end-labeled aptamer by mild alkaline hydrolysis (26) and partitioned into PrP-binding and nonbinding fractions using Strata-CleanTM (Stratagene), as described before (20). The products were analyzed by denaturing 18% PAGE.

Enzymatic Probing and Footprinting—32P end-labeled RNA was digested in 1x HMNK, pH 7.2, buffer (20 mM HEPES, pH 7.2, 100 mM sodium chloride, 50 mM potassium chloride, 5 mM magnesium chloride, 0.04% Nonidet P-40) containing 1 µg of tRNA at 20 °C for 5 min with either nuclease V1 (Pierce; 5 x 10-3 unit) or S1 (Amersham Biosciences; 0.05 unit). Footprinting was achieved by incubating an identical aptamer preparation with prion protein for 1 h at 25 °C and then subjecting the mixture to appropriate nuclease digestion. The digestions were terminated by phenol extraction. The RNA was precipitated using ethanol, then dissolved in formamide buffer, and analyzed by denaturing PAGE (see below).

Urea-denaturing PAGE of 32P-Labeled RNA—All of the samples for truncation and footprinting analysis were heated to 95 °C for 3 min in formamide loading buffer and run on 40-cm-long 18% or 20% polyacrylamide, 8 M urea gels at 25 watts for 3 h. A partial alkaline hydrolysate of the RNA along with a G residue ladder was run alongside analysis samples to aid sequence identification. The G residue ladder was generated by digestion of 50,000 cpm (Cerenkov) denatured RNA at 55 °C in 10 µl of 20 mM sodium citrate, 1 mM EDTA, 7 M urea, pH 4.6, with RNase T1 (Amersham Biosciences; 5 x 10-3 units).

Binding Affinity of Aptamers—End-labeled aptamer (5000 cpm; Cerenkov) was heat-denatured at 95 °C for 2 min in water, cooled to room temperature, and refolded in 1x AMNK buffer (20 mM sodium acetate, pH 5.2, 100 mM sodium chloride, 50 mM potassium chloride, 5 mM magnesium chloride, 0.04% Nonidet P-40) for 10 min. After refolding, the aptamer was incubated with different concentrations of prion protein in the presence of 1 µg of tRNA in 30 µl for 1 h at 25 °C. The bound RNA was then separated from unbound using 0.1 volume of StrataCleanTM resin and centrifuged at 13,000 rpm for 3 min, and the supernatant (containing unbound RNA) was removed. The resin was washed with 30 µl of 1x AMNK buffer and again centrifuged (the wash was pooled with the unbound RNA). The resin (complexed with PrP-bound RNA) was resuspended in 60 µl of 1x AMNK buffer, and the amounts of radioactivity corresponding to PrP-bound and unbound RNA were measured for each concentration of protein. The data were fitted using nonlinear curve fitting to a hyperbolic equation by Graph Pad Prism.

Identification of Sites at Which Incorporation of Biotin Does Not Interfere with PrP Binding—Transcription of aptamer was done as described above, with the addition of 2 µM biotin-16-dUTP (Roche Applied Science). After completion, the transcription was dialyzed against water for 3 h instead of phenol extraction to remove nonincorporated 5-allyl biotinyl UTP. To isolate molecules that could bind a single streptavidin tetramer, the dephosphorylated, biotinylated RNA was incubated for 30 min with 30 µl of streptavidin (2 mg/ml). The complex was then run on an 8% Tris-borate native polyacrylamide gel, and the band corresponding to aptamer bound to one tetramer of streptavidin was cut out, passively eluted from the gel, and ethanol-precipitated. The gel-purified and biotinylated RNA was then 5' end radiolabeled as described above.

The double-labeled RNA was ethanol-precipitated and complexed with streptavidin as before. The complex was then incubated with 40 µl of {beta}-form PrP90-230 (1 mg/ml) for 30 min at 25 °C. The ternary complex (PrP-aptamer-streptavidin) was then partitioned from the binary complex (aptamer-streptavidin) using StrataCleanTM resin (to which streptavidin does not bind). RNA was recovered from both fractions, partially hydrolyzed using alkali, and analyzed by urea-denaturing PAGE, as described above, along with a transcript without biotin incorporated as a control.

Slot Blots Using Chemically Synthesized Biotinylated Aptamer—30 pmol of full-length PrP was bound to a polyvinylidene difluoride membrane (equilibrated in 0.8% (v/v) acetic acid, 350 mM {beta} alanine) by applying a vacuum. The membrane with protein attached was then washed with Tris-buffered saline-Tween and 10 mM magnesium chloride and then prehybridized in the same buffer with the addition of 5% milk powder and 20 µg/ml tRNA for 1 h. The membrane was then incubated overnight in 20 pmol of SAF-93(1-34,35bU,36-60), (chemically synthesized and biotin derivitized at position 35) that had been refolded in 1x HMNK and added to 20 ml of Tris-buffered saline-Tween, 10 mM magnesium chloride, 5% milk powder, 20 µg/ml tRNA. The membrane was blocked again for 1 h, washed, incubated in presence of streptavidin alkaline phosphatase (0.25 µg/ml) (Sigma) for 30 min, washed, and then developed using enzyme chemifluorescence substrate as described before. All of the incubations were carried out at 25 °C.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Primary and Secondary Structure Determination of SAF-76 and SAF93—To elucidate their secondary structures, SAF-76 and SAF-93 were transcribed in vitro, purified, labeled at the 5' end with 32P, and refolded. They were then subjected to limiting digestion using the structure-sensitive nucleases S1 (preference for single-stranded regions) and V1 (preference for double-stranded regions) (27) and analyzed using denaturing PAGE, as described (see "Experimental Procedures"). The results (see supplemental figures) were used to constrain the MFOLD helix prediction algorithm (28) and to eliminate the suboptimal structures that were inconsistent with experimentally determined structural features. The most empirically compliant structures are illustrated in Fig. 1 (A and B). It must be noted that RNA structures are often dynamic (29). Aptamers, which have been selected for their ability to bind to a target protein with good affinity, may not always be fully structured in the absence of protein. For this reason, even though multiple repetitions of these experiments gave entirely consistent results, we cannot be categorical that these putative secondary structures are as well determined as, say, those of tRNAs.



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 1.
Minimal primary and secondary structure of PrP-binding aptamers. Aptamers SAF-76 (A and C) and SAF-93 (B and D) were subjected to secondary structure analysis and 3' boundary determination. To determine secondary structures (A and B), 5'-labeled RNA was probed with the structure-sensitive nucleases, V1, S1, and T1, and analyzed by denaturing PAGE (see supplemental figures). The results were used to constrain the MFOLD helix prediction algorithm (28) and to eliminate the suboptimal structures that were inconsistent with experimentally determined structural features. The most empirically compliant structures are illustrated, and residues found to be cut with V1 are shown in blue, and those cut by S1 are in red. To determine the 3' boundary of the minimal PrP-binding sequence (C and D), a partial hydrolysate of 5'-labeled full-length aptamer was partitioned into PrP-binding and non-PrP-binding fractions and was analyzed by denaturing PAGE together with a T1 digest (G ladder) and a sample of unfractionated hydrolysate (purine ladder). The arrows indicate the 3' boundary inferred from the gel and relate this to the corresponding portion of the appropriate secondary structure.

 
With these caveats, the aptamers appear to fold into two and three helical domains, respectively, separated by extended, nonhelical regions. That is, SAF-76 appears to comprise two domains consisting of helices 1, 2, and 3 and of helices 4, 5, and 6, respectively, and SAF-93 appears to comprise three domains consisting of helix 1, of helices 2, 3, and 4, and of helix 5, respectively. It seemed very possible that only one of these domains in each case was actually necessary for interaction with PrP, particularly because the aptamers are relatively long (117 and 116 nucleotides, respectively). Accordingly, we determined the 3' boundary of the PrP-binding regions of each by separating a partial hydrolysate of 5'-labeled full-length aptamer into PrP-binding and non-PrP-binding fractions and analyzing each by urea-denaturing PAGE (Fig. 1, C and D).

In each case, an approximate 3' boundary could be identified as the point at which the intensity of fragments in the lane of nonbinding RNA became greater than that in the lane of PrP-binding RNA, as indicated in Fig. 1. Again, it should be noted that even very short fragments retain some binding to PrP, probably through the nonspecific interaction with the N-terminal tail of the protein. This results in a slight blurring of the boundary between the essential and inessential regions of aptamer. Nevertheless, in both cases, it seemed possible to eliminate a substantial 3' portion of the aptamer without loss of PrP binding. When the 3' boundaries were placed on the secondary structure previously determined, a paradox immediately presented itself in both cases. In SAF-76 helix 1 was split by the 3' boundary, and in SAF-93 helix 2 was split by the 3' boundary. That is, in each case, only one-half of a predicted helix remained in the 3'-truncated, PrP-binding aptamer.

Three explanations seemed possible. First, the actual secondary structures might be very different from those presented. This could result from a large number of noncanonical, tertiary interactions stabilizing much weaker canonical helices than had been considered by the MFOLD algorithm. Second, the truncated aptamer might still bind to PrP but with much reduced affinity, as a result of the loss of one helix. Third, the truncated aptamer might fold into an altogether different structure from its full-length parent. To resolve these possibilities, we used the primary and secondary structure information to generate defined, truncated aptamers and analyzed both their affinity for PrP and their secondary structures.

Structure and Affinity of Truncated Aptamers—The double-stranded DNA template used to transcribe the parental aptamers in vitro was amplified using the standard 5' SELEX primer together with a 3' primer corresponding to the 3' boundary determined above. The parental aptamers and their truncated derivatives are hereinafter named to indicate the nucleotides retained from the parent.

These truncated derivatives were then analyzed by enzymatic probing as described previously. Again the experiments were repeated a number of times to ensure that the cleavages shown were consistent. SAF-76(1-38) showed anomalous nuclease sensitivity when compared with its parent, SAF-76(1-117) (Fig. 2A). Nucleotides that had formerly appeared to be single-stranded were now sensitive to nuclease V1, whereas those previously showing signs of double-strandedness were now sensitive to nuclease S1, making the parent-like structure shown in Fig. 2A untenable. When we constrained the modeling of SAF-76(1-38) by MFOLD with the new data, the structure shown in Fig. 2B emerged. Intriguingly, this contains secondary structural features found within the 5' region of SAF-93, with which it shows some sequence homology. When we compared the affinity of the truncated aptamer with its parent (Fig. 2C), it appeared that removal of the 3' portion reduced the affinity of SAF-76 for {alpha}-form PrP but maintained its affinity for {beta}-form PrP. This is consistent with the idea that binding to the {alpha}-form is dominated by nonspecific nucleic acid interactions in the unstructured N terminus of PrP, which would lessen as the length of the RNA decreased. Although the conformational specificity of SAF-76(1-38) to {beta}-oligomeric form PrP was improved compared with the full-length aptamer, its affinity was still weaker than that of aptamer SAF-93.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 2.
Structures and binding properties of truncated PrP aptamers. Truncated aptamers were designed using the information from the boundary experiments (Fig. 2). Subscript figures indicate the parental aptamer nucleotides retained in the truncated aptamer. Secondary structures (A, B, and D) were determined by a combination of enzymatic probing and computer modeling as described. Residues sensitive to V1 are shown in blue, and those to S1 are in red. The affinity for both the {beta}- and {alpha}-forms of PrP of parental and truncated derivatives of aptamer SAF-76 (C) and SAF-93 (E) was determined by equilibrium binding, as described under "Experimental Procedures." Briefly, 5'-labeled aptamer was incubated equilibrated with a range of concentrations of PrP and left, and protein-bound RNA was separated from unbound using StrataCleanTM.

 
We constructed three deletion mutants of SAF-93. The first, SAF-93(1-60), was based on the 3' boundary determined above. The second, SAF-93(3G,28-60), was, in addition, truncated at the 5', in accordance with indications from a 5' boundary experiment (data not shown), thereby removing helix 1. The third, SAF-93(1-33,43-45,48-51), was designed to preserve helix 1, shorten helix 3, and remove sequences corresponding to the "half-helix" 2. Nuclease sensitivity experiments, together with computer modeling, indicated that the secondary structure of each of these truncated aptamers was as anticipated (Fig. 2D). Interestingly, nucleotides 31 and 32 of SAF-93(1-60), which had appeared to be in helix 2 of the full-length SAF-93(1-116), were still sensitive to nuclease V1 in the truncated version, in which helix 2 could not form. This is strong evidence for stable, noncanonical interactions between this region and some other portion within the 5' half of SAF-93.

When we measured the affinity of the truncated versions of SAF-93 for both conformations of PrP, we found that removal of the 3' half to generate SAF-93(1-60) had little effect on the binding to the {alpha}-form while slightly improving the affinity for the {beta}-oligomeric form (Fig. 2E). The shortening of helix 3 and removal of region 2 in SAF-93(1-33,43-45,48-51), reduced the binding affinity for the {beta}- oligomeric form of PrP. However, destruction of helix 1 in SAF-93(3G,28-60) and disruption of region 2, comprising presumptively tertiary structure, effectively destroyed the specificity of binding to {beta}-form PrP. These results correlate with the footprinting studies on SAF-93(1-60), which showed that much of the sequence was protected by binding to {beta}-form PrP (see supplemental figures). Taken together, the results show that SAF-93(1-60) defines a minimal, conformationally selective aptamer for PrP and comprises two helices bounding a region of tertiary structure.

Generation of an Internally Derivitized Aptamer for PrP Detection—One consequence of the minimization procedure above was that relatively little of SAF-93(1-60) was free to interact with bulky molecules such as streptavidin once it was bound to {beta}-form PrP, as indicated by the footprinting results (see supplemental figures). This was disadvantageous in many potential applications of aptamer technology. Indeed, introduction of a biotin at the 3' end, the most convenient position for site-specific, post-transcriptional incorporation, resulted in a ligand that, when complexed with streptavidin, had very poor PrP binding characteristics (data not shown). Consequently, we undertook a systematic search for internal uracils that could be so derivitized without detriment.

We exploited the fact that all of the uracils in SAF-93(1-60) were part of the nonhydrolyzable nucleotide, 2'-fluoro-2'-deoxyuridine. This means that, in the unmodified SAF-93(1-60), a partial alkaline hydrolysate of 5'-labeled aptamer gives a purine ladder, and bands corresponding to U are absent. On the other hand, biotinylated uracil was associated with 2' OH 5-allyl-biotinyl uridine, which is readily cleaved by alkali, producing an additional band at any U position into which it is incorporated. We spiked transcription of SAF-93(1-60) with 2 µM 5-allyl-biotinyl UTP to produce an average incorporation rate of one biotinylated uracil/molecule, presumably at random, at the nine available positions. We then complexed the partially biotinylated RNA with streptavidin (SA) and purified the RNA that was bound to one SA tetramer from that which bound none, two, or more molecules of SA. We then incubated the SA-aptamer complex with PrP and partitioned the PrP-binding complexes using the resin StrataCleanTM, which fortuitously binds PrP but not SA. We subjected the PrP-binding and nonbinding complexes to partial alkaline hydrolysis and analyzed them on denaturing PAGE (Fig. 3, A and B).



View larger version (99K):
[in this window]
[in a new window]
  		FIG. 3.
PrP footprinting and internal biotinylation of SAF-93(1-60). The secondary structure prediction consistent with both enzymatic probing and footprinting results is presented for SAF 93(1-60). Nucleotides showing a PrP footprint are in red. Experiments were then carried out on this aptamer to find a residue that could be substituted with a biotin without affecting affinity. All of the uracils and cytosines in SAF-93(1-60) are modified with fluoro at the 2' hydroxyl. This means that, in the parent SAF-93(1-60), a partial alkaline hydrolysate of 5'-labeled aptamer gives a purine ladder, and bands corresponding to U are absent. Therefore if a biotinylated uracil is incorporated with 2' OH, this will be readily cleaved by alkali, producing an additional band at any U position into which it is incorporated. SAF-93(1-60) was transcribed under conditions where only one biotinylated uracil was incorporated, 5'-radiolabeled, and incubated with streptavidin (SA), and the complex of RNA binding one tetramer was isolated. The SA-aptamer complex was then incubated with PrP, and the ternary complexes comprising SA-aptamer-PrP were partitioned from the remaining PrP-unbound SA-aptamer using StrataCleanTM. A shows denaturing PAGE analysis of a partial alkaline hydrolysate of control SAF-93(1-60) along with the biotinylated SAF-93(1-60) isolated from the SA-aptamer-PrP complexes. B shows a magnification of the same gels. The lines indicate corresponding residues in the aptamer. The position of U35, at which a biotin appears to be incorporated in PrP-binding aptamers, is indicated with a blue asterisk. C shows a representation of the secondary structure of SAF-93(1-60) in which U35 is highlighted in blue, and those residues found to protected by PrP in footprinting experiments (see supplemental figures) are shown in red.

 
The results show that the most 5'-proximal uracil that can be derivitized with biotin-streptavidin while retaining its PrP binding properties is U35. In the 5' region of U35, the hydrolysis ladder is identical in both the biotin-SA-PrP-binding sample and the control sample. However, in the 3' end of U35, there is an additional cleavage event in the biotin-SA-PrP-binding sample, the bands migrate more slowly (by approximately one nucleotide equivalent) and are less distinct than in the control sample. This is consistent with the incorporation of an additional 487 Da (corresponding to allyl-biotin) and the replacement of 2'-fluoro with 2'OH at position 35. Because of the resolution of the gel, it is not possible to exclude the possibility that uracils 3' to U35 could also be derivitized in this way without loss of function.

To test whether our interpretation of this experiment was correct, we chemically synthesized an aptamer corresponding to the sequence identified above, namely SAF-93(1-34,35bioU,36-60) and determined its affinity for the two isoforms of PrP when complexed with streptavidin. The results show that, as anticipated, the affinity of the aptamer for {beta}-form PrP was the same in the absence as in the presence of streptavidin: Kd = 8.4 ± 1.4 and 6.8 ± 2.9 nM, respectively (Fig. 4A).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 4.
The PrP binding properties of a synthetic internally biotinylated, truncated derivative of aptamer SAF-93. A, the Kd of SAF-93(1-34,35bU,36-60) for the {beta} form of PrP in the presence ({blacksquare}) and absence ({blacktriangleup}) of streptavidin was determined by equilibrium binding assays to be 8.4 ± 1.4 and 6.8 ± 2.9 nM, respectively. B shows a slot blot assay in which 30 pmol of the {beta}- and {alpha}-forms of PrP and of human serum albumin (HSA) were applied in duplicate to a polyvinylidene difluoride membrane and then probed with SAF-93(1-34,35bU,36-60). The blots were processed with streptavidin-alkaline phosphatase and ECF substrate and imaged using a STORM 840 system.

 
This aptamer was then used to probe slot blots of PrP in either the {beta}-oligomeric or {alpha}-monomeric conformation (Fig. 4B). The aptamer detected the {beta}-form much more effectively than the {alpha}-form and did not bind to an equivalent amount of human serum albumin.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have recently described aptamers that bind a region of PrP that is intimately involved in the conformational shifts that occur in the generation of prions and can inhibit conversion in cell-free assays systems (23). Although the original aptamers had many promising features, for example, their resistance to degradation conferred by 2'-fluoro substitution, they were not ideally suited to analytical or therapeutic applications because of their length and complexity. Accordingly, we have used a range of experimental and computational tools to define the essential functional structures of two prion-binding aptamers.

By analyzing the functional primary and secondary structure of the original 116-nucleotide-long aptamer using a range of techniques, we have been able to preserve its conformational specificity while reducing it to a size suitable for chemical synthesis. Moreover, using a new method for screening internal modifications, we have been able to derivitize it with biotin to generate a ligand that can readily be applied in multiple detection formats. The synthetic aptamer described here (SAF-93(1-34,35bioU,36-60)) is a novel and promising tool for the investigation of PrP isoforms in vivo.

For example, it could be used as a detection reagent in slot or dot blotting applications, as shown here. Specifically, one could envisage applying multiple samples of blood, cerebro-spinal fluid, brain homogenate, or other biological materials in parallel to a polyvinylidene difluoride or similar support membrane. SAF-93(1-34,35bioU,36-60) could then be used to develop the membrane, followed by detection using enzyme-conjugated streptavidin and chemoluminescent, chemofluorescent, or chromogenic imaging. Perhaps more conveniently, high throughput plate format assays could be developed in which aptamer-derivitized assay plates are used to capture abnormal PrP from biological samples. Detection of the captured PrP could again by through the use of SAF-93(1-34,35bioU,36-60) and any of the proprietary streptavidin-based systems.

More biologically informative uses include the replacement of anti-PrP antibodies with SAF-93(1-34,35bioU,36-60) in histochemical analysis of biopsy and post mortem samples of experimentally infected model animals or suspect cases. Certain investigations that are not possible using currently available antibodies might become possible using the aptamer. For example, the specificity of SAF-93(1-34,35bioU,36-60) for abnormally folded PrP does not depend on the resistance of the protein to proteinase K. This means that putative forms of PrP that are abnormally folded and either infectious or pathogenic but not fully resistant to PK could be sought directly. Moreover, because the selective reactivity of abnormally folded PrP for aptamer is expressed under native conditions and does not require the presence of denaturants, as is commonly the case with antibody-based systems, the aptamer approach opens the way for a simpler exploration of the appearance of infectious material in vivo under conditions in which classic PrPSc is not presently detectable.


   FOOTNOTES
 
* This work was funded by a grant from the Biotechnology and Biological Sciences Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains supplemental figures. Back

{ddagger} Supported by a studentship from the Medical Research Council of Great Britain and supported by the Cellular Immunology Unit Hybridoma Fund and Brasenose College (Oxford). Back

§ Supported by the Biotechnology and Biological Research Council. Back

Fellow of Brasenose College (Oxford). To whom correspondence should be addressed. Tel.: 44-1865-275548; Fax: 44-1865-285756; E-mail: William.james{at}path.ox.ac.uk.

1 The abbreviations used are: TSE, transmissible spongiform encephalopathy; SA, streptavidin. Back


   ACKNOWLEDGMENTS
 
We thank James Hope for advice and constructive discussion at early stages of this work and Andrew Gill for help with protein characterization.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hur, K., Kim, J. I., Choi, S. I., Choi, E. K., Carp, R. I., and Kim, Y. S. (2002) Mech. Ageing Dev. 123, 1637-1647[CrossRef][Medline] [Order article via Infotrieve]
  2. Prusiner, S. B., McKinley, M. P., Bowman, K. A., Bolton, D. C., Bendheim, P. E., Groth, D. F., and Glenner, G. G. (1983) Cell 35, 349-358[CrossRef][Medline] [Order article via Infotrieve]
  3. Prusiner, S. B., Groth, D., Serban, A., Koehler, R., Foster, D., Torchia, M., Burton, D., Yang, S. L., and DeArmond, S. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10608-10612[Abstract/Free Full Text]
  4. Bueler, H., Aguzzi, A., Sailer, A., Greiner, R. A., Autenried, P., Aguet, M., and Weissmann, C. (1993) Cell 73, 1339-1347[CrossRef][Medline] [Order article via Infotrieve]
  5. Prusiner, S. B. (1982) Science 216, 136-144[Abstract/Free Full Text]
  6. Pan, K. M., Baldwin, M., Nguyen, J., Gasset, M., Serban, A., Groth, D., Mehlhorn, I., Huang, Z., Fletterick, R. J., Cohen, F. E., and Prusiner, S. B. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10962-10966[Abstract/Free Full Text]
  7. Prusiner, S. B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13363-13383[Abstract/Free Full Text]
  8. Paramithiotis, E., Pinard, M., Lawton, T., LaBoissiere, S., Leathers, V. L., Zou, W. Q., Estey, L. A., Lamontagne, J., Lehto, M. T., Kondejewski, L. H., Francoeur, G. P., Papadopoulos, M., Haghighat, A., Spatz, S. J., Head, M., Will, R., Ironside, J., O'Rourke, K., Tonelli, Q., Ledebur, H. C., Chakrabartty, A., and Cashman, N. R. (2003) Nat. Med. 9, 893-899[CrossRef][Medline] [Order article via Infotrieve]
  9. Rubenstein, R., Gray, P. C., Wehlburg, C. M., Wagner, J. S., and Tisone, G. C. (1998) Biochem. Biophys. Res. Commun. 246, 100-106[CrossRef][Medline] [Order article via Infotrieve]
  10. Bieschke, J., Giese, A., Schulz-Schaeffer, W., Zerr, I., Poser, S., Eigen, M., and Kretzschmar, H. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5468-5473[Abstract/Free Full Text]
  11. Safar, J. G., Scott, M., Monaghan, J., Deering, C., Didorenko, S., Vergara, J., Ball, H., Legname, G., Leclerc, E., Solforosi, L., Serban, H., Groth, D., Burton, D. R., Prusiner, S. B., and Williamson, R. A. (2002) Nat. Biotechnol. 20, 1147-1150[CrossRef][Medline] [Order article via Infotrieve]
  12. Brown, P., Cervenakova, L., McShane, L. M., Barber, P., Rubenstein, R., and Drohan, W. N. (1999) Transfusion 39, 1169-1178[CrossRef][Medline] [Order article via Infotrieve]
  13. Brown, P., Rohwer, R. G., Dunstan, B. C., MacAuley, C., Gajdusek, D. C., and Drohan, W. N. (1998) Transfusion 38, 810-816[CrossRef][Medline] [Order article via Infotrieve]
  14. Shaked, G. M., Shaked, Y., Kariv-Inbal, Z., Halimi, M., Avraham, I., and Gabizon, R. (2001) J. Biol. Chem. 276, 31479-31482[Abstract/Free Full Text]
  15. Saborio, G. P., Permanne, B., and Soto, C. (2001) Nature 411, 810-813[CrossRef][Medline] [Order article via Infotrieve]
  16. Ingrosso, L., Vetrugno, V., Cardone, F., and Pocchiari, M. (2002) Trends Mol. Med. 8, 273-280[CrossRef][Medline] [Order article via Infotrieve]
  17. Tuerk, C., and Gold, L. (1990) Science 249, 505-510[Abstract/Free Full Text]
  18. Kraus, E., James, W., and Barclay, A. N. (1998) J. Immunol. 160, 5209-5212[Abstract/Free Full Text]
  19. Tahiri-Alaoui, A., Frigotto, L., Manville, N., Ibrahim, J., Romby, P., and James, W. (2002) Nucleic Acids Res. 30, 1-9[Abstract/Free Full Text]
  20. Sayer, N., Ibrahim, J., Turner, K., Tahiri-Alaoui, A., and James, W. (2002) Biochem. Biophys. Res. Commun. 293, 924-931[CrossRef][Medline] [Order article via Infotrieve]
  21. Khati, M., Schumann, M., Ibrahim, J., Sattentau, Q., Gordon, S., and James, W. (2003) J. Virol. 77, 12692-12698[Abstract/Free Full Text]
  22. James, W. (2001) Curr. Opin. Pharmacol. 1, 540-546[CrossRef][Medline] [Order article via Infotrieve]
  23. Rhie, A., Kirby, L., Sayer, N., Wellesley, R., Disterer, P., Sylvester, I., Gill, A., Hope, J., James, W., and Tahiri-Alaoui, A. (2003) J. Biol. Chem. 278, 39697-39705[Abstract/Free Full Text]
  24. Baskakov, I. V., Legname, G., Baldwin, M. A., Prusiner, S. B., and Cohen, F. E. (2002) J. Biol. Chem. 277, 21140-21148[Abstract/Free Full Text]
  25. Heidenreich, O., Kang, S. H., Brown, D. A., Xu, X., Swiderski, P., Rossi, J. J., Eckstein, F., and Nerenberg, M. (1995) Nucleic Acids Res. 23, 2223-2228[Abstract/Free Full Text]
  26. Aurup, H., Williams, D. M., and Eckstein, F. (1992) Biochemistry 31, 9636-9641[CrossRef][Medline] [Order article via Infotrieve]
  27. Ehresmann, C., Baudin, F., Mougel, M., Romby, P., Ebel, J. P., and Ehres-mann, B. (1987) Nucleic Acids Res. 15, 9109-9128[Abstract/Free Full Text]
  28. Zuker, M. (2003) Nucleic Acids Res. 31, 3406-3415[Abstract/Free Full Text]
  29. Nguyen, D. H., DeFina, S. C., Fink, W. H., and Dieckmann, T. (2002) J. Am. Chem. Soc. 124, 15081-15084[Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
M. P. B. Gomes, T. A. Millen, P. S. Ferreira, N. L. C. e Silva, T. C. R. G. Vieira, M. S. Almeida, J. L. Silva, and Y. Cordeiro
Prion Protein Complexed to N2a Cellular RNAs through Its N-terminal Domain Forms Aggregates and Is Toxic to Murine Neuroblastoma Cells
J. Biol. Chem., July 11, 2008; 283(28): 19616 - 19625.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. H. J. Bunka, B. J. Mantle, I. J. Morten, G. A. Tennent, S. E. Radford, and P. G. Stockley
Production and Characterization of RNA Aptamers Specific for Amyloid Fibril Epitopes
J. Biol. Chem., November 23, 2007; 282(47): 34500 - 34509.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Tahiri-Alaoui, V. L. Sim, B. Caughey, and W. James
Molecular Heterosis of Prion Protein beta-Oligomers: A POTENTIAL MECHANISM OF HUMAN RESISTANCE TO DISEASE
J. Biol. Chem., November 10, 2006; 281(45): 34171 - 34178.
[Abstract] [Full Text] [PDF]

Home page
 RNAHome page
A. Rentmeister, A. Bill, T. Wahle, J. Walter, and M. Famulok
RNA aptamers selectively modulate protein recruitment to the cytoplasmic domain of {beta}-secretase BACE1 in vitro
RNA, September 1, 2006; 12(9): 1650 - 1660.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
ERRATA
Experimental Biology and Medicine, April 1, 2006; 231(4): 485 - 485.
[Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
K. Takemura, P. Wang, I. Vorberg, W. Surewicz, S. A. Priola, A. Kanthasamy, R. Pottathil, S. G. Chen, and S. Sreevatsan
DNA Aptamers That Bind to PrPC and Not PrPSc Show Sequence and Structure Specificity
Experimental Biology and Medicine, February 1, 2006; 231(2): 204 - 214.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Tahiri-Alaoui, A. C. Gill, P. Disterer, and W. James
Methionine 129 Variant of Human Prion Protein Oligomerizes More Rapidly than the Valine 129 Variant: IMPLICATIONS FOR DISEASE SUSCEPTIBILITY TO CREUTZFELDT-JAKOB DISEASE
J. Biol. Chem., July 23, 2004; 279(30): 31390 - 31397.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
279/13/13102    most recent
M310928200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sayer, N. M.
Right arrow Articles by James, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sayer, N. M.
Right arrow Articles by James, W.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today </