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Sensitivity and Specificity of Photoaptamer Probes

2003; Elsevier BV; Volume: 2; Issue: 1 Linguagem: Inglês

10.1074/mcp.m200059-mcp200

1535-9484

Drew Smith, Brian D. Collins, James Heil, Tad H. Koch,

Advanced Biosensing Techniques and Applications

The potential of photoaptamers as proteomic probes was investigated. Photoaptamers are defined as aptamers that bear photocross-linking functionality, in this report, 5-bromo-2′-deoxyuridine. A key question regarding the use of photoaptamer probes is the specificity of the cross-linking reaction. The specificity of three photoaptamers was explored by comparing their reactions with target proteins and non-target proteins. The range of target/non-target specificity varies from 100- to >106-fold with most values >104-fold. The contributions of the initial binding step and the photocross-linking step were evaluated for each reaction. Photocross-linking never degraded specificity and significantly increased aptamer specificity in some cases. The application of photoaptamer technology to proteomics was investigated in microarray format. Immobilized anti-human immunodeficiency virus-gp120 aptamer was able to detect subnanomolar concentrations of target protein in 5% human serum. The levels of sensitivity and specificity displayed by photoaptamers, combined with other advantageous properties of aptamers, should facilitate development of protein chip technology. The potential of photoaptamers as proteomic probes was investigated. Photoaptamers are defined as aptamers that bear photocross-linking functionality, in this report, 5-bromo-2′-deoxyuridine. A key question regarding the use of photoaptamer probes is the specificity of the cross-linking reaction. The specificity of three photoaptamers was explored by comparing their reactions with target proteins and non-target proteins. The range of target/non-target specificity varies from 100- to >106-fold with most values >104-fold. The contributions of the initial binding step and the photocross-linking step were evaluated for each reaction. Photocross-linking never degraded specificity and significantly increased aptamer specificity in some cases. The application of photoaptamer technology to proteomics was investigated in microarray format. Immobilized anti-human immunodeficiency virus-gp120 aptamer was able to detect subnanomolar concentrations of target protein in 5% human serum. The levels of sensitivity and specificity displayed by photoaptamers, combined with other advantageous properties of aptamers, should facilitate development of protein chip technology. Proteomics, the study of protein expression at the scale of cell, tissue, or organism (1.Blackstock W.P. Weir M.P. Proteomics: quantitative and physical mapping of cellular proteins.Trends Biotechnol. 1999; 17: 121-127Google Scholar, 2.Dutt M.J. Lee K.H. Proteomic analysis.Curr. Opin. Biotechnol. 2000; 11: 176-179Google Scholar), has been defined by a single technology: two-dimensional gel separation followed by mass spectrometric analysis (3.Gevaert K. Vandekerckhove J. Protein identification methods in proteomics.Electrophoresis. 2000; 21: 1145-1154Google Scholar, 4.Anderson N.L. Matheson A.D. Steiner S. Proteomics: applications in basic and applied biology.Curr. Opin. Biotechnol. 2000; 11: 408-412Google Scholar). Although this technology is mature, powerful, and wonderfully sophisticated, it suffers from evident limitations in speed and sensitivity. Several days are required to process a single sample, and only ∼1000 of the most abundant proteins can be detected (5.Gygi S.P. Corthals G.L. Zhang Y. Rochon Y. Aebersold R. Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9390-9395Google Scholar). The ideal proteomic technology would process samples in minutes or hours and be able to quantify even the most weakly expressed proteins. Two-dimensional gels and chromatographic methods separate and identify proteins on the basis of their physical characteristics. An alternative approach is to identify proteins by specific recognition. The potential advantage of this approach is that proteins that have similar size and charge but which differ in sequence and conformation can be resolved and assayed independently with minimal cross-talk. Various strategies for high density arraying and multiplexing of oligonucleotides are in advanced stages of development; hence, a protein chip, analogous to a gene chip, is a logical step in proteomics. The demands on a successful protein probe technology are considerable. First, probes must be generated very rapidly, thousands or tens of thousands in a few years. Second, probes for different proteins must function under similar assay conditions. Third, the manufacture and arraying of the probes must be standardized. Fourth, the probes must demonstrate high levels of sensitivity and specificity toward their targets. The requirements for specificity will be especially rigorous: the limits of detection of existing instrumentation are not set by inherent machine sensitivity but by assay background and noise. For example, the confocal scanners used as gene chip readers can detect a few hundred fluorophores on a 100-μm feature, but probe detection limits are thousands-fold higher due to nonspecific binding to both probe and substrate. Nucleic acids can be selectively amplified to overcome these limitations, but proteins cannot. Monoclonal antibodies have many of the desirable features for protein probes and have the benefit of decades of technological development (6.Borrebaeck C.A. Antibodies in diagnostics—from immunoassays to protein chips.Immunol. Today. 2000; 21: 379-382Google Scholar). Aptamer technology is considerably less mature but has features that may be of advantage in the development of protein probe technology (7.Brody E.N. Willis M.C. Smith J.D. Jayasena S. Zichi D. Gold L. The use of aptamers in large arrays for molecular diagnostics.Mol. Diagn. 1999; 4: 381-388Google Scholar, 8.Brody E.N. Gold L. Aptamers as therapeutic and diagnostic agents.J. Biotechnol. 2000; 74: 5-13Google Scholar, 9.Golden M.C. Collins B.D. Willis M.C. Koch T.H. Diagnostic potential of photoSELEX-evolved ssDNA aptamers.J. Biotechnol. 2000; 81: 167-178Google Scholar). Among the principal advantages of aptamers are the facts that they are synthetic molecules and are identified entirely in vitro by the SELEX 1The abbreviations used are: SELEX, systematic evolution of ligands by exponential enrichment; A, aptamer; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; NHS, N-hydroxysuccinimide; PDGF, platelet-derived growth factor; T, target protein; HIV, human immunodeficiency virus. process (10.Tuerk C. Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.Science. 1990; 249: 505-510Google Scholar, 11.Ellington A.D. Szostak J.W. In vitro selection of RNA molecules that bind specific ligands.Nature. 1990; 346: 818-822Google Scholar). The former feature will facilitate manufacture and arraying, while the latter has facilitated automation for high throughput probe generation. The synthetic nature of aptamers bestows another potentially critical advantage: the ability to introduce desired chemical functions into libraries and select probes that have novel and compatible activities. We have argued that photoactivable cross-linking is a desirable function for a protein probe (7.Brody E.N. Willis M.C. Smith J.D. Jayasena S. Zichi D. Gold L. The use of aptamers in large arrays for molecular diagnostics.Mol. Diagn. 1999; 4: 381-388Google Scholar, 8.Brody E.N. Gold L. Aptamers as therapeutic and diagnostic agents.J. Biotechnol. 2000; 74: 5-13Google Scholar, 9.Golden M.C. Collins B.D. Willis M.C. Koch T.H. Diagnostic potential of photoSELEX-evolved ssDNA aptamers.J. Biotechnol. 2000; 81: 167-178Google Scholar) because it allows proteins to be covalently captured onto an array surface in a controllable manner. This capture allows washing, labeling, and reading steps to be performed under the harshest and most stringent conditions necessary to reduce background and improve signal. What is not established is the effect of photocross-linking on the specificity of the capture step. We set out to characterize, systematically and quantitatively, a set of photocross-linking aptamers, photoaptamers, with regard to their sensitivity and specificity. The photoreactive unit incorporated into our photoaptamers is 5-bromodeoxyuridine (BrdUrd), used for decades in protein-nucleic acid cross-linking studies. Rather than use short wave (254 or 266 nm) UV light for cross-linking, however, we irradiate at 308 nm using a XeCl excimer laser. This technique was developed by Koch and colleagues (12.Jensen K.B. Atkinson B.L. Willis M.C. Koch T.H. Gold L. Using in vitro selection to direct the covalent attachment of human immunodeficiency virus type 1 Rev protein to high-affinity RNA ligands.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12220-12224Google Scholar, 13.Gott J.M. Willis M.C. Koch T.H. Uhlenbeck O.C. A specific, UV-induced RNA-protein cross-link using 5-bromouridine-substituted RNA.Biochemistry. 1991; 30: 6290-6295Google Scholar, 14.Hicke B.J. Willis M.C. Koch T.H. Cech T.R. Telomeric protein-DNA point contacts identified by photo-cross-linking using 5-bromodeoxyuridine.Biochemistry. 1994; 33 (Correction (1994) Biochemistry 33, 7744): 3364-3373Google Scholar, 15.Willis M.C. LeCuyer K.A. Meisenheimer K.M. Uhlenbeck O.C. Koch T.H. An RNA-protein contact determined by 5-bromouridine substitution, photocrosslinking and sequencing.Nucleic Acids Res. 1994; 22: 4947-4952Google Scholar, 16.Meisenheimer K.M. Koch T.H. Photocross-linking of nucleic acids to associated proteins.Crit. Rev. Biochem. Mol. Biol. 1997; 32: 101-140Google Scholar) and has been shown to result in specific and high yield cross-linking reactions. Light at 308 nm induces photoelectron transfer from a nearby electron donor to the bromouracil base via either excitation of the BrdUrd, excitation of the electron donor, or excitation of a BrdUrd-electron donor charge transfer state (17.Norris C.L. Meisenheimer K.M. Koch T.H. Mechanistic studies relevant to bromouridine enhanced nucleoprotein photocrosslinking. Possible involvement of an excited tyrosine residue of the protein.Photochem. Photobiol. 1997; 65: 201-207Google Scholar, 18.Meisenheimer K.M. Meisenheimer P.L. Koch T.H. Nucleoprotein photo-cross-linking using halopyrimidine-substituted RNAs.Methods Enzymol. 2000; 318: 88-104Google Scholar). Amino acid residues that can serve as electron donors in BrdUrd photocross-linking include Tyr, Trp, His, Phe, Cys, Cys-Cys, and Met of which only Tyr and Trp are excited at 308 nm (16.Meisenheimer K.M. Koch T.H. Photocross-linking of nucleic acids to associated proteins.Crit. Rev. Biochem. Mol. Biol. 1997; 32: 101-140Google Scholar, 17.Norris C.L. Meisenheimer K.M. Koch T.H. Mechanistic studies relevant to bromouridine enhanced nucleoprotein photocrosslinking. Possible involvement of an excited tyrosine residue of the protein.Photochem. Photobiol. 1997; 65: 201-207Google Scholar, 18.Meisenheimer K.M. Meisenheimer P.L. Koch T.H. Nucleoprotein photo-cross-linking using halopyrimidine-substituted RNAs.Methods Enzymol. 2000; 318: 88-104Google Scholar, 19.Dietz T.M. Koch T.H. Photochemical coupling of 5-bromouracil to tryptophan, tyrosine and histidine, peptide-like derivatives in aqueous fluid solution.Photochem. Photobiol. 1987; 46: 971-978Google Scholar, 20.Dietz T.M. Koch T.H. Photochemical reduction of 5-bromouracil by cysteine derivatives and coupling of 5-bromouracil to cystine derivatives.Photochem. Photobiol. 1989; 49: 121-129Google Scholar). Cross-linking results from subsequent reaction of the resulting radical ion pair. In the absence of an electron donor the BrdUrd efficiently relaxes back to ground state (17.Norris C.L. Meisenheimer K.M. Koch T.H. Mechanistic studies relevant to bromouridine enhanced nucleoprotein photocrosslinking. Possible involvement of an excited tyrosine residue of the protein.Photochem. Photobiol. 1997; 65: 201-207Google Scholar). We hypothesized that photocross-linking via photoelectron transfer would actually enhance the specificity of the aptamer-protein capture reaction: although a protein might bind an aptamer nonspecifically, the probability that an appropriate amino acid would be positioned to cross-link with a BrdUrd residue would be low. Some evidence for this view has been presented by Golden and co-workers (9.Golden M.C. Collins B.D. Willis M.C. Koch T.H. Diagnostic potential of photoSELEX-evolved ssDNA aptamers.J. Biotechnol. 2000; 81: 167-178Google Scholar), who showed that basic fibroblast growth factor (bFGF) photoaptamers could cross-link picomolar concentrations of target in the presence of serum with very little nonspecific cross-linking. Using these bFGF photoaptamers and a new photoaptamer raised against the HIV coat protein gp120MN we evaluated both the equilibrium binding constant and the relative rate of cross-linking to target proteins. We then compared these values to the values for a set of non-target proteins. These non-target proteins were chosen to provide an exacting test of specificity: 1) aFGF and gp120SF2 are the commercially available proteins most closely related to the target proteins; 2) platelet-derived growth factor (PDGF) is a highly basic heparin-binding growth factor that is notorious for its nonspecific DNA binding; and 3) thrombin is another heparin-binding protein. These experiments confirm the specificity of the photocross-linking reaction in the solution phase. We extend these results to microarray format by measuring cross-linking of immobilized photoaptamers to target protein. We find that the sensitivity and specificity of photocross-linking are maintained in this format: target proteins can be detected at subnanomolar concentrations in buffer and at nanomolar concentrations when spiked into serum. Photoaptamers 0615 and 0650 were discovered by Golden and co-workers (21.Golden M.C. Resing K.A. Collins B.D. Willis M.C. Koch T.H. Mass spectral characterization of a protein-nucleic acid photocrosslink.Protein Sci. 1999; 8: 2806-2812Google Scholar). Their sequences are shown as follows with X representing BrdUrds. 0615: GGG AGG ACG ATG CGG GCG AAG GCA CAC CGA GXX CAX AGX AXC CCA CAG ACG ACG AGC GGG A; 0650: GGG AGG ACG ATG CGG XGA CGX AAG AGX GXA AXC GAX GCA GCC XGG CAG ACG ACG AGC GGG A. Photoaptamer 0518 was discovered using a procedure analogous to that described by Golden and co-workers (9.Golden M.C. Collins B.D. Willis M.C. Koch T.H. Diagnostic potential of photoSELEX-evolved ssDNA aptamers.J. Biotechnol. 2000; 81: 167-178Google Scholar, 21.Golden M.C. Resing K.A. Collins B.D. Willis M.C. Koch T.H. Mass spectral characterization of a protein-nucleic acid photocrosslink.Protein Sci. 1999; 8: 2806-2812Google Scholar), and its sequence is as follows, again with X representing BrdUrd. 0518: GGG AGG ACG ATG CGG AAX GCG CGA GCX XCC GAA AAG GAA AXX ACG CAG ACG ACG AGC GGG A. Photoaptamers were synthesized enzymatically by replication of a complementary synthetic DNA sequence using the Klenow fragment of Escherichia coli DNA polymerase I and a synthetic DNA primer. The reaction mix included BrdUTP (TriLink) in place of TTP with all NTPs at 0.5 mm. The complementary strand contained several biotin residues at its 5′ end. The aptamer and template were resolved by denaturing polyacrylamide gel electrophoresis, and the aptamer was eluted from the gel by standard methods. To avoid exposing the BrdUrd-containing DNA to UV light, the gels were stained with SYBR I (Molecular Probes) and visualized using a blue light Dark Reader transilluminator (Clare Chemical Research, Denver, CO). Aptamers were radiolabeled with 32P at their 5′ end with polynucleotide kinase to a specific activity of 2–5 × 106 dpm/pmol. Aptamers were mixed with excess protein in a buffer of Dulbecco's phosphate-buffered saline supplemented with 1.5 mm MgCl2, 0.1 mm dithiothreitol, 0.01% bovine serum albumin (=PDB buffer) in a total volume of 40–100 μl and allowed to equilibrate for 10 min at room temperature. Samples were filtered under vacuum through 2.5-cm nitrocellulose disks, pore size 0.45 μm (Millipore HAWP 0025), and rinsed immediately with 1 ml of phosphate-buffered saline. Retained radioactivity was determined by Cerenkov scintillation, and the fraction bound was determined relative to an unfiltered control. It can be readily shown from the definition of the equilibrium dissociation constant, Kd, given in Fig. 1 that the fraction of aptamer bound to the target protein, i.e. [A:T]/[A], is given by Equation 1,fraction bound= [A:T][A]=[T][T]+Kd(eq 1) where [T] = target protein concentration. A value for Kd can be estimated by using a non-linear least-squares program (Kaleidagraph, Synergy Software) to fit a plot [A:T]/[A] versus [T]. From the upper panel in Fig. 2, it is evident that the maximum fraction of bound aptamer 0518 reached a level significantly below 1.0, so the Kd value was determined using Equation 1 multiplied by a term for the plateau value; the resulting plateau value is 0.57.Fig. 2Photoaptamer equilibrium binding to target and non-target proteins without cross-linking. The non-covalent complexes were detected by nitrocellulose filter binding as described under "Experimental Procedures." ▪, target protein (gp120MN for aptamer 0518 and bFGF for aptamers 0615 and 0650); □, nearest non-target protein (gp120SF2 for aptamer 0518 and aFGF for aptamers 0615 and 0650); ○, PDGF; ▿), thrombin.View Large Image Figure ViewerDownload (PPT) Radiolabeled photoaptamers were mixed with excess protein in PDB buffer and allowed to equilibrate at room temperature for 10 min. The mixtures were irradiated in 0.6-ml disposable methacrylate cuvettes. The laser used was a TuiLaser S-200 XeCl excimer laser that emits 308 nm light. The beam size measured 6 × 6 mm, and the cuvette position was adjusted so that the entire sample was within the beam. The standard settings produced 2.0 mJ/pulse with a repetition rate of 200 Hz. As discussed in a separate paper 2T. H. Koch, D. Smith, and D. A. Zichi, manuscript in preparation. the cross-linking extent was governed strictly by the total light dose; neither the peak power nor the pulse frequency affected cross-linking. Aliquots were withdrawn from the cross-linking reaction and mixed with denaturing formamide gel buffer. Cross-linked DNA was resolved from free DNA by electrophoresis on denaturing gels (8% acrylamide (19:1 acrylamide:bisacrylamide), Tris borate-EDTA, 7 m urea, 0.05% SDS). Gels were visualized by phosphorimaging on a Fuji FLA-3000 instrument. Our kinetic model assumes three possible fates for an irradiated photoaptamer after absorption of a photon: it can reversibly decay back to ground state, cross-link irreversibly to the target protein, or undergo an irreversible photodegradation reaction. The photoaptamer target complex can be excited at the BrdUrd or BrdUrds involved in cross-linking or at the BrdUrds remote from the cross-linking site or sites. Fig. 1 diagrams these possible fates. In this scheme, A is the photoaptamer, T is the target protein, A:T is the ground state complex, A-T is the cross-linked nucleoprotein, and X is photoinactivated A such that it no longer binds to T. The rate constants kxl and ki are the rate constants for cross-linking and for inactivation. The rate constant for inactivation is assumed to be the same for bound and unbound A. Each rate constant actually represents the product of the rate constant for absorption and the respective quantum yield for the subsequent reaction of the excited state where the rate constant for absorption is the product of the incident light intensity and the probability of absorption. Evidence for this model and derivation of the kinetic equations used in this paper are given in a separate paper. 2T. H. Koch, D. Smith, and D. A. Zichi, manuscript in preparation. The dependence of cross-linking extent on light dose has been observed to follow a monotonic increase to a plateau. The fraction cross-linked as a function of light dose is thus given by Equation 2,fraction cross−linked=fxl=fxlpl(1−e−kobsJ)(eq 2) where fxlpl is a pre-exponential factor equal to the maximal amount cross-linked at a given protein concentration (the plateau value in a plot of fraction cross-linked versus light dose), kobs is the observed first-order rate constant for cross-linking in units of reciprocal light dose, and J is the light dose in units of J/cm2. The parameter kobs is a complex function of kxl, ki, Kd, and [T].2 In the work reported here, cross-linking reactions were evaluated by determining the fraction cross-linked as a function of light dose for a given protein concentration with the protein concentration large relative to the photoaptamer concentration consumed in cross-linking. Hence, the concentration of T remained effectively constant. The best fit value for kobs was then determined by plotting fraction photoaptamer cross-linked versus light dose and solving Equation 2. Two other metrics are used in evaluating photoaptamer activity: fxlmax and Kxl. The parameter fxlmax is the plateau of a plot of fxlpl versus protein concentration; it represents the maximal fraction of aptamer cross-linked at saturating light and saturating protein. Kxl is the protein concentration at which fxlpl = fxlmax/2 and is equal to (1 − fxlmax)Kd. The relationship between fxlpl and Kd and Kxl is shown in Equation 3.fxlpl= fxlmax[T](1−fxlmax)Kd+[T]=fxlmax[T](Kxl+[T])(eq 1) The quotient fxlmax/Kxl is then a second measure that relates cross-linking activity to protein concentration. The parameter fxlpl will be linearly dependent upon protein concentration for [T] << Kxl. Under these conditions, fxlpl/[T] should be a good approximation of fxlmax/Kxl; we use this approximation in evaluating cross-linking to non-target proteins where determination of fxlmax is impractical. A derivation of Equation 3 will be included in a separate paper. 2T. H. Koch, D. Smith, and D. A. Zichi, manuscript in preparation. Photoaptamers 0518, 0615, and 0650 were synthesized with a 5′ C6-amino substituent using standard DNA synthesis methods. The DNA was immobilized on N-hydroxysuccinimide-activated slides (Surmodics) by spotting 1 nl (Gene Machines) of a 20 μm solution in 150 mm phosphate, pH 8.5, 20 μm polyethylene glycol-NH2 (Mr = 2000). Feature diameter is ∼100 μm. After an overnight coupling reaction, unreacted amines were blocked with 0.1 m Tris, 50 mm ethanolamine, pH 9.0 at 50 °C for 15 min. The slides were washed two times with water, then washed with 4X SSC, 0.1% SDS at 50 °C for 15 min, and then rinsed two times with water. Residual amines, which might react with NHS-Cy3 dye (below), were capped by reaction with 0.1 mg/ml sulfo-NHS-acetate in 100 mm Na2HCO3, pH 8.4 at room temperature for 30 min and were then rinsed three times with SELEX buffer (40 mm HEPES, pH 7.5, 111 mm NaCl, 5 mm KCl, 1 mm CaCl2, 1 mm MgCl2, 0.05% Tween 20). Protein assays were performed in 50-μl-volume perfusion chambers (Grace BioLabs), eight chambers/slide. The slides were blocked for 30 min in 1× SELEX buffer, 0.05% Tween 20, 200 μg/ml λ carrageenan (Sigma), 200 μg/ml Ficoll Type 400 (Amersham Biosciences), and 1 mg/ml dimethylated casein (Sigma). This buffer was removed, and fresh buffer containing test proteins and/or human defibrinated serum (SeraCare) was added and incubated for 1 h at room temperature. The protein solutions were then removed, and the slide was washed three times with SELEX buffer. Photocross-linking was performed with 308 nm excimer laser light at a total dose of 5 J/cm2. Uncross-linked protein was removed by washing in 500 ml of SDS wash buffer (20 mm sodium phosphate, pH 7.4, 1 mm EDTA, 150 mm NaCl, 0.1% SDS) for 20 min with stirring and then with water for 5 min. Bound proteins were then stained by addition of 0.1 mg/ml Cy3-NHS (Amersham Biosciences) in 0.1 m Na2HCO3, pH 8.4 for 30 min at room temperature. Free dye was removed by repeating the SDS wash followed by washes in methanol and 20 mm NaOH. Salt residues were removed by rinsing with water, and the slides were dried under a stream of N2. Image data were collected in an ArrayWorx scanner (Applied Precision) in the Cy3 channel at 0.2-s exposure, 5-μm pixel resolution. To begin our analysis of photoaptamer specificity, we determined equilibrium binding constants for target and non-target proteins. To provide a stringent test of binding specificity, we chose a set of non-target proteins likely to cross-react with the aptamers. Using a BLAST search, we identified the proteins most similar to the target proteins. The most closely related protein to bFGF is aFGF, which shares 55% identity. The closest relative to gp120MN is HIV coat protein from the SF2 strain, gp120SF2, which is 81% identical. To these proteins we added two proteins known to have very high nonspecific affinity for nucleic acids, PDGF and thrombin. Both contain polyanion (that is, heparin) binding sites. In addition, PDGF is highly basic, having a predicted pI of ∼9.5, and the AB dimer is expected to have a net charge at neutral pH of +20. Using the nitrocellulose filter binding method, affinities of the photoaptamers for target and non-target proteins were determined (Fig. 2). With the exception of PDGF, the affinities of the photoaptamers for the non-cognate proteins are so weak that detection of binding is difficult. Kd values are compared in Table I.Table IPhotoaptamer binding and cross-linking constantsAptamerProteinKdfxlmaxKxlfxlmax/Kxlmmm−10518gp120MN8.3 × 10−80.309.7 × 10−83.1 × 106gp120SF2>1 × 10−41 × 103PDGF5.7 × 10−77 × 104Thrombin>1 × 10−4 1 × 10−43 × 104PDGF2.0 × 10−76 × 105Thrombin1 × 10−43 × 1040650bFGF4.5 × 10−100.811.4 × 10−105.8 × 109aFGF>1 × 10−44 × 104PDGF2.2 × 10−73 × 105Thrombin>1 × 10−42 × 104 Open table in a new tab Aptamer specificity can also be gauged by contrasting the extent of cross-linking to the target protein with that to related target proteins. The measure we use is fxlmax/Kxl. This term is useful because, when multiplied by protein concentration, the product is the fraction aptamer cross-linked at saturating light dose for protein concentrations significantly less than Kxl (see Equation 3). Except when the fxlmax is high, Kxl is typically close to the equilibrium binding constant Kd (Table I). For cross-linking of each photoaptamer to its target protein, we performed light dose titrations over a full series of protein concentrations. The maximal fraction cross-linked at a given protein concentration (fxlpl) was evaluated using Equation 2. Parameters fxlmax and Kxl were determined by fitting Equation 3 to a plot of fxlpl as a function of protein concentration. For non-target proteins, we quickly recognized that the use of saturating protein concentrations to determine fxlmax or Kxl individually was impractical. Instead we determined fxlpl at protein concentrations less than Kd, assuming that these protein concentrations would also be less than Kxl. Under these conditions fxlpl/[T] should approximate fxlmax/Kxl. Fig. 3 shows plots of fxlpl versus protein concentration. The values for fxlmax/Kxl estimated from these data are reported in Table I. Non-target proteins (the open symbols) are displaced far down and to the right as compared with target proteins. As with the filter binding experiments, detection of non-target cross-linked complexes is quite difficult, requiring high protein concentrations and light doses (Fig. 4). The lower ranges of these values are probably no more accurate than 2–5-fold.Fig. 4Gel assay of cross-linking. Radiolabeled 0650 was mixed with excess protein at the indicated concentrations and irradiated to the doses shown. The photoaptamer-DNA product is identified by its slower migration in denaturing polyacrylamide gel electrophoresis. No-light (left lanes) and no-protein (not shown) controls identify these bands as the cross-link product.View Large Image Figure ViewerDownload (PPT) Our goal is to use photoaptamers as probes in microarray format. Having evaluated their activity and specificity in solution phase, we extended our analysis to the solid phase. The photoaptamers 0518, 0615, and 0650 were synthesized with a 5′ C6-amino linker and immobilized by spotting on NHS-activated slides (see "Experimental Procedures"). The aptamers were then assayed for photocross-linking activity by dose response to their targets. Protein dilutions were applied to subarrays in perfusion chambers for 1 h at room temperature, rinsed briefly, and then cross-linked with 5 J/cm2 308 nm laser light. Most protein not covalently bound was removed by successive washes in 0.1% SDS and 20 mm NaOH. Captured proteins were stained via their primary amines by NHS-Cy3 reactive dye. Excess dye was removed by repeating the denaturing washes. After scanning in a slide reader, the capture reactions were evaluated as a function of feature intensity minus local background (Imagene). Fig. 5 shows plots of fluorescence intensity versus protein concentration. As both bFGF and gp120 aptamers were present on the arrays, cross-reactivity could also be evaluated. These results show that the aptamers are as well behaved on surfaces as they are in solution: subnanomolar sensitivity with little or no cross-reactivity. A previous study (9.Golden M.C. Collins B.D. Willis M.C. Koch T.H. Diagnostic potential of photoSELEX-evolved ssDNA aptamers.J. Biotechnol. 2000; 81: 167-178Google Scholar) demonstrated specific cross-linking of the bFGF aptamers in serum. We extended these results by measuring the dose response for gp120 in serum. There should be no endogenous gp120 in normal serum, which might confound the interpretation of results. gp120 was diluted into 5% defibrinated delipidated human serum (total protein, ∼3 mg/ml), and the response was assayed as described under "Experimental Procedures." Fig. 6 shows a definite dose response to gp120 protein from its cognate aptamer and none from the non-cognate aptamer. The sensitivity of the response is less than in buffer, presumably due to interference with serum components. Three photoaptamers bearing between five and seven BrdUrd nucleotides in place of T nucleotides have recently been identified through the photoSELEX methodology: two, 0650 and 0615, show specific affinity and cross-linking to bFGF, and one, 0518, shows specific affinity and cross-linking to HIV gp120MN. We have proposed photoaptamers as probes for highly multiplexed protein assays (7.Brody E.N. Willis M.C. Smith J.D. Jayasena S. Zichi D. Gold L. The use of aptamers in large arrays for molecular diagnostics.Mol. Diagn. 1999; 4: 381-388Google Scholar, 8.Brody E.N. Gold L. Aptamers as therapeutic and diagnostic agents.J. Biotechnol. 2000; 74: 5-13Google Scholar), an application which requires an extremely high degree of specificity. Previous studies of aptamer equilibrium binding have shown good specificity of binding (e.g. Refs. 23.Zimmermann G.R. Wick C.L. Shields T.P. Jenison R.D. Pardi A. Molecular interactions and metal binding in the theophylline-binding core of an RNA aptamer.RNA. 2000; 6: 659-667Google Scholar, 24.Romig T.S. Bell C. Drolet D.W. Aptamer affinity chromatography: combinatorial chemistry applied to protein purification.J. Chromatogr. B Biomed. Sci. Appl. 1999; 731: 275-284Google Scholar, 25.Jenison R.D. Jennings S.D. Walker D.W. Bargatze R.F. Parma D. Oligonucleotide inhibitors of P-selectin-dependent neutrophil-platelet adhesion.Antisense Nucleic Acid Drug Dev. 1998; 8: 265-279Google Scholar; see Ref. 26.Hirao I. Spingola M. Peabody D. Ellington A.D. The limits of specificity: an experimental analysis with RNA aptamers to MS2 coat protein variants.Mol. Divers. 1998; 4: 75-89Google Scholar for a counter example). Our studies of the photocross-linking mechanism2 indicate the requirement that cross-linking occurs within a complex of aptamer and protein. This requirement suggests that photocross-linking should enhance probe specificity; our work tests and quantifies this hypothesis. Nucleic acid-protein photocross-linking reactions generally occur through formation of a highly reactive species, independent of prior nucleic acid-protein complexation (18.Meisenheimer K.M. Meisenheimer P.L. Koch T.H. Nucleoprotein photo-cross-linking using halopyrimidine-substituted RNAs.Methods Enzymol. 2000; 318: 88-104Google Scholar). In contrast, photocross-linking with a BrdUrd photoaptamer to its cognate protein is initiated predominantly via photoelectron transfer between the bromouracil chromophore of the nucleic acid and an electron-donating chromophore of the protein, frequently the phenolic group of a Tyr residue. In the absence of an electron donor, excited bromouracil decays back to the electronic ground state. Hence, BrdUrd photoaptamers exhibit covalent molecular recognition. This mechanism should not only maintain but increase the specificity of target capture by an aptamer: binding of a non-target protein is less likely to result in the appropriate geometry for photoelectron transfer-initiated cross-linking. We tested this model by measuring the specificity of binding and cross-linking reactions. Specificity of photoaptamers was measured by determining Kd values and cross-linking activities for a few proteins that might interfere with detection of the cognate protein (Table I). These included proteins with high sequence homology to the cognate proteins (aFGF and gp120SF2) and proteins that have high nonspecific affinity for nucleic acids because of their polyanionic binding sites (PDGF and thrombin). Although aFGF has 55% sequence homology with bFGF, it shows 5–6 orders of magnitude lower affinity for the bFGF aptamers 0650 and 0615. Similarly, gp120SF2 has 81% sequence homology with gp120MN but shows 3 orders of magnitude lower affinity for aptamer 0518. PDGF, which has a very high positive charge, shows much higher affinity for the aptamers (Kd 200–500 nm) than do any of the other non-cognate proteins. Despite the presence of a heparin binding site, thrombin bound very weakly to all aptamers. Cross-linking activities, defined as fxlmax/Kxl, were determined for non-cognate proteins, and these are also reported in Table I. These activities are substantially smaller for non-cognate proteins than for cognate proteins. The ratios for aFGF with 0650 and 0615 are smaller by 5 orders of magnitude relative to the ratios for the cognate protein bFGF. Similarly, the ratio for gp120SF2 is 3 orders of magnitude smaller relative to the ratio for the cognate protein gp120MN. PDGF shows the greatest nonspecific cross-linking activity with all three aptamers. A comparison of the specificity of cross-linking activity with that of binding affinity is instructive. PDGF binding is 7-, 2500-, and 500-fold weaker than cognate protein binding for aptamers 0518, 0615, and 0650, respectively (Table I). Correspondingly, cross-linking activity of PDGF is 40-, 5000-, and 20,000-fold lower than cross-linking activity of cognate proteins. The cross-linking reaction, therefore, imparts 5–40-fold greater specificity over affinity binding alone in all three cases. A similar quantitative comparison of affinity and cross-linking specificity for the other non-target proteins (aFGF, gp120SF2, and thrombin) is problematic because the measurements made for these reactions are near the limits of detection for the assays used. Qualitatively, however, it is clear that there is no apparent loss of specificity in the cross-linking reactions as compared with the affinity binding reactions; both are extremely specific. Previous studies have shown that immobilized aptamers are active in affinity purification on a porous support (24.Romig T.S. Bell C. Drolet D.W. Aptamer affinity chromatography: combinatorial chemistry applied to protein purification.J. Chromatogr. B Biomed. Sci. Appl. 1999; 731: 275-284Google Scholar) and in protein assays on beads (22.Lee M. Walt D.R. A fiber-optic microarray biosensor using aptamers as receptors.Anal. Biochem. 2000; 282: 142-146Google Scholar). We extended this work by showing that photoaptamer-based assays are feasible in microarray format. All three aptamers responded to target protein in a near linear fashion over 3 orders of magnitude of protein concentration with subnanomolar limits of detection (Fig. 6). In the presence of 5% serum (∼3 mg/ml protein) we were able to detect ≤5 nm gp120 (∼3 × 10−4 mg/ml), a sensitivity and specificity >1/104. We expect this sensitivity to increase as our assay methods are further developed. It is worth emphasizing that these results were obtained without the use of a secondary reagent. We have argued (7.Brody E.N. Willis M.C. Smith J.D. Jayasena S. Zichi D. Gold L. The use of aptamers in large arrays for molecular diagnostics.Mol. Diagn. 1999; 4: 381-388Google Scholar) that covalent capture by photoaptamers would allow the use of reactive dyes in protein detection, and these results confirm this expectation. One of the goals of our study was to assess the suitability of photoaptamers as probes for highly multiplexed protein assays. A chief concern is the specificity of the photocross-linking reaction: although aptamer binding is generally quite specific, photocross-linking might be unselective. Our work shows that, in the three examples explored here, the cross-linking reaction significantly enhanced probe specificity in the case where affinity specificity was weakest and had a small but positive effect when affinity specificity was strongest. It thus seems plausible that a single photoaptamer can be used as a capture agent. The current standard in protein measurement is the sandwich assay, which requires matching pairs of antibodies with compatible affinities and non-overlapping epitopes. This process is burdensome when developing single protein assays and small panels; it will become a major challenge when the goal is to measure hundreds or thousands of protein levels simultaneously. We have shown that arrayed photoaptamers can function as sensitive and specific single detection reagents. The sensitivity and specificity of photoaptamers, combined with the ability to automate and scale up their selection and the ability to use them on solid surfaces, indicate that they could become an important factor in the development of proteomic technology. We thank Michael Lochrie for cloning and sequencing experiments associated with the selection of 0518, Jeff Carter and Matt Otis for oligonucleotide synthesis, Chad Greef for development of microarray spotting techniques, and Larry Gold for useful discussions and suggestions.