SomaLogic, Inc., Boulder, Colorado 80301
¶ Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215
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ABSTRACT |
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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). Aptamer technology is considerably less mature but has features that may be of advantage in the development of protein probe technology (79). Among the principal advantages of aptamers are the facts that they are synthetic molecules and are identified entirely in vitro by the SELEX1 process (10, 11). 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 (79) 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 (1216) 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, 18). 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 (1620). 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).
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), 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.
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EXPERIMENTAL PROCEDURES |
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Photoaptamer 0518 was discovered using a procedure analogous to that described by Golden and co-workers (9, 21), 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).
Equilibrium Dissociation Constant Measurements
Aptamers were radiolabeled with 32P at their 5' end with polynucleotide kinase to a specific activity of 25 x 106 dpm/pmol. Aptamers were mixed with excess protein in a buffer of Dulbeccos 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 40100 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,
![]() | (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.
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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.
Photocross-linking Kinetic Analysis
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.2
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,
![]() | (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.
![]() | (Eq. 3) |
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.2
Detection of Target Protein with Photoaptamer on Slides
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 1x SELEX buffer, 0.05% Tween 20, 200 µg/ml 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.
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RESULTS |
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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.
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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.
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DISCUSSION |
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We have proposed photoaptamers as probes for highly multiplexed protein assays (7, 8), 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. 2325; see Ref. 26 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). 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 56 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 200500 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 540-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) and in protein assays on beads (22). 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 x 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) that covalent capture by photoaptamers would allow the use of reactive dyes in protein detection, and these results confirm this expectation.
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CONCLUDING REMARKS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published, MCP Papers in Press, December 11, 2002, DOI 10.1074/mcp.M200059-MCP200
1 The 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.
2 T. H. Koch, D. Smith, and D. A. Zichi, manuscript in preparation.
* 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.
|| Supported by the Council for Tobacco Research and recipient of a faculty fellowship from the University of Colorado Council on Research and Creative Work. To whom correspondence may be addressed: Dept. of Chemistry and Biochemistry, 215UCB, University of Colorado, Boulder, CO 80309-0215. Fax: 303-492-5894; E-mail: tad.koch{at}colorado.edu.
To whom correspondence may be addressed. Fax: 303-545-2525; E-mail: drew.smith{at}somalogic.com
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REFERENCES |
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