Histochemical Application Of A Peroxidase DNAzyme with a

Feb 18, 2010 - Histochemical Application Of A Peroxidase DNAzyme with a Covalently .... The Analyst 2015 140 (8), 2556-2572 ... The Analyst 2014 139, ...
0 downloads 0 Views 4MB Size
Download PDF
Anal. Chem. 2010, 82, 2498–2504

Histochemical Application Of A Peroxidase DNAzyme with a Covalently Attached Hemin Cofactor Derek Thirstrup and Geoffrey S. Baird* University of Washington, Seattle, Department of Laboratory Medicine The enzyme horseradish peroxidase is routinely used in immunohistochemistry to facilitate the chromogenic oxidation of 3,3′-diaminobenzidine, producing an insoluble brown precipitate marking the location and quantity of a tissue protein. In an effort to develop non-protein reagents for tissue diagnostics, we have developed a peroxidase DNAzyme construct that can be used as a chromogenic functional group in immunohistochemistry assays. The DNAzyme is based on a reported 18-mer G-quadruplexforming DNA sequence, PS2.M, and has been covalently linked to its catalytically active moiety, hemin, to avoid the high background signal associated with the use of free hemin in histochemical studies. The activity of the covalent construct is maintained under conditions where G-quadruplex formation is unfavored and where the noncovalent DNAzyme-hemin complex has no activity. In the diagnostic pathology laboratory, immunohistochemistry (IHC) is used to localize tissue proteins. After an antibody binds a protein of interest, additional antibodies and biotin-avidin or polymer reagents are used to bring a chromogenic enzyme to the site of the protein of interest. Most commonly, this chromogenic enzyme is horseradish peroxidase (HRP), used because it catalyzes the oxidative polymerization of 3,3′-diaminobenzidine (DAB) into an insoluble brown precipitate.1 In an effort to develop alternative reagents for tissue protein detection assays, we have been investigating oligonucleotide aptamers. Aptamers are short single-stranded oligonucleotides, DNA or RNA, that are selected through an iterative process (SELEX) from random sequence RNA/DNA pools, and that can bind to other molecules with high affinities and selectivity. DNA aptamers have numerous practical advantages over protein reagents like antibodies and enzymes, such as highly parallel and automated discovery and production, relatively simple derivatization chemistries, and stability to dry storage under ambient conditions. When aptamers are used rather than antibodies to identify target molecules histochemically, protein visualization is accomplished by detecting and localizing a DNA molecule. There are many commercialized methods to detect endogenous DNA in solid tissue, such as in situ hybridization, but these are usually * To whom correspondence should be addressed. Mailing address: Box 359743, 325 9th Avenue, Seattle, WA 98105. E-mail: [email protected]. (1) Seligman, A.; Karnovsky, M.; Wasserkrug, H.; Hanker, J. J. Cell Biol. 1968, 38, 1–14.

2498

Analytical Chemistry, Vol. 82, No. 6, March 15, 2010

multistep procedures that require lengthy incubations and multiple reagents to achieve the required degree of sequence specificity. In the case of aptamer histochemistry, the problem of DNA detection is much simpler to solve because the DNA to be detected is exogenous, does not need to be detected by sequencedirected hybridization, and can be chemically modified in any way desired prior to addition to the tissue. In this study, we have investigated whether or not the DNA of an aptamer construct could itself be modified to act as a chromogenic catalyst, thus obviating the need for secondary or tertiary detection reagents. We have chosen to investigate chromogenic detection methods because the overwhelming majority of diagnoses made by anatomic pathologists are made by morphologically examining chemically stained tissues with a simple transmitted light microscope. Fluorescent or luminescent signals may be more sensitive or quantitative, but most anatomic pathologists do not have the equipment to detect these signals routinely, and thus the most preferred readout of a routine assay for a protein in tissue is currently a chromogenic signal that can be seen by eye under transmitted light. Furthermore, since all anatomic pathologists are familiar with identifying the brown product of DAB oxidation localized into subcellular compartments, we sought a solution that would permit a DNA construct to produce a brown DAB signal in tissue in the same manner as HRP. Our interests were therefore directed toward the DNAzyme, PS2.M, reported by Travascio, et al.2 PS2.M has been described as an 18-base G-rich DNA sequence that can form a noncovalent complex with the Fe3+-containing porphyrin hemin. In complex with hemin, PS2.M can catalyze the chromogenic oxidation of 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), in a manner similar to peroxidase enzymes. Many other investigators have engineered this noncovalent DNAzyme complex into novel biosensors,3 and some have investigated the complex’s reactivity toward substrates other than ABTS.4 To our knowledge, however, no previous studies have reported attaching hemin to PS2.M covalently, nor have any examined the ability of this DNAzyme to catalyze the oxidation of DAB to localize a protein spatially in a tissue section. (2) Travascio, P.; Li, Y.; Sen, D. Chem. Biol. 1998, 5, 505–17. (3) (a) Elbaz, J.; Shlyahovsky, B.; Li, D.; Willner, I. Chembiochem 2008, 9, 232–9. (b) Elbaz, J.; Moshe, M.; Shlyahovsky, B.; Willner, I. Chemistry 2009, 15, 3411–8. (c) Li, T.; Wang, E.; Dong, S. Chem. Commun. 2009, 580–2. (4) (a) Li, B.; Du, Y.; Li, T.; Dong, S. Anal. Chim. Acta 2009, 651, 234–40. (b) Rojas, A.; Gonzalez, P.; Antipov, E.; Klibanov, A. Biotechnol. Lett. 2007, 29, 227–32. 10.1021/ac902887j  2010 American Chemical Society Published on Web 02/18/2010

Table 1. All DNA Constructs Reported in This Worka construct name

sequence

thrombin aptamer Chlamydomonas telomere PS2.M 5′Bio-PS2.M PS2.M-3′Bio 5′Bio-A5-PS2.M 5′Bio-(A5-PS2.M)2 5′Bio-(A5-PS2.M)3

5′-\5BiosG\GGT TGG TGT GGT TGG-3′ 5′-TTT TAG GGT TTT AGG GTT TTA GGG TTT TAG GG-3′ 5′-GTG GGT AGG GCG GGT TGG-3′ 5′-\5Biosg\GTG GGT AGG GCG GGT TGG-3′ 5′-GTG GGT AGG GCG GGT TGG \3Bio\-3′ 5′-\5Biosg\AA AAA GTG GGT AGG GCG GGT TGG-3′ 5′-\5Biosg\A AAA AGT GGG TAG GGC GGG TTG GAA AAA GTG GGT AGG GCG GGT TGG-3′ 5′-\5Biosg\AAA AAG TGG GTA GGG CGG GTT GGA AAA AGT GGG TAG GGC GGG TTG GAA AAA GTG GGT AGG GCG GGT TGG-3′ 5′-GCA GTT GAT CCT TTG GAT ACC CTG GAA AAA AAA AAA AAA AGT GGG TAG GGC GGG TTG G-3′ 5′-\5BiosG\TTT TTT TTT TTT TTT-3′ 5′-\5BiosG\TTT TTT TTT TTT TTT\3AmM\-3′ 5′-\5Biosg\AAA AAA AAA AAA AAA GTG GGT AGG GCG GGT TGG\3AmM\-3′

MUC1-A15-PS2.M 5′Bio-T15 5′Bio-T15-NH2 5′Bio-A15-PS2.M-NH2

a Aside from the bases, modifications are indicated with the codes used when purchasing from IDT (www.idtdna.com). The exact structures of the modifications (5Biosg ) 5′-biotin and 3AmM ) 3′-amino) are proprietary; however, IDT does disclose that these modifications are attached through 6-carbon linkers.

EXPERIMENTAL SECTION All DNA constructs were purchased from IDT (www.idtdna.com), with sequences shown in Table 1. Hemin was obtained from Frontier Scientific (Logan, UT), and all other salts, buffers and solvents, as well as 3,3′-diaminobenzidine (DAB), hydrogen peroxide, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), dicyclohexyl carbodiimide (DCC), N-hydroxy succinimide (NHS), N,N′-diisopropylethylamine (DIPEA), and dextran sulfate (sodium salt, molecular weight 6500-10 000 Da) were purchased from Sigma-Aldrich (St. Louis, MO). Avidin-agarose beads, neutravidin, peroxidase suppressor buffer, and d-biotin were purchased from Pierce (ThermoScientific, Rockford, Il). Salmon sperm DNA was purchased from Invitrogen (Carlsbad, CA). The monoclonal antiPSA antibody [ER-PR8 + PA05] was obtained from Labvision (Fremont, CA), and the goat anti-mouse-HRP antibody conjugate 115-035-146 was purchased from Jackson ImmunoResearch (West Grove, PA). The tyramide signal amplification kit “TSA Biotin System” was purchased from Perkin-Elmer Life Sciences (Waltham, MA). All human tissue samples were obtained with approval of the institutional review board of the University of Washington. Kinetics. Kinetic analyses were performed in a 96-well plate format on a Tecan Safire2 spectrophotometer at 24 ± 1 °C, using 50 mM HEPES/NH4OH pH 8.0, 20 mM KCl, 0.025% Tween-20, 10 mM H2O2, and 1 mM DAB, 270 nM DNAzyme-hemin conjugate unless otherwise indicated. Analysis of noncovalent DNA-hemin complexes were performed with 500 nM DNA, 500 nM free hemin, and identical buffer conditions unless otherwise indicated. DNA-hemin complexes (either covalent DNA-hemin complexes or 1:1 mixtures of DNA and free hemin) were prepared as stocks in 50 mM HEPES/NH4OH pH 8.0, 20 mM KCl, 0.025% Tween-20 prior to use by heating to 88 °C for 5 min and cooling on the benchtop to room temperature. Reactions were initiated by mixing 100 µL of DNA solution with 100 µL of peroxide/DAB solution in multiple wells simultaneously prior to initiating absorbance reads at 465 nm. Using known amounts of DAB oxidized to completion, we found that 1 OD unit in the microwell plate format was equivalent to 316 µM oxidized DAB reaction product. The pH dependence of catalysis was tested using MOPS, HEPES, and glycine buffers spanning the range of pH 6.1-9.5, all balanced with NH4OH. For cation dependence studies with

added KCl or ammonium acetate, pH 8.0 HEPES buffer was balanced with NaOH rather than NH4OH. To measure the dependence of DAB oxidation rate on H2O2 concentration, a lower covalent DNAzyme-hemin conjugate concentration of 96 nM was used because at higher DNA concentrations the reaction proceeded too quickly with H2O2 >50 mM to observe the linear phase with the available instrumentation. To attach hemin-DNAzyme complexes to avidin-agarose beads, hemin-DNA complexes were premade as indicated above and then mixed with beads in the standard buffer (50 mM HEPES/NH4OH pH 8.0, 20 mM KCl, 0.025% Tween-20) at stoichiometries predicted to saturate the beads (according to the manufacturer’s stated binding capacity). Beads were then exposed to 10 mM H2O2 and 1 mM DAB in this same buffer to assay staining. Hemin Conjugation. The activated ester of hemin was prepared by adding 2.8 equiv each of DCC, NHS, and DIPEA in 5 mL anhydrous DMF to 500 µmol hemin and letting the reaction proceed with gentle agitation in a sealed 15 mL tube. After overnight incubation at room temperature, a 10-fold volume of isopropanol was added to the reaction mixture, and the insoluble black solid was collected by centrifugation and washed again with isopropanol. The active ester was stored dry at -70 °C and dissolved into anhydrous DMSO prior to use. Electrospray positive ion mass spectrometry of hemin(NHS)2 demonstrated a major peak at m/z 805.6 (predicted [M - Cl]+ m/z 805.6), with no contaminating underivatized hemin. Hemin-DNA conjugates were prepared by adding hemin(NHS)2 in DMSO in a 40-fold molar excess to 50 nmol amino-functionalized DNA, either 5′-biotin-A15PS2.M-NH2 or 5′biotin-T15-NH2, in 200 µL of 100 mM NaHCO3, pH 8.5, and incubating with gentle mixing at room temperature for 24 h. Hemin-DNA conjugates were initially purified by two precipitations from ethanol. First, 1/10 volume of 3 M sodium acetate and 2.5 volumes of ice-cold absolute ethanol were added to the reaction mixture, and the mixture was incubated at -20 °C for 1 h. The precipitate was collected by centrifugation for 10 min at 21 000 g and redissolved in water, and the precipitation was repeated substituting 1/2 volume of 7.5 M ammonium acetate for sodium acetate. The DNA-hemin conjugate was Analytical Chemistry, Vol. 82, No. 6, March 15, 2010

2499

then resuspended in water and purified with a NAP-5 size exclusion column (GE Healthcare, Piscataway, NJ) to desalt and remove unbound hemin; no further purification was performed. Utilizing the extinction coefficients at 260 nm supplied by IDT and the reported extinction coefficient of DNAcomplexed hemin at 405 nm2, the absorbance spectra of the covalent derivatives were consistent with a 1:1 DNA-hemin complex (see Supporting Information Figure 1). Histochemistry. Tissue staining was performed on 5 µm thick sections of formalin-fixed, paraffin-embedded prostate tissue on charged glass slides, after heat-induced epitope retrieval consisting of microwaving slides for 20 min on power level 3 while immersed in pH 6.0 10 mM sodium citrate buffer. Endogenous peroxidase activity was quenched in a 30 min incubation in peroxidase suppressor buffer. The endogenous biotin background was blocked by a 1 h 0.05% w/v neutravidin incubation followed by a 1 h incubation in 0.05% w/v d-biotin. The monoclonal anti-PSA antibody was supplied in a prediluted, ready-to-use format, and applied directly to the tissue sections for 1 h, followed by a 30 min incubation with goat anti-mouse-HRP antibody conjugate. A commercial tyramide signal amplification kit was used according to the manufacturer’s guidelines, with the exception that the biotintyramide reagent was applied at a 1:200 dilution for 15 min. HRP added in the tyramide signal amplification step was destroyed by 30 min incubation in 3% H2O2, 150 mM NaN3, and 80% v/v Pierce peroxidase suppressor; this incubation was verified to destroy HRP with a no-DNA control on every run. A PS2.M-heminavidin complex was prepared by incubating 2 equivalents of 5′-Biotin-A15PS2.M-hemin with 1 equivalent of neutravidin for 1 h, and this complex was applied to tissue for 90 min in a sealed chamber at 62 °C in 600 mM NaCl, 60 mM sodium citrate, pH 7.0, supplemented with 40 µg/mL salmon sperm DNA, 10% dextran sulfate and 50% formamide. DAB was added to the sections at room temperature in 50 mM HEPES/NH4OH pH 8.0, 2.5 mM DAB, 200 mM H2O2, 20 mM KCl, 150 mM NaCl, and 0.025% Tween-20, and sections were inspected by light microscopy to assess the completeness of staining. After DAB staining, sections were counterstained with Mayer’s hematoxylin. RESULTS Reactivity of PS2.M toward DAB. Under conditions identical to those known to facilitate PS2.M activity toward the standard chromogenic substrate ABTS, the noncovalent PS2.M-hemin complex oxidizes DAB in solution to form a stable brown precipitate whose production can be monitored spectrophotometrically at 465 nm5 (Figure 1). Alternate DNA Constructs. Because PS2.M would likely need to be attached to another molecule to localize it in tissue, we investigated the ability of noncovalent hemin-PS2.M complexes to tolerate nucleotide and linker attachments to the DNA 5′ and 3′ ends. In addition, we investigated noncovalent complexes between hemin and alternate catalytic DNA sequences, as well as constructs with multiple concatenated PS2.M sequences, in an effort to determine if the catalytic activity per DNA construct could (5) Deerinck, T.; Martone, M.; Lev-Ram, V.; Green, D.; Tsien, R.; Spector, D.; Huang, S.; Ellisman, M. J. Cell Biol. 1994, 126, 901–10. (6) Bock, L.; Griffin, L.; Latham, J.; Vermaas, E.; Toole, J. Nature 1992, 355, 564–6.

2500

Analytical Chemistry, Vol. 82, No. 6, March 15, 2010

Figure 1. Absorbance vs time for DAB oxidized by a noncovalent DNA:Hemin complex. Conditions: 50 mM HEPES/NH4OH pH 8.0, 20 mM KCl, 0.025% Tween-20, 10 mM H2O2, and 1 mM DAB, 500nM Ps2.M, 500nM hemin.

be enhanced. When DNA constructs based on PS2.M were assayed at 10-fold excess over free hemin concentrations, only small differences in initial rates of DAB oxidation were noted between most constructs (Figure 2). The published DNA aptamer to thrombin,6 known to form a G-quadruplex, showed only mild catalytic enhancement over hemin alone, and a telomeric sequence from Chlamydomonas7 appeared to have similar activity to PS2.M. This corroborates earlier observations2 that not all G-quadruplexes bind hemin and promote peroxidase activity to comparable extents. Biotins and 5-adenine repeats attached to PS2.M appeared to increase the catalytic activity mildly, while a larger increase in activity was noted for PS2.M attached through 15 adenines to the published aptamer to MUC1,8 thus confirming that PS2.M retains activity when attached to other sequences. Constructs with multiple copies of PS2.M spaced apart by 5 base adenine linkers showed no improvement in rate enhancement over PS2.M alone, even in conditions in which hemin was present at a significant molar excess (not shown). Localized Staining with PS2.M. To determine whether or not PS2.M could direct localization of the colored DAB product, we first prepared a noncovalent complex between free hemin and 5′- biotinylated PS2.M, and attached this complex to avidin-agarose beads at saturating concentrations. On incubation with 1 mM DAB and 10 mM H2O2 (Figure 3), the beads became dark brown in several seconds. Avidin-agarose beads (not shown), beads containing biotin-oligo-dT (shown), and beads containing biotinylated random oligonucleotides (not shown) were preincubated with hemin and stained with DAB under identical conditions, and none turned brown, even over several hours. Once prepared, PS2.M-labeled agarose beads demonstrated essentially identical staining behavior after 3 weeks of storage at room temperature in an aqueous buffer or incubation at 95 degrees for 10 min. Limiting dilution experiments indicated that PS2.M bound to beads at 2 h of incubation, indicating a limit of detection in this system of approximately 2 × 109 PS2.M molecules per bead (data not shown). Initial efforts to use the 5′-biotin-(A)15-PS2.M and free hemin in the place of HRP in traditional immunohistochemistry assays were hampered by the high background caused by free hemin. Standard immunohistochemistry requires preincubating tissue in a concentrated H2O2 solution to destroy endogenous peroxidases from cytochromes and other heme proteins, but even as low as 1 µM hemin added to tissue after destroying endogenous peroxidase activity would generate a dark brown color in all cells in the presence of DAB and H2O2 (data not shown). To avoid the need to add free hemin, therefore, we sought to create a covalent hemin-PS2.M construct by attaching an activated N-hydroxysuccinimide diester of hemin to a 3′amino derivatized PS2.M construct. PS2.M-Hemin Conjugate Studies. Covalent PS2.M-hemin was found to have nearly equivalent activity to the noncovalent PS2.M-hemin complex in standard conditions, but while the

noncovalent complex demonstrated oxidative activity only at DNA/hemin concentrations above 100nM, activity of covalent PS2.M-hemin constructs was observable at concentrations as low as 10nM. The covalent PS2M-hemin showed activity in the agarose-avidin bead staining assay described previously, although beads labeled with a covalent oligo-dT-hemin complex did not, indicating that the bound hemin moiety alone does not possess significant catalytic activity unless linked to an appropriate DNA sequence. Kinetic analysis of the covalent (Figures 4-8) and noncovalent PS2.M-hemin complexes (Supporting Information Figure 2) yielded similar results. Both covalent and noncovalent constructs demonstrated that a low concentration of nonionic surfactant and a pH near 8.0 were optimal for catalysis, similar to prior reports. In contrast to the noncovalent DNAzyme, however, the covalent DNAzyme-hemin construct had essentially equivalent activities in the presence or absence of either potassium or ammonium cations (Figure 5). DNAzyme-catalyzed DAB oxidation rates were monitored as a function of DAB concentration (Figure 6). While the activity of covalent or noncovalent PS2.M-hemin complexes toward ABTS had no dependence on ABTS concentrations from 0.25-5 mM (data not shown), the rate of DAB oxidation depended markedly on DAB concentration, showing an increase in rate with increasing DAB at low DAB concentrations, and a significant inhibition of rate with higher DAB concentrations. The dependence of the DAB oxidation rate of both covalent and noncovalent complexes on H2O2 concentration also differs from previous reports,2 in that, while apparent kcat values were comparable to previous reports (∼500 s-1), the apparent Km of the of both covalent and noncovalent complexes for H2O2, approximately 50-100 mM, is much higher (Figure 7). From 0 to 200 mM H2O2, the rate of DAB oxidation by hemin alone was negligible compared to the rateobservedwitheithercovalentornoncovalentDNAzyme-hemin complexes (not shown), indicating that oxidation catalyzed by hemin alone is not responsible for this phenomenon. Analytical Chemistry, Vol. 82, No. 6, March 15, 2010

2501

Figure 4. Effects of pH (A) and surfactant concentration (B) on catalyzed DAB oxidation rate.

Figure 7. Michaelis-Menten plot of catalyzed oxidation rate vs H2O2 concentration. Figure 5. Effect of added cations (potassium, open diamonds; Ammonium, dark squares) on catalyzed DAB oxidation rate.

Figure 6. Substrate inhibition in PS2.M-hemin catalyzed oxidation of DAB.

Immunohistochemistry. Once characterized, the covalent PS2.M-hemin construct was used in place of HRP in an immunohistochemistry experiment. A monoclonal antibody to PSA was 2502

Analytical Chemistry, Vol. 82, No. 6, March 15, 2010

applied to formalin-fixed, paraffin embedded prostate tissue, followed by a biotinylated secondary antibody, avidin, and then 5′-biotin-(A)15-PS2.M-hemin. The DNAzyme was visualized with DAB and H2O2. Despite an appropriately positive control using HRP, no specific DAB signal was detected in the tissue. Therefore, a commercial tyramide signal amplification kit was used to amplify the biotin signal prior to application of avidin and 5′-biotin-A15-PS2.M-hemin. Tyramide signal amplification is a standard, commercialized protocol that involves labeling tissue biotins first with an avidin-linked peroxidase, and then using that peroxidatic activity to catalyze attachment of hundreds or thousands more biotinyl moieties to nearby molecules via a highly reactive oxidized biotin-tyramide intermediate. The newly deposited biotins can then be used to attach additional avidin-peroxidase complexes to the site of interest, thus creating a greatly amplified signal. Under these conditions, unambiguous DAB staining was visible in 30 min with 5′-biotin(A)15-PS2.M-hemin (Figure 8A), whereas only 3 min were required for a dark stain reaction using HRP instead of the DNAzyme (Figure 8B). Of note, because tyramide signal amplification requires HRP, care was taken to inactivate the HRP used for tyramide amplification prior to DNAzyme application. All negative controls, including a no-primary negative control and a no DNA-hemin control, lacked staining appropriately.

Figure 8. Immunohistochemistry for prostate specific antigen in prostate tissue, developed with the hemin-PS2.M covalent construct for 30 min (A) or HRP for 3 min (B).

DISCUSSION Aptamer histochemistry (AHC) is a novel conceptual approach to addressing some of the methodologic shortcomings of IHC. However, current aptamer and DNAzyme technology has not focused on the problems inherent to solid tissue diagnosis. In this study, we report the first attempted use of a covalent DNAzymehemin complex to detect a protein in solid tissue sections. While the focus of our efforts has been to optimize a reagent for use in tissue, and the focus of this report is to describe the efforts taken to optimize an application, we collected data during the characterization of the reagent that raise questions about the nature of the DNAzyme structure and function. First, we have found that the covalent PS2.M-hemin construct does not require added potassium or ammonium ions to achieve catalytic activity, which is a significant difference from what we found working with the noncovalent complex and DAB, as well as from prior reports on PS2.M.2 PS2.M has been shown to adopt a potassium- or ammonium-dependent G-quadruplex structure that is essential for catalytic activity,9 and prior studies have G quadruplex structure can be greatly affected by the potassium, ammonium, and sodium counterions.10 It is possible that enough ammonium to facilitate proper folding was associated with the covalent construct during purification (ethanol/ammonium acetate precipitation), but because the construct was desalted several times during purification, the total concentration of any cation carried over from synthesis into the kinetics reactions would be many fold lower than what has previously reported as necessary for activity. Therefore, we postulate that either the attached hemin stabilizes the G-quadruplex structure or else that the free energy benefit of solvating the hydrophobic hemin moiety in the DNA complex rather than in the surrounding water is sufficient to drive G-quadruplex formation even in the absence of favorable cations. Further detailed studies of the structure of this complex, including whether or not it functions as a monomer or oligomer, may help explain this behavior. The kinetic analysis of DAB oxidation by PS2.M in both covalent and noncovalent complexes with hemin cannot adequately be described with a simple Michaelis-Menten model, because several of the assumptions made in this model do not hold. Thus, interpretations of the kinetic parameters derived in (9) Travascio, P.; Witting, P.; Mauk, A.; Sen, D. J. Am. Chem. Soc. 2001, 123, 1337–48. (10) Smargiasso, N.; Rosu, F.; Hsia, W.; Colson, P.; Baker, E.; Bowers, M.; De Pauw, E.; Gabelica, V. J. Am. Chem. Soc. 2008, 130, 10208–16.

this work in light of those found in other works must be done with caution. The primary source of this complexity is probably the mechanism by which DAB forms its colored polymeric precipitate, which is the substance that we detect in our assay. DAB, unlike ABTS, polymerizes upon oxidation to form its colored product, so it is unsurprising that the rate of oxidation initially increases with increasing DAB concentration. However, the suppression of the DAB signal at very high DAB concentrations is not necessarily expected. Mechanistically, substrate inhibition of this sort might occur if the readout signal (measured absorbance at 465 nm) depended on both the size and number of polymeric DAB particles formed in a reaction. Since the absorbance at 465 nm is probably caused by light scatter, there should be some concentration of DAB above or below which the preference for forming relatively few large DAB polymer particles would switch to a preference for creating numerous small DAB polymer particles with different light scattering properties. While this could explain the observed substrate inhibition phenomenon, it is also possible that increased rates of DAB polymer production serve to trap active catalytic sites within polymer networks that sterically hinder the deliver of additional hydrogen peroxide or DAB monomers; further studies are warranted to investigate these possibilities. Yet another alternative explanation of the observed substrate inhibition is that DAB radicals might themselves destroy the DNA-hemin complex. The fact that ABTS shows no substrate inhibition indicates that not all radicals can destroy the DNA-hemin complex, but we are currently investigating the structural changes that occur in the DNA-hemin complex during catalysis to address this question. CONCLUSION As a histochemical reagent, the covalent PS2.M-hemin conjugate demonstrates both promise and problems. On the plus side, it is technically feasible to use the construct in the place of HRP in an otherwise standard IHC experiment. In addition, the conjugate maintains near-maximal activity in buffer conditions that are similar to physiological conditions, it retains activity when attached to different DNA sequences, and it avoids the highbackground problems seen with adding free hemin to tissue. Furthermore, it is smaller than a protein, easy to totally synthesize chemically, and stable to long storage and temperatures that denature many proteins. While the cost of the reagent produced on the scales used in this study are not a significant advantage Analytical Chemistry, Vol. 82, No. 6, March 15, 2010

2503

over a protein reagent, economies of scale could easily reduce this cost, and the opportunity cost of creating additional variants of this construct in an optimization effort would likely be much less costly than protein engineering efforts. The major drawback of this detection reagent currently, however, is that it is still a few orders of magnitude less sensitive than HRP. This is not surprising, after all, because HRP has evolved over millions of years into the highly efficient, diffusion-limited enzyme that it is today whereas PS2.M has undergone very little in terms of mutation and selection over the few years it has existed. A primary advantage of working with aptamers and DNAzymes, however, is that the methodology for optimizing reagents through in vitro evolution is so robust. There is no reason to believe, therefore, that the catalytic activity of PS2.M and, thus, its utility as a diagnostic reporter element in tissue assays or other diagnostic tests requiring a localized colorimetric readout cannot be signifi-

2504

Analytical Chemistry, Vol. 82, No. 6, March 15, 2010

cantly enhanced through some combination of rational or random mutagenesis, concatenation, amplification via nanoparticle conjugation, or the use of other metal and porphyrin cofactors. ACKNOWLEDGMENT The authors thank Dr. Dipankar Sen for helpful comments. The authors disclose research funding for other work from SomaLogic, Inc. a biotechnology company that produces aptamers. SUPPORTING INFORMATION AVAILABLE Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 17, 2009. Accepted February 3, 2010. AC902887J