Poly(dG) Spacers Lead to Increased Surface Coverage of DNA

Jun 12, 2008 - Department of Chemistry, University of Florida, Gainesville, Florida 32611-7200, Laboratoire de Université de Nantes, CNRS, UMR 6230, ...
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Langmuir 2008, 24, 7394-7399

Poly(dG) Spacers Lead to Increased Surface Coverage of DNA Probes: An XPS Study of Oligonucleotide Binding to Zirconium Phosphonate Modified Surfaces Sarah M. Lane,† Julien Monot,‡ Marc Petit,‡ Charles Tellier,§ Bruno Bujoli,*,‡ and Daniel R. Talham*,† Department of Chemistry, UniVersity of Florida, GainesVille, Florida 32611-7200, Laboratoire de UniVersite´ de Nantes, CNRS, UMR 6230, Chimie et Interdisciplinarite´: Synthe`se Analyse Mode´lisation (CEISAM), UFR Sciences et Techniques, 2 rue de la Houssinie`re, BP 92208, 44322 Nantes Cedex 3, and Nantes Atlantique UniVersite´s, CNRS, UMR 6201, Laboratoire de Biotechnologie, Biocatalyse et Biore´gulation, 2 rue de la Houssinie`re, BP 92208, 44322 Nantes Cedex 3, France ReceiVed December 30, 2007. ReVised Manuscript ReceiVed March 5, 2008 A spacer is often employed between the surface linking group and the probe sequence to improve the performance of DNA microarrays. Previous work demonstrated that a consecutive stretch of guanines as a spacer increased target capture during hybridization relative to probes with either no spacer or a similar stretch of one of the other nucleotides. Using zirconium phosphonate modified surfaces with 5′-phosphorylated ssDNA probes, the present study compares the surface coverage of ssDNA probes containing either a poly(dG) spacer or a poly(dA) spacer. Surface coverages are quantified by XPS using a modified overlayer model. The results show that after treatment to mimic conditions of the passivation and hybridization steps the probe with the poly(dG) spacer has about twice the surface coverage as the probe with the poly(dA) spacer, indicating that increased target capture is due to higher probe coverage. When monitoring the surface coverage after each rinsing step, it is observed that the probe with the poly(dA) spacer is more susceptible to rinsing, suggesting the interaction with the surface is different for the two probes. It is suggested that the formation of G quadruplexes causes an increased avidity of the probe for the zirconium phosphonate surface.

Introduction DNA microarrays are a recently developed tool that allows high-throughput, highly parallel investigation of DNA structure. One of the many features often employed to improve the performance of DNA microarrays is the placement of a spacer between the probe sequence and the surface-linking group.1–4 The spacer, which creates distance between the linking group and the probe sequence, is thought to lift the probe off the surface to provide better contact with the solution phase. A number of different functional groups have been studied as spacers. For DNA microarrays on gold, a simple alkyl chain, often six carbons long, is frequently used.5 In addition, a poly(ethylene glycol) spacer on DNA has been used for arrays on gold to study DNA-protein interactions.6 On the other hand, a stretch of nonparticipating nucleotides can also be employed. For example, Guo et al. studied arrays of 3 mm diameter spots of 5′-aminefunctionalized probes on an isocyanate-modified surface with * To whom correspondence should be addressed. Tel: +1 352 392 9016. Fax: +1 352 392 3255. E-mail: [email protected], Bruno.Bujoli@ univ-nantes.fr. † University of Florida. ‡ Chimie et Interdisciplinarite´: Synthe`se Analyse Mode´lisation. § Laboratoire de Biotechnologie, Biocatalyse et Biore´gulation. (1) Halperin, A.; Buhot, A.; Zhulina, E. B. Langmuir 2006, 22, 11290–11304. (2) Hong, B. J.; Oh, S. J.; Youn, T. O.; Kwon, S. H.; Park, J. W. Langmuir 2005, 21, 4257–4261. (3) Shchepinov, M. S.; Case-Green, S. C.; Southern, E. M. Nucleic Acids Res. 1997, 25, 1155–1161. (4) Nonglaton, G.; Benitez, I. O.; Guisle, I.; Pipelier, M.; Le´ger, J.; Dubreuil, D.; Tellier, C.; Talham, D. R.; Bujoli, B. J. Am. Chem. Soc. 2004, 126, 1497– 1502. (5) Georgiadis, R.; Peterlinz, K. P.; Peterson, A. W. J. Am. Chem. Soc. 2000, 122, 3166–3173. (6) Brockman, J. M.; Frutos, A. G.; Corn, R. M. J. Am. Chem. Soc. 1999, 121, 8044–8051.

poly(dT) spacers 0, 3, 6, 9, 12, or 15 nucleotides long.7 No hybridization was observed without a spacer, but starting with 9 nucleotides an increase in hybridization was seen, which continued up to 15 nucleotides. During the course of investigation aimed at developing inorganic surfaces exhibiting specific DNA probe binding for array applications, we observed enhanced target capture when using poly(dG) segments as spacers relative to other homooligomers. For example, with a zirconium phosphonate surface and fluorescence detection we observed that a stretch of at least five guanines leads to increased hybridization relative to other spacers in model studies using 33-mer probe sequences and corresponding 33-mer targets.4 It is reasonable that this “poly-G effect” is related to the ability of oligomers of guanines to form quadruplex structures, as illustrated in Figure 1, and we identified two possible consequences of quadruplex formation that would give an increase in target capture. One possibility is that the poly(dG)-modified probes bind to the surface more effectively, leading to an increase in the probe density, thus increasing the number of binding sites available for the target. The other possibility is that the poly(dG) spacer orients the probe for more efficient hybridization. The zirconium phosphonate modified surfaces used in these studies are known to efficiently bind organophosphonates and organophosphates. For example, alkylphosphonates and other phosphonate-modified molecules, including porphyrins, azobenzenes, and others, have been successfully immobilized on zirconium phosphonate-derivatized substrates, and for many cases organized self-assembled monolayers are achieved.8,9 Similarly, (7) Guo, Z.; Guilfoyle, R. A.; Thiel, A. J.; Wang, R.; Smith, L. M. Nucleic Acids Res. 1994, 22, 5456–5465. (8) Benitez, I. O.; Bujoli, B.; Camus, L. J.; Lee, C. M.; Odobel, F.; Talham, D. R. J. Am. Chem. Soc. 2002, 124, 4363–4370. (9) Wu, A. P.; Talham, D. R. Langmuir 2000, 16, 7449–7456.

10.1021/la704049h CCC: $40.75  2008 American Chemical Society Published on Web 06/12/2008

Oligonucleotide Binding to Modified Surfaces

Figure 1. Illustration of how the G quadruplex might hold together the probe DNA, raising the avidity of the complex for terminal phosphate binding to the surface relative to a single strand.

Langmuir, Vol. 24, No. 14, 2008 7395

is a powerful surface analytical technique which provides qualitative and quantitative information about the elements on a surface. Recently, XPS was shown to be an efficient, label-free method for studying DNA surface coverage.15–21 Petrovykh et al. showed that with XPS data the density of thiol-modified DNA on gold could be determined with an overlayer model.20,21 Similarly, we demonstrated that XPS can be used to determine the DNA surface density on zirconium phosphonate surfaces.22 The XPS comparison of the probe layers containing poly(dG) and poly(dA) spacers reported here demonstrates that the poly(dG)-containing probes achieve higher surface coverage, which is the likely cause of the poly-G effect seen previously. In the course of these studies we investigated several rinsing conditions with the poly(dA) and poly(dG) spacers. The effect of rinsing has been discussed on limited occasions in the literature.23,24 Yuak et al. demonstrated that automated rinsing compared to manual decreases variability across mouse cDNA microarrays spotted onto UltraGAPS slides (amine-coated slides manufactured by Corning Inc.).23 Han et al. varied the stringency of the washing conditions of rat oligonucleotide microarrays made by Clontech and found improved signal-to-background ratios after hybridization.24 In this current study, by monitoring the surface coverage following successive rinsing steps, it is observed that the poly(dG) probes and poly(dA) probes respond differently to rinses of increasing stringency, indicating that probe molecules with poly(dG) and poly(dA) spacers interact differently with the zirconium phosphonate surface.

Experimental Section

Figure 2. Illustration of the zirconium phosphonate surface used to immobilize phosphorylated DNA followed by hybridizing with the complementary strand.

zirconium phosphonate surfaces can be used to immobilize DNA,4,10 and we have found that the zirconium phosphonate surface selectively binds phosphorylated DNA over nonphosphorylated DNA, attributable to the specific binding of the terminal phosphate in preference to weaker binding of the phosphodiester backbone (Figure 2). Although there are a number of different methods for preparing zirconium phosphonate substrates to be used for self-assembly of phosphate containing molecules,11–14 we have found that zirconium phosphonate monolayers made using the Langmuir-Blodgett technique provide a robust, densely zirconated surface, which we have observed to work best for the 5′-phosphorylated DNA microarrays.4 To determine if the poly(dG) effect is caused by an increase in probe density, we report here on an X-ray photoelectron spectroscopy (XPS) study comparing the DNA surface coverage of zirconium phosphonate slides modified with probe sequences containing poly(dG) and poly(dA) homooligomer spacers. XPS (10) Xu, X.-H.; Yang, H. C.; Mallouk, T. E.; Bard, A. J. J. Am. Chem. Soc. 1994, 116, 8386–8387. (11) Mitzi, D. B. Chem. Mater. 2001, 13, 3283–3298. (12) Lee, H.; Kepley, L. J.; Hong, H. G.; Akhter, S.; Mallouk, T. E. J. Phys. Chem. 1988, 92, 2597–2601. (13) Lee, H.; Kepley, L. J.; Hong, H. G.; Mallouk, T. E. J. Am. Chem. Soc. 1988, 110, 618–620. (14) Byrd, H.; Whipps, S.; Pike, J. K.; Talham, D. R. Thin Solid Films 1994, 244, 768–771.

Glass substrates were purchased from Gold Seal Products. Oligonucleotides were ordered lyophilized with HPLC purity from Invitrogen (Carlsbad, CA). Reagents were of analytical grade and used as received from commercial sources unless indicated otherwise. Hydrophobic glass slides were made using octadecyltrichlorosilane (OTS) following a method by Sagiv.25 The zirconium phosphonate Langmuir-Blodgett monolayers were prepared on the hydrophobic slides as described previously.8 The ssDNA sequence used was 5′-H2O3PO-(X)9,11-CCGCCGGTAACCGGAGGTTAAGATCGAGATCCA. In the sequence, X represents either guanine or adenine, which was either 11 or 9 nucleotides long. The ssDNA solutions were prepared in a 1 × SSC (saline sodium citrate) buffer, pH 6 at a concentration of 40 µM. To create a spot large enough for XPS analysis, 30 µL of the DNA was pipetted onto the rinsed and dried zirconium phosphonate surface. The slides were placed in Petri dishes overnight at room temperature. Several different rinsing conditions were employed before XPS was taken. For rinsing set 1, the slide was submerged successively in 2 × SSC, 0.1% SDS (sodium dodecylsulfate) (2 min), 1 × SSC (2 min), and 0.2 × SSC (2 times, 2 min), followed by dipping twice in water. Rinsing set 2 began with the spotted slide being immersed (15) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916–8920. (16) Saprigin, A. V.; Thomas, C. W.; Dulcey, C. S.; Patterson, C. H.; Spector, M. S. Surf. Interface Anal. 2005, 37, 24–32. (17) Shen, G.; Anand, M. F. G.; Levicky, R. Nucleic Acids Res. 2004, 32, 5973–5980. (18) Kimura-Suda, H.; Petrovykh, D. Y.; Tarlov, M. J.; Whitman, L. J. J. Am. Chem. Soc. 2003, 125, 9014–9015. (19) Leavitt, A. J.; Wenzler, L. A.; Williams, J. M.; Thomas, P.; Beebe, J. J. Phys. Chem. 1994, 98, 8742–8746. (20) Petrovykh, D. Y.; Kimura-Suda, H.; Tarlov, M. J.; Whitman, L. J. Langmuir 2004, 20, 429–440. (21) Petrovykh, D. Y.; Kimura-Suda, H.; Whitman, L. J.; Tarlov, M. J. J. Am. Chem. Soc. 2003, 125, 5219–5226. (22) Lane, S. M.; Monot, J.; Petit, M.; Bujoli, B.; Talham, D. R. Colloids Surf., B 2007, 58, 34–38. (23) Yauk, C.; Berndt, L.; Williams, A.; Douglas, G. R. J. Biochem. Biophys. Methods 2005, 64, 69–75. (24) Han, T.; Melvin, C. D.; Shi, L.; Branham, W. S.; Moland, C. L.; Pine, P. S.; Thompson, K. L.; Fuscoe, J. C. BMC Bioinformatics 2006, 7(Suppl 2), S17. (25) Frydman, E.; Cohen, H.; Maoz, R.; Sagiv, J. Langmuir 1997, 13, 5089– 5106.

7396 Langmuir, Vol. 24, No. 14, 2008 for 1 h in 3.5 × SSC, 0.3% SDS at 42 °C, followed by rinsing five times with nanopure water and spin-drying. Rinsing set 3 used the 1 h submersion in 3.5 × SSC, 0.3% SDS at 42 °C, followed by rinsing five times with nanopure water and spin-drying. In rinsing set 3, the slides then underwent a mock hybridization, which should give the true probe concentration as if they underwent hybridization, by treatment with 25µL per spot of 3 × SSC, 0.1% SDS overnight at 42 °C. Finally, the slides were rinsed in 2 × SSC, 0.1% SDS (2 min), 1 × SSC (2 min), and 0.2 × SSC (2 times, 2 min), followed by rinsing in water five times. At least four spots were examined at each different rinsing condition. XPS was performed using a UHV XPS/ESCA PHI 5100 system. Survey scans and multiplex scans (Zr 3d, P 2p, and N 1s) were taken with a Mg KR X-ray source using a power setting of 300 W and a take-off angle of 45° with respect to the surface. Survey scans were taken for all samples with a pass energy of 89.4 eV, and multiplex scans were taken with a pass energy of 22.36 eV. Using commercial XPS analysis software and Shirley background subtraction, the peak areas were determined. Four different spots were analyzed to determine the scatter of the data. The analyzer transmission function, which is necessary for the surface coverage calculations, was determined using a method by Weng.26 To find the analyzer transmission function with this method, the XPS intensities of the C 1s, O 1s, Zr 3d, and Zr 3p3 peaks of a zirconium phosphonate substrate were analyzed at nine different pass energies: 179.0, 143.0, 89.5, 71.5, 44.7, 35.7, 22.4, 18.0, and 11.2 eV.

Results The concept of using an inorganic surface for DNA microarrays is different than most other strategies in that it uses a coordinatecovalent bond to link the probe to the surface. One other example, aside from the zirconium phosphonate system, that employs a coordinate-covalent linkage is the use of thiol-modified DNA to bind to gold. Similar to gold’s selectivity for thiol-modified DNA, the zirconium phosphonate surfaces preferentially bind phosphorlyated DNA over nonphosphorylated DNA through a coordinate covalent bond between the terminal phosphate on the DNA and the zirconium ions on the surface.4 When phosphorylated and nonphosphorylated probes were spotted onto zirconium phosphonate modified slides and exposed to fluorescently labeled target, intense fluorescence was observed at phosphorylated probe spots but only minimal fluorescence for the nonphosphorylated probes.4 Initial experiments using XPS22 to quantify DNA probe binding onto the zirconium phosphonate surface indicated surface coverages on the order of 1011 molecules/ cm2, significantly less than a close-packed monolayer. The low surface density suggests that, along with specific binding of the phosphate linker, there may be some nonspecific adsorption along the DNA strand, which ultimately limits the final surface coverage by blocking access of additional probes. However, it is seen from the fluorescence data that the surface coverage obtained is sufficient for microarray experiments. XPS Determination of Surface Coverage. XPS is often used to determine the thickness and surface coverage of thin films by quantifying the attenuation of substrate photoelectrons as they pass through an overlayer thin film on their way to the analyzer. A simple substrate overlayer model generally assumes an infinitely thick substrate and a finitely thick overlayer. We use this approach to quantify the coverage of the oligonucleotide overlayer on zirconium phosphonate modified supports. However, the calculation is slightly different because the zirconium ions from the substrate are not infinitely thick, but rather arranged in a monolayer, which can be assumed not to attenuate itself (illustrated in Figure 3). A thorough discussion of these (26) Weng, L. T.; Vereecke, G.; Genet, M. J.; Bertrand, P.; Stone, W. E. E. Surf. Interface Anal. 1993, 20, 179–192.

Lane et al.

Figure 3. Illustration of the EALs used in the modified overlayer model for the zirconium phosphonate/DNA calculations. As illustrated, LNA is the average EAL of the N 1s photoelectron as it travels through the DNA overlayer, LNQ is the EAL for quantitative analysis of the N 1s electron A as it travels through the DNA overlayer, and LZr is the average EAL of the Zr 3d photoelectron as it travels through the DNA overlayer.

calculations is given in a previous paper.22 Briefly, the intensity of the zirconium XPS peak attenuated by an overlayer is given by A IZr ) CnZrσZrTZrexp(-t ⁄ (LZr sinθ))

(1)

for which C is a constant which accounts for instrumental parameters such as the X-ray flux and analyzed sample area; nZr is the surface coverage of zirconium ions; σZr is the photoelectric cross section of the Zr 3d photoelectron; TZr is the analyzer transmission function of the spectrometer for the Zr 3d photoA electron; t is the thickness of the overlayer or DNA; LZr is the average effective attenuation length (EAL) of the zirconium photoelectron as it travels through the DNA layer; and θ is the angle of the photoelectron detection with respect to the sample surface. Scofield coefficients are usually used for the photoelectric cross section.27 To calculate the EALs needed for these equations, the NIST SRD-82 software was used.28 The EALs, as stated in the manual for the software, are calculated from analytical expressions derived from the solution of the kinetic Boltzman equation within the transport approximation. There is an excellent paper on EALs written by Powell and Jablonski, which also briefly discusses the NIST SRD-82 software.29 Equation 1 can be expressed in terms of the intensity from the bare substrate, 0 IZr , attenuated by the DNA layer: 0 A IZr ) IZr exp(-t ⁄ (LZr sinθ))

(2)

Using this equation and the intensity of the zirconium peak before and after DNA coverage, the thickness of the DNA film can be calculated. Similarly, the intensity of the peak coming from nitrogen in the DNA overlayer, IN, can be written as

IN ) CNNσNTNLNQ sinθ(1 - exp(-t ⁄ (LNA sinθ)))

(3)

With NN being the atomic density of nitrogen, σN the photoelectric cross section of the N 1s photoelectron and TN the analyzer transmission function of the N 1s photoelectron. The parameter LNA is the average practical EAL of the nitrogen photoelectrons as they travel through the DNA layer and LNQ is the EAL for quantitative analysis. The terminology used here matches the EAL output values in the NIST SRD-82 software. An in-depth discussion of the difference in the definitions of the EALs for quantitative analysis and the average EALs is given in a review (27) Scofield, J. H. J. Electron Spectrosc. Relat. Phenom. 1976, 8, 129–137. (28) Powell, C. J.; Jablonski, A. NIST Electron EffectiVe-Attenuation-Length Database, Version 1.0 (NIST SRD-82), U.S. Department of Commerce National Institute of Standards and Technology: Gaithersburg, 2001. (29) Powell, C. J.; Jablonski, A. Surf. Interface Anal. 2002, 33, 211–229.

Oligonucleotide Binding to Modified Surfaces

Langmuir, Vol. 24, No. 14, 2008 7397 Table 1. N 1s/Zr 3d Peak Area Ratio and Surface Coverage of the Probe Molecules after Different Rinsing Conditionsa IN/IZr(peak area ratio)

surface coverage(ssDNA/cm2)

poly-A poly-G

Rinsing Set 1a 0.185 ( 0.025 12.7 × 1011 ( 1.3 × 1011 0.0929 ( 0.011 6.35 × 1011 ( 0.70 × 1011

poly-A poly-G

Rinsing Set 2b 0.0673 ( 0.014 4.78 × 1011 ( 0.92 × 1011 0.0421 ( 0.016 4.81 × 1011 ( 1.1 × 1011

poly-A poly-G

Rinsing Set 3c 0.0345 ( 0.0078 2.53 × 1011 ( 0.60 × 1011 0.0688 ( 0.012 5.01 × 1011 ( 0.91 × 1011

a Rinsing set 1, the least stringent, consists of a several brief rinsings in SSC/SDS buffer. b Rinsing set 2, which mimics a passivation step, involves submersion of the slide in an SSC/SDS buffer at 42°C. c Rinsing set 3, which mimics a passivation step followed by hybridization, includes rinsing set 2 followed by several SSC/SDS rinsings, overnight treatment with hybridization buffer, and several more brief rinsings with an SSC/SDS buffer.

Figure 4. XPS N 1s peak coming from the DNA film and the Zr 3d peak coming from the zirconium phosphonate monolayer of the poly-Acontaining probe after rinsing set 1. The intensities of the N 1s peaks and Zr 3d peaks were used to calculate the DNA surface coverage.

paper by Jablonski and Powell.30 Alternatively, the P 2p peak could have been used for the surface coverage calculations instead of the N 1s peak, but phosphorus is present in the zirconiumphosphonate monolayer, it has a lower sensitivity factor and, compared to nitrogen, phosphorus is less abundant in DNA. For these reasons the N 1s peak instead of the P 2p was used in the calculations and, in fact, it was difficult to discern a difference in the P 2p peak intensity before and after DNA immobilization. The atomic surface coverage, nN (atoms/cm2) can be expressed as the product of the atomic density (atoms/cm3) and the overlayer thickness,

nN ) NNt

(4)

and using this relationship and eqs 1 and 3, the ratio of the surface coverage of nitrogen to zirconium can be found A INσZrTZrtexp(-t ⁄ (LZr nN sinθ)) ) nZr I σ T LQ sinθ(1 - exp(-t ⁄ (LA sinθ))) Zr N N N N

(5)

The ratio allows the constant instrumental parameters such as the X-ray flux to cancel out. Using the number of nitrogens in the DNA sequence and the known surface coverage of the zirconium ion layer, 4.2 × 1014 atoms/cm2,31 the DNA coverage can be calculated. An example of a N 1s peak, originating from the DNA film, and a Zr 3d peak, originating from the zirconium phosphonate monolayer, which are used in the calculations of the DNA surface coverage, are shown in Figure 4. Samples and Rinsing Protocols. The probe surface coverage was investigated using the two different spacers and several rinsing protocols. Rinsing, although not a heavily investigated parameter, is a key step in the DNA microarray process. After (30) Jablonski, A.; Powell, C. J. Surf. Sci. Rep. 2002, 47, 33–91. (31) Byrd, H.; Pike, J. K.; Talham, D. R. Chem. Mater. 1993, 5, 709–715.

the DNA is spotted onto a surface, it is generally rinsed before hybridization. The rinsing solutions often contain a buffer, such as SSC, and a detergent such as SDS, and a solution that contains less salt and more surfactant is considered more stringent. The slide may be heated during the rinsing process, also increasing stringency. A passivation step can be used in between the rinsing and hybridization. Passivation employs a protein, such as bovine serum albumin (BSA), which binds to the surface where there is no probe to prevent target molecules from physisorbing to the surface away from the probe, ultimately improving the signalto-noise ratio. After the initial rinsing step, the passivation step and hybridization process can also rinse off more probe. Three different rinsings with increasing stringency, representing typical treatments during each step in the microarray process, were investigated in this study. Rinsing set 1, normally employed after the initial spotting of probes, involves dipping the spotted slide in an SSC/SDS solution, in several SSC solutions, and several times in water. In rinsing set 2, typical of the passivation step, the slide is submerged in an SSC/SDS solution at 42 °C for 1 h. Rinsing set 2 is more stringent than rinsing set 1 because of the increased surfactant concentration, the heating, and the increased time the slide stays in the solution. Also, the increased number of water rinsings at the end of rinsing set 2 raises the stringency. In rinsing set 3, the slide is again submerged in an SSC/SDS solution at 42 °C for 1 h, and then the spotted area is subjected to conditions which mimic a hybridization step. The blank hybridization step involves treating the spot area with hybridization buffer but containing no target DNA. The blank hybridization is followed by several more rinsings in SSC/SDS solutions. Due to the increased length of the rinsings and heating, rinsing 3 is more stringent than rinsing sets 1 and 2. Surface Coverage Results. A comparison of the XPSdetermined surface coverages of the poly(dA)-containing probe and poly(dG)-containing probe is shown in Table 1. The surface coverage of the poly(dG)-containing probe stayed relatively constant with the different rinsing procedures, but the surface coverage of the probe containing the poly(dA) spacer decreased with increasing rinsing stringency. After spotting and with the least stringent SSC rinsing conditions (rinsing set 1), the calculated surface coverage of the poly(dA), 12.7 × 1011 ( 1.3 × 1011 ssDNA molecules/cm2, is twice that of the poly(dG)-containing probe, 6.3 × 1011 ( 0.7 × 1011 ssDNA molecules/cm2. After rinsing set 2, the surface coverages are essentially equal, 4.8 × 1011 ( 1.1 × 1011 ssDNA molecules/cm2 for the poly(dA) containing probe and 4.8 × 1011 ( 0.9 × 1011 ssDNA molecules/ cm2 for the poly(dG)-containing probe. Finally, after treatment with rinsing set 3, the surface coverage of the poly(dG)-containing

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probe remains unchanged, 5.0 × 1011 ( 0.9 × 1011 ssDNA molecules/cm2, but is now twice that of the poly(dA)-containing probe, 2.5 × 1011 ( 0.6 × 1011 ssDNA molecules/cm2. Also shown in Table 1 are the peak area ratios for the N 1s and Zr 3d photoelectrons, which are used in eq 5, from which the surface coverage was calculated. The peak area ratios show a little more fluctuation than the surface coverages but, in general correspond well with the surface coverage data. The uncertainty shown with the data is the standard deviation of the mean from measurements of at least four different spots for each probe under each set of conditions.

Discussion Earlier studies that used fluorescence imaging to monitor target capture in microarrays of 33-mer probes compared the responses at probes containing 11-mer homooligonucleotide spacers of either thymine, guanine, cytosine, or adenine between the probe sequence and the 5′-phosphate to probes with no spacer.4 It was seen that, irrespective of probe sequence and concentration, the poly(dG) spacer resulted in the highest target capture, roughly two to three times that of the probe with no spacer. The probes containing the poly(dA) spacer gave the same target capture as the probes with no spacer, and those containing the poly(dT) and poly(dC) spacer actually led to slightly less target capture than the controls with no spacer. The influence of the length of the spacer was also investigated, and enhanced target capture was seen for spacers of more than five guanines, with the highest response for spacers of 7-11 guanines. The XPS results shed light on the reason for the increased target capture when a poly(dG) spacer is used. The likely causes are that the probes with a poly(dG) spacer either bind the substrate better, leading to higher probe density, or better position the probe sequence for hybridization, leading to enhanced hybridization efficiency. The XPS analysis indicates that after the mock hybridization, rinsing set 3, the final probe coverage of the poly(dG)-containing probe is approximately twice that of the poly(dA)-containing probe. This increased probe coverage nearly mirrors the increase in target capture previously observed using fluorescence detection. From this result, we can conclude that the reason for increased target capture when using a poly(dG) spacer is higher surface density of probe molecules. Although this is the first report of a poly(dG) spacer giving increased probe surface density on a zirconium-phosphonate surface, Saprigin et al. recently performed an XPS study of homooligonucleotide attachment to amine-modified surfaces. The 3′-phosphorylated homooligonucleotides were reacted with a carbodiimide to form an O-phosphoryl isourea intermediate which was then attached to the amine-modified surface through a phosphoramidate linkage.16 These authors found that homooligomers of guanine gave a higher surface coverage than the other oligonucleotides and attributed the behavior to the formation of non-Watson-Crick base-pairing resulting in the G quadruplexes. How these DNA quadruplexes might increase the probe surface density at zirconium phosphonate modified surfaces is not yet known, although the G quadruplex most likely forms in solution before the probes are spotted onto the surface. Circular dichroism analysis of dsDNA probes containing poly(dG) spacers investigated for protein binding studies indicates quadruplex formation in the spotting solution.32 One possible explanation for the increased probe density is that the quadruplex allows the DNA to pack more tightly. However, the overall surface coverages are (32) Monot, J.; Petit, M.; Lane, S. M.; Guisle, I.; Le´ger, J.; Tellier, C.; Talham, D. R.; Bujoli, B. J. Am. Chem. Soc. 2008, 130, 6243–6251.

Lane et al.

very low, much less than ∼1013 molecules/cm2 for a close-packed monolayer, suggesting this is not the reason. Alternatively, the quadruplex may do a better job of lifting the probes off the surface, preventing some nonspecific binding of the ssDNA strands that can block access of the linking groups on other molecules to the surface. However, this idea is inconsistent with the observation that the initial coverage of the poly(dA)-containing probe is higher than the initial poly(dG)-containing probe coverage. A third possibility is that the quadruplex, with its four phosphate groups fairly close to each other, increases the affinity of the probes toward the zirconium surface. This explanation is most consistent with observations, supported by the fact that the initially bound poly(dG)-containing probes are less susceptible to rinsing than the poly(dA)-containing probes. Probes adsorb to the surface through a combination of specific and nonspecific binding. We previously demonstrated that probes containing terminal phosphate can covalently bind the zirconium ion surface, whereas those without the phosphate terminus do not.4 Probes can also interact nonspecifically, and given the length of the oligomers, this interaction is likely the initial one with the surface. The covalent linkage can then follow if the end of the molecule gains access to the surface. In some cases, this may not happen and the probes remain physisorbed until rinsed off. For the quadruplexes, the presence of four terminal phosphate groups in the unit greatly increases the chance for covalent linkage. In addition, once one covalent linkage is formed, the chances of the terminal phosphate groups of the other strands in the quadruplex binding to the surface increases. The reported width of a poly dG quadruplex is about 21-23 Å, with the interphosphate distances being about 12 Å,33 and the distance between zirconium atoms in the monolayer is a little over 5 Å,31 which would allow the possibility of more than one, if not all four, phosphates of the quadruplex to bind to the substrate. The observation that the probes with the poly(dA) sequence start at a higher surface coverage but rinse off the surface with more stringent rinsing conditions, while the surface coverage of the probes with the poly(dG) sequence remains fairly constant, indicates that the nature of the interaction with the surface is different. The poly(dA)-containing probes appear to experience stronger nonspecific binding with the surface. Other studies of the specific and nonspecific binding of homo-oligomers of oligonucleotides have reported strong nonspecific interactions of poly(dA) segments on surfaces. Wolf et al. compared the binding of thiolated and nonthiolated 25-mers of homooligomers on gold34 and observed considerable nonspecific binding, with the nonthiolated poly(dA) exhibiting more nonspecific binding than nonthiolated poly(dC) and poly(dT). With thiolated poly(dA), -(dT), and -(dC), which form specific thiol-gold linkages, these authors found that the strands with the most nonspecific binding, poly(dA), bound at a slower rate. Furthermore, during rinsing more of the poly(dA) was removed. Unfortunately, they did not look at poly(dG) due to the inefficient synthesis of homooligomers of guanines caused by the non-Watson-Crick base-pairing of the guanines. Other reports have demonstrated adenine self-assembled monolayers on metallic surfaces, including copper and silver.35,36 The adenine was found to adsorb in a slightly tilted orientation, with the amino group and in some reports the N7 interacting with the metal surface.35,36 (33) Neidle, S.; Balasubramanian, S. Quadruplex Nucleic Acids; Royal Society of Chemistry: London, 2006. (34) Wolf, L. K.; Gao, Y.; Georgiadis, R. M. Langmuir 2004, 20, 3357–3361. (35) Chen, Q.; Frankel, D. J.; Richardson, N. V. Langmuir 2002, 18, 3219– 3225. (36) Giese, B.; McNaughton, D. J. Phys. Chem. B 2002, 106, 101–112.

Oligonucleotide Binding to Modified Surfaces

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The metallic gold surface and the zirconium phosphonate surface are different, of course, but the nature of the weaker interaction may not be that different. Gold prefers softer ligands such as sulfur, but the nitrogen donors of the nucleobases also interact. It should be remembered that the surface of the zirconium phosphonate slides are not bare zirconium ions, but rather, they are terminated in oxide and hydroxide groups. The terminal phosphate or phosphonate groups displace the oxide and hydroxide to form metal-oxygen covalent linkages, although the phosphodiester backbone is not sufficiently basic to form the same bonds. Therefore, the nonspecific interactions of the oligonucleotides with the surface are most likely hydrogenbonding in nature. The amines of the adenine groups that better bind to the metal surfaces may also be better arranged for hydrogen binding to the oxide/hydroxide groups at the zirconium phosphonate surface. Our studies indicate that the poly(dA) groups exhibit higher nonspecific binding to the zirconium surface, perhaps through hydrogen bonding. This interaction limits the specific binding and ultimately reduces the final surface coverage after all the rinsing steps.

insight into this behavior, the effect of a poly(dG) spacer and poly(dA) spacer on the surface coverage of a probe molecule was studied. The probe molecule was immobilized on a zirconium phosphonate surface through a 5′-phosphate linker, and after immobilization, several different rinsing conditions were investigated. Using the XPS nitrogen peak intensity from the DNA and the zirconium peak intensity from the substrate, the probe surface coverage was calculated. With less stringent rinsing conditions, the probe with the poly(dA) spacer had a higher surface density than the probe with the poly(dG) spacer. However, it was found that with more stringent rinsing conditions, which simulated hybridization, the poly(dG)-containing probe had a surface coverage about twice that of the poly(dA)-containing probe. These results indicate that probes containing a poly(dG) spacer produce a higher target capture because of a higher probe surface density. On the basis of the rinsing results, which showed that the probe with the poly(dG) spacer was less susceptible to the stringency of the solutions, we suspect the increased surface density is due to an increase in the avidity of the probe for the zirconium surface.

Conclusions

Acknowledgment. Support from the U.S. National Science Foundation through Grant No. CHE-0514437 (D.R.T.) is acknowledged. This work was partially supported by the CNRS (Programme “Puces a` ADN” and Action CNRS-Etats-Unis 2005 no. 3310)

Oligonucleotide probes containing a poly(dG) spacer immobilized on a zirconium phosphonate surface through a 5′phosphate lead to higher target capture, compared to probes with either no spacer or a different nucleotide spacer, during hybridization with the complement of the probe.4 To provide

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