Direct Detection Of Dna By Hybridization Adsorption

The direct analysis of genomic DNA and RNA in an array format without labeling or PCR amplification would be extremely advantageous for a variety of biological applications. The detection and identification of ssDNA oligonucleotides by sequence specific adsorption onto an ssDNA microarray element to form a double-stranded duplex is called hybridization adsorption. If the target oligonucleotide is labeled with a fluorophore, fluorescence imaging can be used to detect DNA with the microarray. If the DNA has no fluorescent tag, then SPRI can be used to directly detect DNA hybridization adsorption. SPR imaging measurements of DNA microarrays has been demonstrated previously by a number of research groups (Guedon et al., 2000; Nelson et al., 2001; Livache et al., 2003; Rella et al., 2004; Shumaker-Parry et al., 2004; Okumura et al., 2005; Wark et al., 2005). However, the detection limit for DNA or RNA analysis via hybridization adsorption of untagged target molecules typically lies in the nanomolar range (Guedon et al., 2000; Nelson et al., 2001; Livache et al., 2003; Okumura et al., 2005; Wark et al., 2005). A typical sample of genomic DNA (35 Mg/mL) has a ssDNA concentration of around 20 fM. What can be done to improve the SPRI methodology to increase its sensitivity into the femtomolar range?

To answer this question, we must carefully examine the thermodynamic and kinetic limitations of surface bioaffinity measurements. An example of the detection of DNA by hybridization adsorption onto DNA microarrays with SPRI is shown in Figure 8.4. A four-component ssDNA microarray is exposed to two of the complementary DNA target sequences, with only the perfectly matched array elements forming duplexes via hybridization adsorption (Figure 8.4b). A positive increase in percent reflectivity (A%R) due to selective hybridization adsorption is observed; Fresnel calculations and experimental evidence show that if the SPRI response remains below 10%, then it is directly proportional to the relative surface coverage (0) of complementary DNA (Nelson et al., 2001), where 0 = r/Ttot and r represents the molecular surface density. 0 is related to the bulk concentration (C) of ssDNA by the Langmuir adsorption isotherm:

where KAds is the Langmuir adsorption coefficient. A typical Langmuir isotherm for 16mer adsorption is shown in Figure 8.4c. At a bulk concentration of C0 5 = 1/KAds, the relative surface coverage is 0.5. For a 16mer DNA, KAds = 2 x 107 M-1, so that C0.5 is at 50 nM.

Figure 8.4. (a) Schematic representing DNA hybridization adsorption onto DNA or RNA microarrays. (b) An example of an SPR difference image showing the sequence-specific hybridization/adsorption of target DNA onto a four-component ssDNA microarray. This was obtained by exposing the ssDNA array to a 500 nM solution containing two different target DNA sequences with duplex formation occurring only at the complementary probe array elements. A maximum of 130 individually addressable array elements on a single chip with a total surface area of 0.8 cm2 can be created using a 500-^.m2 photopatterning mask. (c) An example showing a plot of the relative surface coverage (9) as a function of target complementary DNA concentration. The solid line represents a Langmuir isotherm fit to the data. A value of Kads = 2.0 (± 0.2) x 107 M-1 was determined from the fit.

Figure 8.4. (a) Schematic representing DNA hybridization adsorption onto DNA or RNA microarrays. (b) An example of an SPR difference image showing the sequence-specific hybridization/adsorption of target DNA onto a four-component ssDNA microarray. This was obtained by exposing the ssDNA array to a 500 nM solution containing two different target DNA sequences with duplex formation occurring only at the complementary probe array elements. A maximum of 130 individually addressable array elements on a single chip with a total surface area of 0.8 cm2 can be created using a 500-^.m2 photopatterning mask. (c) An example showing a plot of the relative surface coverage (9) as a function of target complementary DNA concentration. The solid line represents a Langmuir isotherm fit to the data. A value of Kads = 2.0 (± 0.2) x 107 M-1 was determined from the fit.

Comparable KAds values have been observed with both fluorescence imaging and SPR measurements (Liebermann et al., 2000; Nelson et al., 2001; Peterson et al., 2002; Lee et al., 2005a; Levicky and Horgan, 2005; Wark et al., 2005).

At low surface coverages, the Langmuir isotherm depends linearly on the target DNA concentration:

Equation (8.2) shows one reason why it is difficult to measure 1 fM target concentrations with a surface bioaffinity measurement: As C goes to zero, 0 goes to zero as well! If one wants to detect ssDNA at a bulk concentration of 1 fM, then one must be able to detect a relative surface coverage of 0 = 2 x 10-8! As mentioned in the previous section, the surface density of DNA probes on a microarray element is typically 5 x 1012 molecules/cm2, so that the surface density of dsDNA is V = 105 molecules/cm2 at 1 fM. For a 500-^m array element, this corresponds to 250 molecules; for a 50-^m array element, this surface density corresponds to 2.5 molecules!! SPRI typically has a detection limit of 1 nM for 16mer oligonucleotide adsorption, which corresponds to a lowest relative surface coverage of 0 ~ 2 x 10-2 or V ~ 1011 molecules/cm2. If a sandwich assay is used with a DNA-coated nanoparticle, a relative surface coverage of 0 ~ 2 x 10-4 corresponding to a detection limit of approximately 10 pM is observed (He et al., 2000). For comparison, a typical lowest surface coverage that can be observed above background for the detection of DNA in a sandwich assay with a fluorescently tagged DNA molecule is V ~ 108 molecules/cm2, corresponding to a detection limit of approximately 1 pM (Zammatteo et al., 2000; Lehr et al., 2003; Livache et al., 2003).

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Figure 8.5. Representative real-time SPRI curves showing sequence specific hybridization adsorption of complementary DNA onto a surface probe element at concentrations of 1 nM (O) and 10 nM (■). The figure inset is an SPR difference image obtained by subtracting images acquired before and after the hybridization adsorption of 100 nM target DNA onto DNA array elements corresponding to a final A%R change of 2%. [Reprinted with permission from Anal. Chem. 77:5096-5100, Copyright 2005, American Chemical Society.]

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Figure 8.5. Representative real-time SPRI curves showing sequence specific hybridization adsorption of complementary DNA onto a surface probe element at concentrations of 1 nM (O) and 10 nM (■). The figure inset is an SPR difference image obtained by subtracting images acquired before and after the hybridization adsorption of 100 nM target DNA onto DNA array elements corresponding to a final A%R change of 2%. [Reprinted with permission from Anal. Chem. 77:5096-5100, Copyright 2005, American Chemical Society.]

A second reason that it is difficult to detect a 1 femtomolar solution of ssDNA with SPRI hybridization adsorption measurements is the kinetics of hybridization adsorption. The hybridization adsorption rate (ka) for a 16mer was measured to be approximately 105M-1s-1 from the data in Figure 8.5. Since the velocity of the adsorption reaction is kaC, this rate decreases significantly as the concentration is lowered. Moreover, there is also a diffusional contribution that becomes significant at lower concentrations due to the time required for the molecules to reach the surface. These two effects are observed in Figure 8.5, which shows the time-dependent SPRI responses upon exposure of an ssDNA array to 10 and 1 nM solutions of complementary 16mer DNA. The initial adsorption rate, as determined from the initial slopes, decreases by a factor of 10 as the target DNA concentration is decreased from 10 to 1 nM, as expected from simple Langmuir kinetics. Thus, due to both Langmuir adsorption equilibrium arguments and adsorption kinetics, the detection limit for the direct detection of ssDNA oligonucleotides with SPRI is estimated to be 1 nM. To analyze genomic DNA at a concentration of 1 fM with SPRI, an amplification methodology is required. While an ELISA-based amplification scheme may provide sufficient sensitivity (Kawai et al., 1993; Morata et al., 2003), we will choose a different strategy for DNA detection, employing RNase H in conjunction with RNA microarrays to create a new type of surface enzyme amplification scheme.

8.4. RNase H SURFACE ENZYME KINETICS

RNase H is an endoribonuclease that selectively hydrolyzes the RNA strand of an RNA-DNA heteroduplex (Nakamura et al., 1991; Katayanagi et al., 1993; Zamaratski et al., 2001). Figure 8.6 shows a schematic drawing of the two possible ways that RNase H can react with surface-bound RNA-DNA heteroduplexes. The heteroduplex is first formed on

Figure 8.6. Schematic representation of RNase H activity at surface-bound RNA-DNA heteroduplexes formed by hybridization adsorption of (a) target RNA onto ssDNA microarrays and (b) target DNA onto ssRNA microarrays.

the surface of either a DNA or RNA microarray (see Figures 8.6a and 8.6b, respectively) by hybridization adsorption of the complementary oligonucleotide from solution. After hybridization, the RNA strand of the heteroduplex will be hydrolyzed and destroyed by RNase H. In the case of the DNA microarray (Figure 8.6a), hydrolysis leaves the original ssDNA still attached to the surface and capable of hybridization adsorption. In contrast, the RNase H hydrolysis of the RNA microarray results in both the destruction of the surface array element and the release of the target DNA back into solution (Figure 8.6b).

To demonstrate this surface RNase H activity, a series of real-time SPR imaging measurements were obtained for the reaction of RNase H with RNA-DNA heteroduplexes formed on DNA and RNA microarrays. Figure 8.7 plots the real-time SPRI signal for both cases (labeled a and b, respectively). A rapid rise in A%R to a steady-state value of + 1.6% was observed upon the hybridization adsorption of DNA or RNA onto the surfaces from 500 nM complement solutions. Subsequent rinsing of the DNA and RNA microarrays with buffer followed by exposure to RNase H resulted in a rapid decrease in A% R from the hydrolysis of the RNA on the surface. For the case of the DNA microarray, a A% R of -1.6% was observed; this decrease brought the SPRI signal back to the original reflectivity level prior to hybridization adsorption and suggests that the RNase H completely removed all surface-bound RNA. In contrast, for the case of the heteroduplexes formed using RNA microarrays, a decrease of -3.2% was observed corresponding to the complete loss of both the target DNA and probe RNA from the surface. No hybridization adsorption was observed upon subsequent exposure of the RNA microarray to complementary ssDNA. Moreover, if the experiments were repeated after the creation of either surface-bound dsDNA or dsRNA, no surface enzymatic hydrolysis was observed. These additional measurements confirmed the selectivity and specificity of the surface RNase H reaction to the RNA-DNA heteroduplex.

How does one quantify the reaction rate of the RNase H surface hydrolysis process? The characterization of the surface enzyme reactions utilized in biosensing processes is extremely important for optimization of the array-based biosensor, because an enzyme reaction can be orders of magnitude slower on a surface as compared to solution due to

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