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Contents : Published on Web 03/11/2004 Direct Detection of Genomic DNA by Enzymatically Amplified SPR Imaging Measurements of RNA Microarrays Terry T. Goodrich Hye Jin Lee and Robert M. Corn* Department of Chemistry UniVersity of Wisconsin Madison Wisconsin 53706 Received November 26 2003 E-mail: corn@chem.wisc.edu The direct analysis of genomic DNA and RNA in an array format without labeling or PCR amplification would be extremely useful for the identification of viral1 and bacterial samples 2 gene expression analysis 3-5 and biological warfare detection.6 The label-free technique of surface plasmon resonance (SPR) imaging is in principle an excellent candidate for these direct detection measurements but to date has not yet been extensively applied to DNA and RNA analysis due to a nanomolar detection limit 7 typical genomic DNA samples are approximately 20 fM (35 g/mL). In this paper we demonstrate a novel surface enzymatic amplification process that utilizes RNase H and RNA microarrays to sufficiently enhance the sensitivity of SPR imaging for the detection of DNA oligonucleotides down to a concentration of 1 fM corresponding to a remarkable 106 enhancement in sensitivity. The utility of this method is further demonstrated by the direct detection of the TSPY gene in human genomic DNA samples. The enzymatic amplification process presented in this Communication is based on the unique selectivity of RNase H which specifically destroys RNA in RNA-DNA heteroduplexes but does not digest single-stranded DNA (ssDNA) double-stranded DNA (dsDNA) ssRNA or dsRNA. An overview of the surface enzymatic amplification process used in these SPR imaging experiments is shown in Scheme 1. A single-stranded RNA microarray is exposed to a solution that contains a complementary ssDNA sequence and the enzyme RNase H. The DNA will bind to its RNA complement on the surface forming an RNA-DNA heteroduplex (step 1). The enzyme RNase H will then specifically hydrolyze only the RNA strand in this heteroduplex 8 releasing the DNA molecule back into solution (step 2). This DNA molecule can then bind to another RNA molecule on the surface causing the hydrolysis of a second RNA probe. This cyclic process results in an amplification effect whereby a very small number of DNA molecules can initiate the hydrolysis of many RNA molecules from the surface (step 3). This loss of RNA probes from the surface results in a significant negative change in percent reflectivity. The decrease in percent reflectivity will become larger with time until all of the available RNA probes on the surface are destroyed. SPR imaging measurements were first performed on a threecomponent microarray to demonstrate the selectivity of RNase H for the hydrolysis of surface-bound RNA-DNA heteroduplexes. A three-component array (see Figure 1 inset) composed of two noninteracting RNA probe molecules (with sequences denoted RA and RB) and a third DNA probe molecule (DA) with the same sequence as RA was created by attaching thiol-modified RNA and DNA oligonucleotides onto an alkanethiol-modified gold thin film using a fabrication methodology described previously.9 This array was exposed to a 500 nM solution of target DNA that was complementary to sequences RA and DA the SPR reflectivity difference image was obtained by subtracting the images taken before and after exposure (see Figure 1a). The DNA target bound specifically to both the RA and the DA array elements on the surface and nonspecific adsorption was not observed to either the mismatched RB 4086 9 J. AM. CHEM. SOC. 2004 126 4086-4087 Scheme 1. Schematic Presentation of a Surface Enzymatic Reaction for the Amplification of SPR Signal by Selective RNA Probe Removal (See Text for Details) sequence or the array background. This array then contained array elements with heteroduplexed RNA-DNA dsDNA and ssRNA. Next the array was exposed to a solution of RNase H and Figure 1b shows the SPR reflectivity difference image obtained after approximately a 5-min reaction time. A large decrease in percent reflectivity was obtained only for the RA array elements which were RNA-DNA heteroduplexes prior to exposure to RNase H. This change in SPR signal is attributed to the selective removal of these RNA probes from the surface. Note that both the dsDNA and the ssRNA array elements show no change in SPR signal after exposure to RNase H demonstrating the specificity and selectivity of this enzyme. To demonstrate the sensitivity of this enzymatically amplified SPR imaging method a second three-component RNA microarray was constructed and exposed to target DNA. The three sequences chosen for this array R1 R2 and R3 are 20mers two of which (R1 and R2) were selected to bind to the human TSPY gene (testisspecific protein Y-encoded) located on the Y chromosome and the other is a negative control. Figure 2a shows an SPR reflectivity difference image of the three-component array obtained after approximately a 4 h e

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