Detection of Specific Sequences in RNA Using Differential Adsorption

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Anal. Chem. 2005, 77, 6229-6233

Detection of Specific Sequences in RNA Using Differential Adsorption of Single-Stranded Oligonucleotides on Gold Nanoparticles Huixiang Li and Lewis Rothberg*

Department of Chemistry, University of Rochester, Rochester, New York 14627

We have used the disparity in adsorption rates for singleand double-stranded RNA on ionically coated gold nanoparticles suspended in a colloid to design a rapid sequence identification assay. Unlabeled target RNA and a probe sequence are mixed prior to exposure to the gold nanoparticles to enable efficient hybridization. We have designed assays based on either color changes or fluorescence that are sensitive to a few picomoles of target. Single-base mutations on RNA sequences can be detected even in complex oligonucleotide mixtures. The assay requires less than 10 min so that RNA degradation problems are avoided. Ribonucleic acid (RNA) plays many important roles in biological processes,1 and RNA sequence detection is consequently an indispensable tool in many fields of current research including molecular biology, toxicology, physiology, pharmacology, and biochemistry. The relationship between RNA sequence and protein expression also makes RNA sensing important for proteomics.2 Identifying the presence of specific RNA sequences is also useful for early clinical diagnosis of deadly diseases such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV).3 Although RNA has structure and recognition chemistry similar to that of DNA, the hydroxyl group at position 2 of RNA makes RNA much less stable than DNA so that many DNA detection methods are too harsh or time-consuming to practically apply to RNA. Commonly used methods for RNA detection include Northern blot (NB),4 ribonuclease protection assay (RPA),5 and reverse-transcription polymerase chain reaction (RT-PCR).6 Each * To whom correspondence should be addressed. E-mail: rothberg@ chem.rochester.edu. (1) Barciszewski, J., Clark, B. F. C., Eds. RNA Biochemistry and Biotechnology; Nato Science Series 3 Vol. 70; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1998. (2) Hecker, M., Mu ¨ llner, S., Eds. Advances in biochemical engineering/biotechnologies; Springer: New York, 2003; Vol. 83. (3) Levy, J. A.; Fraenkel-Conrat, H.; Owens, R. A. Virology; Prentice Hall: Englewood Cliffs, NJ, 1994. (4) Brown, T. In Current Protocols in Molecular Biology; Ausubel, E. M., Brent, R., Kingstorn, R. E., Moore, D. D., Siedman, J. G., Smith J. A., Struhl, K., Eds.; John Wiley and Sons: New York, 1993; pp 4.9.1.-4.9.14. (5) Gilman, M. In Current Protocols in Molecular Biology; Ausubel, E. M., Brent, R., Kingstorn, R. E., Moore, D. D., Siedman, J. G., Smith, J.A., Struhl, K., Eds.; John Wiley and Sons: New York, 1993; pp 4.7.1.-4.7.8. (6) Erlich, H. A., Ed. PCR Technology: Principles and Applications for DNA Amplification; Stockton Press: London, 1989. 10.1021/ac050921y CCC: $30.25 Published on Web 08/26/2005

© 2005 American Chemical Society

of these methods has its drawbacks.7 NB and RPA use unsafe reagents such as formaldehyde and radioactively labeled materials. RT-PCR requires reverse transcription of RNA to DNA followed by polymerase chain reaction amplification of the resulting DNA. A simpler, safer, faster, and more cost-effective method to detect RNA sequences would be likely to expand the use of RNA detection in research and clinical practice. In our previous work, we have developed colorimetric8 and fluorescent9 detection methods for rapid detection of DNA oligonucleotides or genomic DNA10 by exploiting the differential affinity of single-stranded (ss) DNA and double-stranded (ds) DNA for unmodified gold nanoparticles (Au-nps). Large disparities in adsorption rates for ss-DNA and ds-DNA onto Au-nps result from a difference in the electrostatic interactions of DNA with unfunctionalized Au-nps that are stabilized in a colloidal suspension by citrate ion coatings. Rapid adsorption of short DNA oligonucleotides onto the Au-np stabilizes them against salt-induced aggregation and concomitant color changes, thereby allowing detection of fewer than 100 fmol of target by visual inspection.8,10 Use of fluorescently tagged oligonucleotides as probes does not substantially change the adsorption behavior and results in fluorescence quenching when the probes do not hybridize to DNA in the analyte. Exploiting the preferential quenching when fluorescent probes do not hybridize with target sequences in the analyte enabled detection of 0.1 fmol of DNA target even in complex oligonucleotide mixtures.9 For both the colorimetric and fluorescent assays, adaptation of the method for detection of single-base mismatches is straightforward. In the present work, we demonstrate that it is possible to apply analogous methods to RNA sequence detection. Some differences in protocol are helpful due to the instability of RNA. For example, we use fluorescently tagged DNA probes to implement the fluorescent detection assay since RNA is more difficult to modify and has short shelf life. We show that our approach is appropriate to sequence detection on long strands with secondary structure even in the presence of substantial amounts of nontarget RNA. This represents a first step toward direct detection of genomic RNA in complex samples. (7) Dvorak, Z.; Pascussi, J.-M.; Modriansky, M. Biomed. Pap. 2003, 147, 131135. (8) Li, H. X.; Rothberg, L. J. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 1403614039. (9) Li, H. X.; Rothberg, L. J. Anal. Chem. 2004, 76, 5414-5417. (10) Li, H. X.; Rothberg, L. J. J. Am. Chem. Soc. 2004, 126, 10958-10961.

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EXPERIMENTAL SECTION Synthesis of Au-nps. Hydrogen tetrachloroaurate(III) (HAuCl4‚ 3H2O), 99.99%, and sodium citrate (Na3C6H5O7‚2H2O), 99%, were purchased from Alfa Aesar (Ward Hill, MA) and used without further purification. Gold colloid, an aqueous suspension of Aunps stabilized against aggregation by sodium citrate, was prepared as described elsewhere.11 Briefly, 250 mL of 1 mM HAuCl4 (Alfa Aesar) aqueous solution is heated to its boiling point while stirring. After adding 25 mL of 38.8 mM sodium citrate (Alfa Aesar) in water rapidly to the boiling solution, we continue boiling and stirring for 15 min. The solution is cooled to room temperature for use. The gold nanoparticle diameters are measured by TEM to be 13 nm, and this is consistent with their absorption spectrum, which has a maximum at 520 nm. The concentration of the gold colloid is ∼17 nM. Selection and Preparation of Oligonucleotide Targets and Probes. A 2-o-methyl RNA oligonucleotide (5′-AGG AAU UCC AUA GCU-3′) was synthesized and purified by IDT (Coralville, IA) to be used as a probe sequence for the colorimetric assay. Three RNA sequences with the same length as the probe are used as model targets. These were also synthesized and purified (RNase-free HPLC purification, RNA oligos of greater than 85% full length product) by IDT. One sequence (c-target) was complementary to probe, the second (mc-target: 5′-AGC UAU AGA AUU CCU-3′) had a one-base-pair mismatch with the probe, and the third (nc-target: 5′-CGA UCA CGA GAU CGA-3′) is not complementary to the probe. For the fluorescent assay, we used rhodamine red-labeled DNA as probes (wild-type probe, rhodamine red-5′-AGG AAT TCC ATA GCT-3′, and mutant probe, rhodamine red-5′-AGG AAT GCC ATA GCT-3′). Rhodamine red-labeled DNA sequences were purchased from MWG Biotech (High Point, NC). 2′-ACE-protected 50-mer RNA50a and RNA50b were purchased from Dharmacom RNA Technologies (Lafayette, CO). These two sequences have only a single base difference in their sequences (RNA50a (/RNA50b): 5′-ACU AGG CAC UGU ACG CCA GCU AUG GA(/C)A UUC CUU AGC UAU GAG AUC CUU CG-3′). RNA50a contains a sequence perfectly matched with the wildtype probe while the analogous segment of RNA50b has a singlebase-pair mismatch with the wild-type probe. Conversely, the target sequence on RNA50b is perfectly matched with the mutant probe so that the analogous segment of RNA50a has a singlebase-pair mismatch with the mutant probe. RNA and DNA solutions with concentrations of salt and phosphate buffer as specified in the text were made. The buffer solution, water, pipet tips, and plastic vials were autoclaved, and the RNA samples were kept in the freezer at -20 °C until use. The requisite potassium phosphate (monobasic, anhydrous 99.999%) and sodium phosphate (dibasic, anhydrous, 99.999%) were obtained from Aldrich Chemical (Milwaukee, WI) and used as supplied. Sodium chloride crystals were purchased from Mallinckrodt (Hazelwood, MO). Deprotection of 2′-ACE-Protected RNA. Prior to attempted hybridization, 2′-ACE-protected RNA was deprotected according to the procedure provided by the manufacturer and used without further purification. Deprotection involves centrifugation for 2 min, adding 400 µL of deprotection buffer to the tube of RNA, and (11) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735-743.

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completely dissolving the resulting RNA pellet. This solution is spun for 10 s, centrifuged for 10 s, and incubated at 60 °C for 30 min. The sample is then dehydrated using a SpeedVac before use. Hybridization. A trial hybridization solution containing equal quantities of probe and target sequences is made in 10 mM phosphate buffer solution (PBS) containing 0.3 M NaCl. To break any secondary structure in the RNA target and allow hybridization with the probe, the trial solution was heated to 95 °C for 3 min and then cooled to an appropriate temperature for the desired assay for 1 min. The temperature used for simple sequence detection is typically ambient while single-base-mismatch detection requires that hybridization takes place at a temperature between the melting temperature of the mismatch and that of the perfect match. In performing the assays below, the gold colloid is used at ambient temperature regardless of the temperature of the trial hybridization solution. Assay. For colorimetric detection, 50 µL of gold colloid solution is added to 10 µL of 2 µM trial hybridization solution and the color of the mixture is viewed immediately. Photographs are recorded with a Canon S-30 digital camera. For fluorescence detection, 5 µL of 1 µM trial hybridization solution is added to 500 µL of gold colloid (250 µL of original colloid diluted with 250 µL of water) and then mixed with 500 µL of 10 PBS containing 0.2 M NaCl. The fluorescence spectrum of the mixture is recorded within 2 min after mixing using a fluorometer (Fluorolog 3, Jobin Yvon) set for excitation at 570 nm. Spectrometer slits for both excitation and emission were set for 4-nm band-pass. Traces of photoluminescence versus time were recorded with detection spectrometer set for 590 nm, near the rhodamine emission maximum. The large solution volumes are used to facilitate measurements with a fluorometer designed for centimeter path length cuvettes, and fluorescence is efficiently collected from only ∼1% of the sample volume. The sensitivity of the fluorescent assay is therefore greatly underestimated. RESULTS AND DISCUSSION Mechanistic Basis for the Assays: Interactions between Oligonucleotides and Unfunctionalized Au-nps. Surface plasmon resonance imbues isolated 13-nm-diameter Au-nps with a sharp absorption at ∼520 nm and a corresponding reddish hue.12 Aggregation of these Au-nps leads to interparticle plasmon interactions that substantially change the spectrum to a very broad absorption throughout the visible and give the colloid a corresponding grayish-blue color.13 Colloidal Au-np suspensions are stabilized against Au-np aggregation by adsorption of ions that lead to strong electrostatic repulsion between the nanoparticles.14 Most commonly, sodium citrate is added to gold nanoparticles during their synthesis so that citrate adsorption makes the Au-np surfaces negatively charged. Both our colorimetric and fluorescent detection protocols take advantage of the rapid adsorption of short single-stranded oligonucleotides to the Au-np. This adsorption has been documented using fluorescence quenching and Raman experiments.8 These results are surprising since oligonucleotides are themselves commonly regarded as negatively charged species presenting negatively charged phosphate backbones that would (12) Kreibig, U.; Genzel, L. Surf. Sci. 1985, 156, 678-700. (13) Quinten, M.; Kreibig, U. Surf. Sci. 1986, 172, 557-577. (14) Hunter, R. J. Foundations of Colloid Science; Oxford University Press Inc.: New York, 2001.

be repelled by citrate. We have rationalized the rapid ss-DNA adsorption with a model where single-stranded oligonucleotides can sometimes configure themselves with hydrophobic bases facing the Au-np. In this geometry, dipolar attraction can reduce the barrier to adsorption of ss-DNA and ss-RNA.10 Double-stranded oligonucleotides are unable to achieve an uncoiled geometry with exposed bases and, therefore, experience much larger repulsion by the ions on the Au-np surface. Consequently, they take much longer times to adsorb or do not adsorb to the Au-np at all under some conditions. Similarly, long single-stranded oligonucleotides adsorb slowly though the detailed reasons for that remain unclear. In part, this is probably due to the formation of secondary structure but it may also be related to how oligonucleotide length affects the flexibility of ss-DNA.10 Once adsorbed, the short single-stranded oligonucleotides add negative charge density to the Au-np surface and therefore act to enhance the stability of the colloid against aggregation. It is therefore possible to protect the colloid from aggregation upon exposure to amounts of salt that would ordinarily screen the electrostatic repulsion between Au-np and induce aggregation. Hence, the gold will remain pink upon exposure to salt following exposure to ss-DNA or ss-RNA while it will turn grayish-blue following exposure to double-stranded or long oligonucleotides. This observation forms the basis for the colorimetric hybridization assay. The preferential adsorption of short ss-DNA probe sequences on Au-np can also be exploited to perform a fluorescent assay. When the ss-DNA probe is fluorescently tagged, adsorption to the metallic surface results in fluorescence quenching.15 However, if the probe binds to a target in the analyte solution, it is resistant to adsorption and its fluorescence persists, indicating a match. The fact that these assays rely on the difference in electrostatic properties of ss-DNA and ds-RNA (or ds-DNA) distinguishes them from detection approaches using Au-nps covalently functionalized with oligomers where hybridization is used to link the Au-nps.16,17 In the present work, the trial hybridization is performed separately from the assay and facilitates rapid duplex formation. Colorimetric Detection of RNA Oligonucleotides. Panels A-D in Figure 1 are photographs taken immediately after mixing trial hybridization solutions with gold colloid. The quantity of salt in the hybridization solution would be adequate to cause Au-np aggregation in the absence of RNA. Each vial contains 10 µL of trial hybridization solution that contains 10 mM PBS and 0.3 M NaCl and 50 µL of gold colloid. In the hybridization solution, there are 20 pmol of RNA probe and target. For the probe, we use 2′o-methyl RNA because of its high stability.18 Complementary (c), single-base-mismatched (mc) and unrelated (nc) target sequences are used in the left, center, and right vials, respectively. All trial hybridization solutions were heated at 95 °C for 3 min and then annealed for 1 min at the specified temperatures: (A) 20, (B) 50, (C) 59, and (D) 64 °C. (15) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Kluwer Academic/ Plenum Publishers: New York, 1999. (16) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078-1081. (17) Sato, K.; Hosokawa, K.; Maeda, M. J. Am. Chem. Soc. 2003, 125, 81028104. (18) Majlessi, M.; Nelson, N. C.; Becker, M. M. Nucleic Acids Res. 1998, 26, 2224-2229.

Figure 1. Colorimetric method for RNA sequence detection. Photographs A-D were recorded from the mixtures of trial hybridization solutions and gold colloid. The left vial in each image contains complementary target, the middle vial contains a target with a singlebase mismatch with the probe, and the right vial contains a random noncomplementary target. Each hybridization solution was heated at 94 °C for 3 min and subsequently annealed at a different temperature for 1 min: (A) 20, (B) 50, (C) 59, and (D) 64 °C.

In (A) and (B), the c and mc targets present gray while the nc target appears light pink. This result indicates that c-target and mc-target hybridize with probe and form ds-RNA below 50 °C. The ds-RNA does not adsorb to Au-nps so that the salt in the hybridization solution causes Au-np aggregation and color change. Since the nc target is not complementary to the probe, both nctarget and probe remain single stranded. They therefore adsorb rapidly to the Au-nps and stabilize them against salt-induced aggregation so that the gold colloid remains pink. When we elevated the annealing temperature to 59 °C (Figure 1C) prior to mixing with Au-np, the mixture containing c-target again turns gray but the mixture containing mc-target now also remains pink because 59 °C is above the melting temperature (Tm) of the mctarget but lower than that of the c-target. At 64 °C (Figure 1D), none of the targets can hybridize to the probe and all of the solutions appear pink. We point out that it is practical to heat only the trial hybridization solutions but allow the gold colloid to remain at 20 °C because hybridization under the conditions in the colloid is much slower than adsorption to the Au-np. At the same time, there is adequate salt in the mixture to maintain the stability of the double strand for longer than the Au-nps take to aggregate. The Au-np aggregation is irreversible. Color changes can be more sensitively and quantitatively monitored by absorption spectra (Figure 2). These exhibit the characteristic isolated Au-np spectra in cases where aggregation does not occur and the broad red tail associated with aggregates when the salt is able to cause aggregation. Substantial changes in salt-induced aggregation behavior as observed in Figures 1 and 2 are seen when there are g10 oligonucleotides (15-mers) per Au-np. Remarkably, this corresponds to occupying only ∼1% of the Au-np surface area with ss-RNA. Because of the enormous extinction coefficient (∼107 L mol-1 cm-1) associated with the gold nanoparticles, the color of 17 nM Au-np solutions is easily detected by eye in 10-µL droplets or by using an absorption spectrometer with 100-µm path length sample cells. The data of Figures 1 and 2 are recorded with ∼40 single strands (or 20 double strands) of Analytical Chemistry, Vol. 77, No. 19, October 1, 2005

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Figure 2. Absorption spectra from the mixtures of trial hybridization solutions annealed at two different temperatures after being added to gold colloid. Squares, circles, and triangles from the mixtures containing complementary target, single-base-mismatched target, and noncomplementary target, respectively. In each case, the hybridization solutions were heated at 95 °C for 3 min and then annealed for 1 min prior to addition to gold colloid at 20 °C. Left: annealed at 50 °C. Right: annealed at 60°C.

RNA per Au-np and illustrate that detection of a few picomoles of target RNA by visual inspection is possible. Fluorescent Detection of RNA Long-Mers. There are some limitations on colorimetric detection that can be ameliorated by using the fluorescent assay described briefly above. Since traditional absorption spectroscopy is by its nature not a null experiment, its sensitivity is limited. Moreover, a number of ambiguities arise in the context of using the colorimetric method we have illustrated in the previous section. For example, it is easy to imagine circumstances where the quantities of target and probe differ so that the trial hybridization solution contains both single and double strands. In addition, situations where the length of the probe does not match that of the target leave a single-stranded overhang on a double-stranded complex. Using the fluorescent assay, these practical and conceptual difficulties do not arise. Since only the fate of the fluorescently tagged probe strand is monitored, unmatched targets do not affect the assay. When the probe sequence hybridizes with a sequence in the analyte, it will be protected from adsorption to Au-nps and the concomitant fluorescence quenching. Thus, as long as there is adequate concentration of Au-nps to adsorb all of the probe oligonucleotides, the presence of fluorescence indicates the presence of the target sequence in the analyte. In the absence of target, no fluorescence should be observed. The null character of the measurement, high sensitivity of fluorescence detection, and ability to work in complex mixtures of target make this a powerful assay for DNA detection.9 For RNA sequence detection, we used DNA sequences labeled with rhodamine red as probes because RNA oligonucleotides are more difficult and expensive to fluorescently label. We used long synthetic targets (50 bases) predicted by standard software (mFold, available, for example, at scitools.idtdna.com/mfold/) to have substantial secondary structure to simulate genomic RNA, and we used 15-base probes to assay for a complementary sequence on the targets. The hybridization solution is heated to 94 °C for 3 min to break up secondary structure and annealed for 1 min at a suitable lower temperature. As with any hybridization-based assay, it is important to select probes with melting temperatures higher than that of secondary structure in the target that could otherwise make the complementary sequence unavailable for hybridization. As demonstrated for the colorimetric method, single-base mutations can be detected by judicious choice of annealing temperature for hybridization. The duplex formed from 6232

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Figure 3. Detection of sequences and single-base mutations in RNA targets using fluorescence quenching. Fluorescence spectra of the mixtures of hybridization solution, gold colloid, and buffer/salt solution were recorded within 2 min after mixing. The hybridization solutions all were annealed at 65 °C. Red squares: wild-type RNA target containing a sequence perfectly complementary to the DNA probe. Green up-triangles: mutant RNA target containing a sequence forming a single-base-pair mismatch with the probe. Blue downtriangles: noncomplementary RNA target. DNA probe: rhodamine red-5′-AGG AAT TCC ATA GCT-3′. Wild-type target: 5′-ACU AGG CAC UGU ACG CCA GCU AUG GAA UUC CUU AGC UAU GAG AUC CUU CG-3′. Mutant target: 5′-ACU AGG CAC UGU ACG CCA GCU AUG GCA UUC CUU AGC UAU GAG AUC CUU CG-3′. Noncomplementary target: 5′-CGA UCA CGA GAU CGA-3′.

a probe and mutant target has lower melting temperature than the duplex formed from the probe and wild-type target. Hybridization at a temperature between the melting temperature of these two duplexes will only result in duplex formation for wild-type targets. When the probe hybridizes with the RNA target, it does not adsorb to Au-nps and its fluorescence persists. The result of this experiment is shown in Figure 3 where 15-base probes were used to detect wild-type 50-mers while 50-mers with a single base difference overlapping the probe sequence do not yield appreciable fluorescence. For practical purposes, it is desirable to detect target RNA sequences in a complex mixture of oligonucleotides. Because the fluorescent method is structured so that luminescence will be observed as long as the tagged probes hybridize with some component of the analyte, it is well suited to mixtures. To demonstrate this feature, we added short RNA sequences that

Figure 4. Detection of single-base mutations in RNA sequences in complex mixtures using the fluorescent assay. P and T denote probe and target, respectively, and w and m indicate wild-type and mutant, respectively. Red squares: P(w) + T(w). Green uptriangles: P(w) + T(m). Pink diamonds: P(m) + T(m). Blue downtriangles: P(m) + T(w). All hybridization solutions contain noncomplementary background RNA at 10 times the concentration of the target. Mutant probe: rhodamine red-5′-AGG AAT TCC ATA GCT3′. Other sequences are stated in the caption of Figure 3.

were not complementary to the probes to the trial hybridization solution at concentrations 10 times that of the target. Figure 4 depicts the time course of the luminescence after mixing the trial hybridization solution with Au-nps as monitored at the wavelength maximum of the fluorescence in an experiment analogous to that of Figure 3 but where the hybridization solution contains 5 pmol of probe, 5 pmol of target, and 50 pmol of short RNA noncomplementary segments. We also illustrate each combination of wildtype and mutant probes and targets at an annealing temperature below the wild-type melting temperature. These data verify that choice of the probe sequence perfectly matching the mutant target will, of course, result in much more fluorescence than the wildtype probe sequence when exposed to the mutant target. An important implication of Figure 4 is that the fluorescent assay can tolerate substantial amounts of RNA degradation into short sequences as often occurs. As long as there is an adequate concentration of Au-np, these do not interfere with adsorption of unhybridized probes and the attendant fluorescence quenching essential to the assay. The dynamics reflected in Figure 4 are important in the performance of the assay since the fluorescence must be evaluated at a time long compared to the adsorption of the unhybridized probes but short compared to the lifetime or adsorption rate of the hybridized complex formed between probe and target. Under the conditions of Figure 4, the adsorption of unhybridized probes on the Au-np is very rapid and occurs prior to the beginning of

the trace. The subsequent slow decay seen in Figure 4 has several possible explanations. The complex formed between the probe and target may not be perfectly stable in the gold colloid and may slowly dehybridize. Even if it does not, single-stranded portions of the long target strand may adsorb and bring the probe fluorophore close to the Au-np so that quenching is observed. Finally, there can be slow adsorption of even perfect duplexes onto the gold at the salt concentrations used in the experiment. We know empirically that the duplex sticks rapidly to Au-np at high salt concentrations where the electrostatic repulsion between the citrate coating on the Au-np and the phosphate backbone is heavily screened. Differentiating between these possibilities is nontrivial and will be the subject of future work. We note that slow decay of the fluorescence as shown in Figure 4 also occurs in experiments with DNA targets. CONCLUSIONS We have demonstrated a simple approach to detection of specific RNA sequences based on the differential adsorption rates for single- and double-stranded oligonucleotides onto Au-nps. A colorimetric assay for target RNA sequences with 2-o-methyl RNA probes and a fluorescent assay based on hybridization of target RNA with fluorescently labeled DNA probes have been developed. The assays require only commercially available reagents. A key strength of the methods is that the hybridization step is completed independent of the assay so that it can be performed under optimal conditions for rapid, efficient hybridization. Each assay therefore takes less than 10 min so that issues concerning RNA instability are minimized. Single-base mismatches between probe and target sequences are easily detected with high contrast. The fluorescent assay is particularly promising since it is effective even for complex target mixtures and in cases where the probe and target have quite different lengths. In particular, we are excited about the prospects that it may be directly applicable to searching for target sequences in samples of genomic RNA. We believe that these methods will find wide application in molecular biology and clinical diagnosis. ACKNOWLEDGMENT We are pleased to thank Barbara MacGregor, Doug Turner, Paul Coleman, and Esther Conwell for insightful comments and careful readings of the manuscript as well as Howard Federoff for making this work possible. We are also grateful for support of the early part of this work by a grant from the NIH (AG18231).

Received for review May 26, 2005. Accepted July 28, 2005. AC050921Y

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