Anal. Chem. 2007, 79, 4724-4728
Correspondence
DNA Nanoswitch as a Biosensor Amy H. Buck,† Colin J. Campbell,*,†,‡ Paul Dickinson, † Christopher P. Mountford,§ He´le`ne C. Stoquert,† Jonathan G. Terry,| Stuart A. G. Evans,‡ Lorraine M. Keane,† Tsueu-Ju Su,§ Andrew R. Mount,‡ Anthony J. Walton,| John S. Beattie,† Jason Crain,§ and Peter Ghazal†
Division of Pathway Medicine, School of Physics, School of Chemistry, and Institute for Integrated Micro and Nano Systems, University of Edinburgh, Edinburgh, UK
We present a new type of DNA switch, based on the Holliday junction, that uses a combination of binding and conformational switching to enable specific label-free detection of DNA and RNA. We show that a single RNA oligonucleotide species can be detected in a complex mixture of extracted cellular RNA and demonstrate that by exploiting different aspects of the switch characteristics we can achieve 30-fold discrimination between singlenucleotide mismatches in a DNA oligonucleotide. Molecular machines are a diverse class of construct capable of moving between distinct states.1,2 The transitions between these states can be triggered by electrochemical, optical, thermal, or chemical stimuli to result in changes at a molecular level,3 and their mechanisms can be harnessed to perform a range of useful tasks from computation to actuation. However, none to date has used a switch mechanism as a principle for sequence-specific, nucleic acid recognition. During the past 10 years, protein and small-molecule detection schemes have benefited from the development of probes such as aptamers4 and DNAzymes5 that are based on novel methods of molecular recognition. Nucleic acid detection, however, remains dominated by base pairing, which relies fundamentally on differences in bonding enthalpies between probes and matched/ mismatched targets. In order to ensure a high level of fidelity of sequence detection, many of these assays must incorporate a second-stage recognition process, such as ligation or primer
* To whom correspondence should be addressed. E-mail: colin.campbell@ ed.ac.uk (C.J.C.),
[email protected] (P.G.). † Division of Pathway Medicine. ‡ School of Chemistry. § School of Physics. | Institute for Integrated Micro and Nano Systems. (1) Beckman, R.; Beverly, K.; Boukai, A.; Bunimovich, Y.; Choi, J. W.; DeIonno, E.; Green, J.; Johnston-Halperin, E.; Luo, Y.; Sheriff, B.; Stoddart, J. F.; Heath, J. R. Faraday Discuss. 2006, 131, 9-22. (2) Bedard, T. C.; Moore, J. S. J. Am. Chem. Soc. 1995, 117, 10662-10671. (3) Seeman, N. C. Trends Biochem. Sci. 2005, 30, 119-125. (4) Hamaguchi, N.; Ellington, A.; Stanton, M. Anal. Biochem. 2001, 294, 126131. (5) He, Q. W.; Miller, E. W.; Wong, A. P.; Chang, C. J. J. Am. Chem. Soc. 2006, 128, 9316-9317.
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Figure 1. DNA switch. Assembly (probe and target hybridization) and Mg2+-dependent switching of the device. “D” is the position of the donor and “A” is the position of the acceptor.
extension,6,7 and this enhanced specificity comes at the expense of added time and cost. On this basis, we sought to understand whether the properties of a molecular switch could be exploited to provide, as a single entity, a two-stage molecular recognition device whose switch characteristics have the potential to improve specificity and avoid the need for labeling a sample. The molecular switch described here is structurally based on the Holliday junction (HJ), a fourway DNA junction with a central branch point.8 The switch presented in Figure 1 requires the hybridization of two strands: (1) a labeled probe strand that comprises the majority of the structure and contains two unpaired binding arms and (2) a target strand whose hybridization to the probe completes the assembly of the DNA switch. The nucleotide sequence around the branch point is designed so that the junction is immobile9 and the switch exists in either an open (extended) or closed (coaxially stacked) conformation.10,11 Extensive biochemical data have shown that electrostatics dominate the equilibrium between open and closed states of the HJ,12,13 which can be switched by the presence or absence of (6) Bockisch, B.; Grunwald, T.; Spillner, E.; Bredehorst, R. Nucleic Acids Res. 2005, 33, e101. (7) Kwok, P. Y. Annu. Rev. Genomics Hum. Genet. 2001, 2, 235-258. (8) Holliday, R. A. Genet. Res. 1964, 5, 282-304. (9) Seeman, N. C.; Kallenbach, N. R. Biophys. J. 1983, 44, 201-209. (10) Cooper, J. P.; Hagerman, P. J. J. Mol. Biol. 1987, 198, 711-719. (11) Duckett, D. R.; Murchie, A. I.; Diekmann, S.; von Kitzing, E.; Kemper, B.; Lilley, D. M. Cell 1988, 55, 79-89. (12) Duckett, D. R.; Murchie, A. I.; Lilley, D. M. EMBO J. 1990, 9, 583-590. (13) Liu, J.; Declais, A. C.; Lilley, D. M. J. Mol. Biol. 2004, 343, 851-864. 10.1021/ac070251r CCC: $37.00
© 2007 American Chemical Society Published on Web 05/18/2007
different cations.11,14 By introducing donor and acceptor fluorophores at specific sites in the probe, switching between open and closed states of the probe-target complex can be monitored using FRET,15 thus eliminating the need for target labeling. There are two possible conformations of the closed state, depending on the sequence around the branch point;16 the switch presented here is based on the well-studied junction, “J1”,17,18 which exists predominantly (95%) in the stacking conformation depicted in Figure 1.19 We have previously examined the design properties of the HJ probe and determined the positions of the donor and acceptor moieties that confer the largest differential FRET signal between open and closed states.20 Here we examine the switching properties of this HJ and demonstrate its unique capacity as a sequence-specific biosensor. MATERIALS AND METHODS DNA Switch Assembly. The DNA probe (5′-TGCATAGTGGATTGCATTTTTGCAATCCTGAGCACATTTTTGTGCTCACCGAATCCCA-3′) was synthesized (Eurogentec) with tetramethylrhodamine attached at the 5′ end and carboxyfluorescein attached internally at a thymidine (19 nucleotides from the 5′ end). The probe was assembled in 20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, and 50 mM NaCl in the presence of 0-5-fold excess target DNA oligonucleotide (5′-TGGGATTCGGACTATGCA-3′) or noncomplementary DNA oligonucleotide (5′- GAGCCGTTTAAGTGCGAT-3′); concentrations of each probe/target are indicated in the figure legends. Samples were incubated in a water bath at 80 °C for 30 min, followed by a slow temperature decrease to room temperature. The Mg2+ and Na+ ions were then removed by sequential application to two Sephadex G25 gel filtration columns (Amersham Biosciences UK Ltd.) to buffer exchange the sample into 20 mM Tris-HCl (pH 7.5). This protocol was designed to eliminate any potential alternate conformers or aggregates of the probe/target complex, however, further analysis demonstrated that annealing of the probe and target at room temperature for 85% bound under these conditions (see Supporting Informa(22) Chen, J. H.; Churchill, M. E.; Tullius, T. D.; Kallenbach, N. R.; Seeman, N. C. Biochemistry 1988, 27, 6032-6038. (23) Duckett, D. R.; Lilley, D. M. J. Mol. Biol. 1991, 221, 147-161.
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tion Figure 3), and we therefore conclude that the difference is primarily due to switching. Under the same MgCl2 conditions (100 µM), when FRET is measured at lower concentrations of probe and target (50 nM/100 nM), discrimination is further enhanced (Figure 3 and Supporting Information Figure 4), demonstrating that binding and switching conditions can be optimized to produce up to 30-fold discrimination between matched and mismatched targets. The exact factors that allow switching discrimination between each specific point mutation are currently under investigation. Indeed, mismatches could influence any combination of three factors: the Mg2+ concentration at which the switch closes (as shown in Supporting Information Figure 2), the overall FRET emission intensity due to changes in the equilibrium between the two different closed conformers or the overall FRET emission intensity due to changes in the distance or angle between the helices in the closed state. Nonetheless, as demonstrated in Figures 2 and 3, the switch mechanism provides a novel method for distinguishing single-nucleotide mismatches. Having established the potential for sequence-dependent detection with the HJ switch, we explored its efficacy for detection of an RNA target. In Figure 4, we show that the same probe described above, at 10 nM, can detect the complementary RNA target. In addition, this specific RNA target can be detected in a background of 100 µg/mL complex heterogeneous RNA extracted from murine kidney. Figure 4 shows a plot of the FRET ratios resulting from a titration of the probe with the RNA target in the absence or presence of switching concentrations of Mg2+ (5.0 mM) and in the absence or presence of the complex RNA.
Figure 3. Switching characteristics for maximal specificty. (a) Normalized FRET ratios calculated at 5.0 mM MgCl2 (1 µM/5 µM probe/target) and 100 µM MgCl2 (1 µM/5 µM probe/target and 50 nM/100 nM probe/target). Data were normalized by treating the FRET ratio of the probe in the absence of target as 0 and the FRET ratio of the probe in the presence of matched target as 1. Error bars shown are standard deviations (n ) 3). The unnormalized FRET ratios are shown in Supporting Information Figure 4.
excess of probe concentration, since this ensures that differences in FRET ratio are due to sequence-specific differences and not the kind of concentration-dependent behavior shown in Figure 4.
Figure 4. Detection of RNA target. FRET ratio of 10 nM probe, titrated with 0-5× RNA target in the presence and absence of 5.0 mM MgCl2 and in the presence or absence of 100 µg/mL murine renal total RNA. Error bars shown are standard deviations (n ) 3).
Titration of the probe with its complementary RNA target results in a concentration-dependent increase in the FRET ratio in the presence of 5.0 mM Mg2+, which is preserved in the presence of murine renal total RNA. The HJ switch is therefore capable of detecting target RNA even in the presence of background cellular extract. Such a target-dependent FRET increase establishes a prerequisite for applications of this concept in quantitative transcription detection, and the concentration demonstrated represents detection of a highly abundant RNA transcript. Currently, we would require some sample amplification such as PCR, in order to detect low levels of transcripts, but we have not carried out sufficient assay development to establish the true limits of this technique. Clearly, FRET has been established as a singlemolecule technique,24,25 indicating that detection of lower concentrations of target should be possible, and we are currently investigating how we can move toward such high sensitivity. In Figure 3, we demonstrated that FRET ratio is dependent on target sequence, with a perfect match giving the largest value (Figure 3). We have further shown that the FRET ratio increases in proportion to target concentration (Figure 4). We therefore consider that, when using our nanoswitch to measure sequence variation, it is necessary for the target concentration to be in (24) Joo, C.; McKinney, S. A.; Lilley, D. M. J.; Ha, T. J. Mol. Biol. 2004, 341, 739-751. (25) McKinney, S. A.; Tan, E.; Wilson, T. J.; Nahas, M. K.; Declais, A. C.; Clegg, R. M.; Lilley, D. M. J.; Ha, T. Biochem. Soc. Trans. 2004, 32, 41-45.
CONCLUSIONS We have demonstrated the principle that a DNA switch combining Watson-Crick base pairing with sequence-dependent switching affords molecular recognition precision beyond base pairing and avoids the need for target labeling when using either DNA or RNA targets. A recent report has shown that an HJ of similar size functions as a single-molecule nanometronome. While the authors demonstrate that the rate of switching between conformations can be changed by hybridization of one arm to another, they do not demonstrate the application of their device for sequence-specific detection.26 Other types of probe, such as molecular beacons, aptamers, and DNAzymes, undergo a conformational change on target binding; however, the conformational change is coupled directly to target binding. Our approach is different since binding and switching are separate events. For example, in the case of molecular beacons,27 detection of specific nucleic acid sequences is achieved through hybridization of a target to a hairpin (stemloop) probe where the probe sequence is in the uncomplexed loop. In the absence of binding, the stem is complexed and this constrains two dye molecules (commonly a donor and either an acceptor or a quencher) in proximity leading either to FRET or a low donor emission, respectively. Binding of target to the probe (loop) competes with the stability of the stem, leading to its concerted dissociation and the consequent separation of the dye pair, which alters the fluorescent output. The molecular beacon’s conformational change requires the simultaneous formation and breaking of hydrogen bonds, which is facilitated under tightly defined reaction conditions, and although high degrees of specificity can be achieved, this often necessitates long assay times.27-29 The fundamental features of the switch that we present here are distinct from molecular beacons since binding and detection through switching are separate events. The fact that, in this device, (26) (27) (28) (29)
Buranachai, C.; McKinney, S. A.; Ha, T. Nano Lett. 2006, 6, 496-500. Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303-308. Mhlanga, M. M.; Malmberg, L. Methods 2001, 25, 463-471. Tsourkas, A.; Behlke, M. A.; Rose, S. D.; Bao, G. Nucleic Acids Res. 2003, 31, 1319-1330.
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the probe strands are initially uncomplexed removes any intrinsic energy barrier to hybridization and can thereby increase the kinetics of detection. Similarly, aptamers and DNAzymes are probes, which undergo conformational changes on target binding, but although both types can be designed to be highly specific, binding and switching are directly coupled. Moreover, we note that most other detection strategies for highly specific nucleic acid discrimination employ either secondary enzymatic reactions (such as primer extension) or stringent denaturing conditions that are not necessary when using our DNA switch. For both molecular beacons and the DNA switch, sensitivity is dictated by the properties of fluorophore, fluorescence detection method, background signal, and optical imaging instrumentation. A significant amount of research has gone into developing the design features of molecular beacons, which are reported to be able to detect
