Homogeneous DNA-detection Based on the Non

ANALYTICAL SCIENCES JUNE 2007, VOL. 23 2007 © The Japan Society for Analytical Chemistry

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Reviews

Homogeneous DNA-detection Based on the Non-enzymatic Reactions Promoted by Target DNA Toshihiro IHARA*,**† and Motoko MUKAE* *Department of Applied Chemistry and Biochemistry, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860–8555, Japan **PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332–0012, Japan

Much effort has focused on methods for detecting various genetic differences in individuals, including single nucleotide polymorphisms (SNPs). SNP can be characterized as a substitution, insertion, or deletion at a single base position on a DNA strand. There is expected to be on average one SNP for every 1000 bases of the human genome, and some variations located in genes are suspected to alter both the protein structure and the expression level. Therefore, highly sensitive techniques with a simple procedure would be desirable for a high-throughput screening of millions of SNPs widely dispersed throughout the human genome. In this short review, we consider recently reported unique techniques for genotyping in a homogeneous solution, and organize them in terms of the chemical and physical processes accelerated on DNA. (Received March 22, 2007; Accepted April 9, 2007; Published June 10, 2007)

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Introduction FRET Excimer Complexation with Metals

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1 Introduction Single nucleotide polymorphisms (SNPs) are the most abundant forms of DNA sequence variation in the human genome, and contribute to phenotypic diversity, influencing an individual’s anthropometric characteristics, risk of certain disease, and variable response to drugs and the environment. Due to their dense distribution across the genome, SNPs are used as markers in medical diagnosis and personalized medicines. To date, several methods based on the hybridization of allele-specific oligonucleotide (ASO) on solid supports were proposed for the effective screening of an enormous number of SNPs. Almost all of the proposed methods, however, consist of multi-step procedures. Such conditions as the temperature, pH, and ionic strength were strictly limited, and must be carefully controlled. Moreover, some of these methods require expensive instruments, while others suffer from a high level of mishybridization or a non-specific background resulting from an interaction with the substrate. Therefore, the new SNP genotyping methodologies, chemistries, and platforms that provide a quality signal with simple operation in solution would be quite beneficial for high-throughput gene analyses. For an assay in solution, the signal should be generated by a certain interaction with the targets, because a free probe can not † To whom correspondence should be addressed. E-mail: [email protected]

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Chemical Ligation Other DNA Mediated Reactions Conclusion References

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be washed down from bound. That is, it is not until the probe binds to the target that the signal should be activated (“off-on” switching). Recently, some promising challenges have been reported to address this difficult task by using split-probing or target-induced specific reactions. Here, we briefly review such molecular devices or mechanisms that work on DNAs with certain sequences. We picked up studies on DNA-directed signal transductions through FRET (fluorescence resonance energy transfer), excimer formation, complexation with metal ions, chemical ligation, and reactions with aptamer and DNAzyme (Fig. 1).

2 FRET Solution-phase DNA detection using FRET is not a technique that has risen during the past several years. Competitive hybridization1 and split probing2 were proposed more than 10 years ago, in which two DNA conjugates carrying, respectively, donor and acceptor dye molecule were used as probes. Since the brilliant success of the original molecular beacon (MB) by Tyagi and Kramer,3 a number of various refinements have been made on MB:4–9 MBs used quantum dots (fluorescent semiconductor nanocrystals, QDs) as a donor,4 Au nanoparticle as a dark quencher,5,6 dual MB,7 duplex labeling by concomitant use of PNA (peptide nucleic acid),8 and MB based on quadruplex structure.9 DNA detection based on intermolecular FRET has also been actively studied. For example, Bichenkova

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Fig. 1 Possible reactions promoted on DNA. DNA would be an excellent scaffold for various interactions or chemical reactions, if the systems were designed carefully. Genetic information is transduced to certain detectable signals by the synergistic functions of DNA complexes.

et al. showed mismatch detection by split probing based on FRET between metal complexes immobilized at the ends of different ODNs (oligodeoxyribonucleotide).10 Dyadyusha et al. showed that emission from QD-ODN (5′-end modified ODN with a QD) was effectively quenched by specific hybridization with Au-ODN (3′-end modified ODN with an Au nanoparticle) (QD/Au system).11 FRET in QD/dye or dye/Au systems to probe specific hybridization was shown by Gill et al.12 and Peng et al.13 Ho et al. attained extremely high sensitivity (3 zM) in DNA detection using FRET between ODN-modified dyes and fluorescent cationic polymers.14 Selective aggregation of the polystyrene-based DNA-modified nanoparticles and FRET between them were reported by Ihara et al.15,16

3 Excimer To form an excimer, two molecules have to make contact with each other. The distance required for making an excimer is much closer compared with that for FRET. Therefore, excimermonomer switching could be a very useful measure that covers the sub-nm range. That is, excimer emission is sensitive to a subtle translocation of one of the counterparts induced by local structural turbulence, while FRET is tolerant to such a small change in distance. Pyrenes have been widely used in studies of the excimer formation on DNA platforms because of its distinct difference in monomer/excimer emission spectra and versatility in chemical derivatization. Paris et al.17 and Balakin et al.18 reported excimer formation between pyrenes modified on the ends of different ODNs. Mahara et al. developed bispyrene probes that respond to RNA by excimer formation.19 Kashida et

al. inserted two pyrene into ODNs with one or two intervening nucleotides to make the probes for deletion polymorphisms.20 The ODN conjugate bearing two pyrenes on both of its terminus was synthesized by Fujimoto et al., as the MB based on excimer/monomer transformation.21 Excimer-based peptide beacons were proposed by Oh et al.22 This probe responds to proteins or DNAs that interact with their peptide backbone.

4 Complexation with Metals There are several examples of metal ion-directed DNA recognition.23–34 They are divided roughly into two groups, metal ion-mediated ligand (probe) modification23–28 and base pairing.29–34 These studies triggered their analytical applications. Brunner and Krämer reported MB based on the hairpin-coil transition through metal complexation (Fig. 2a).35 The MB carries a fluorophore and a chelator on each end. Its fluorescence is quenched by intramolecular complexation with Cu2+ at the end of the stem in hairpin form, while hybridization with a complementary target forces them apart, leading to a restoration of fluorescence. This approach could provide more unambiguous signals compared with the traditional MBs based on FRET, because, as stated in the probes based on excimer/monomer transformation, to make a complex, the counterparts have to be in close proximity to each other. Kitamura et al. reported lanthanide ion-mediated split probing using two ODN probes carrying a metal chelating molecule (Fig. 2b).36,37 Since the sequences of the two probes are designed to be complementary to the adjacent sites of the target, both auxiliary units of the two probes face each other to

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Fig. 2 DNA detection based on metal complexation with chelating DNA probes. (a) Copper-quenched DNA probe.35 The target opens the hairpin structure to form a duplex, resulting in a restoration of fluorescence. (b) Split probing by lanthanide luminescence.36,37 Two chelating probes make a lanthanide complex with the assistance of the target.

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Fig. 3 Chemical ligation-based DNA detections. (a) Quenched autoligation.46 A quencher is eliminated as a leaving group when two probes join, resulting in a restoration of fluorescence. (b) Photochemical ligation of anthracene-ODN conjugates.52 The probes are ligated through anthracene dimer formation with the assistance of a target.

reconstruct an integrated ligand form with all of the essential elements required for emission only when the template DNA is added. Multiplex colorimetry for SNP genotyping was carried out with time-gating techniques.

5 Chemical Ligation Because of high fidelity of ligation, enzymatic ligation has proven to be useful in a number of novel gene detection techniques.38–42 There are, however, a number of potential applications in which it would be useful to be able to join DNA strands without the need for ligase enzymes, as long as high fidelity in ligation could be maintained. After the first report of ligation that does not depend on the enzyme,43 several research groups have been engaged in gene detection using ligation based on a variety of chemical reactions.28,44–51 Xu and Kool reported chemical ligation through nucleophilic substitution with high sequence fidelity.44 They applied this techniques to a multiplex analysis of SNPs using FRET between the fluorescent dyes appended to the probes.45 Moreover, they used a quencher as a leaving group in the nucleophilic ligation reaction to analyze SNPs by fluorescence restoration when joining the probe ends (Fig. 3a).46 The signal amplification by self-ligation was also reported using these systems.45,47 Ficht et al. succeeded in PNA ligation in sequence specific manner for the template.48 They also applied the reaction to signal amplification through spontaneous rearrangement following ligation, which reduces the template affinity of the initially formed ligation product.49 Photoligation has several advantages, including the lack of a need for additives, low cost, and ease of reaction control by wavelength, light strength, and irradiation time. Fujimoto et al. reported photochemical ligation through [2+2] photocyclization reaction of modified bases.50,51 Ihara et al. also succeeded in photochemical ligation through [4+4] photodimer formation of anthracenes appended to the ends of the ODN probes (Fig. 3b).52 Since the yields depend on the sequences of the templates (targets), the techniques could be a quick and sensitive method for SNP genotyping combined with MS or HPLC.

Fig. 4 Split aptamer for fluorescent DNA detection.54 By the assistance of the target, the split aptamers for malachite green are integrated to make a complete form, which binds malachite green and gives a fluorescence signal.

6 Other DNA Mediated Reactions Structure-dependent chemical tagging has been developed by John and Weeks.53 They showed that 2′-amine at the site of a mismatch was acylated more rapidly than paired nucleotides. Kolpashchikov split the aptamer for malachite green into two parts (Fig. 4).54 To each part of the aptamer, half of the target binding arm was appended. The target gathered them to make an integrated form of the aptamer, which bound malachite green and gave the fluorescent signal. To improve the sensitivity, several catalytic systems have been developed for signal amplification using DNA-templated reactions or DNAzymes. Grossmann and Seitz showed that the transfer of the reporter group, one of the counterparts of FRET, from one PNA probe to another (acyl transfer) was catalyzed by the template DNA.55 The yield and turnover number of the reaction depended on the sequences of the templates. Cheglakov et al. succeeded in signal amplification by PCR using DNAzymes appended to the primers.56 Stojanovic et al. synthesized the chimera DNA consisting of a DNAzyme and a MB to make the catalytic MB.57 The catalytic activity of the DNAzyme moiety was restored by target binding to the MB moiety, making it possible to cleave fluorescent substrates in a catalytic manner. Sando et al. reported catalytic signal amplification using self-cleaving DNAzyme58 and a switchable cell-free translation system for luciferase synthesis.59

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7 Conclusion DNA is the only material that enables us to design and synthesize, from scratch, the molecules that bind given nucleic acids according to the rule of Watson–Crick base pairing. In addition, the developing techniques of in vitro evolution have extended the molecular library to molecules that recognize and react with non-nucleoside molecules. Importantly, such DNA units could be combined, covalently, or by hybridization, as independent functional modules to form molecular complexes with intended synergy functions. Thus, DNA would be a versatile scaffold to construct molecular devices for analyzing not only nucleic acids, but also other varieties of species in aqueous solution, such as metal ions,60–63 proteins,64–66 and other physiologically active substances.67,68

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