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Available online at www.sciencedirect.com ScienceDirect Technological applications arising from the interactions of DNA bases with metal ions Ki Soo Park and Hyun Gyu Park An intense interest has grown in the unique interactions of nucleic acids with metal ions, which lead to the formation of metal-base pairs and the generation of fluorescent nanomaterials. In this review, different types of metal-base pairs, especially those formed from naturally occurring nucleosides, are described with emphasis also being given to recent advances made in employing these complexes to govern enzymatic reactions. The review also contains a comprehensive description of DNA-templated inorganic nanomaterials such as silver nanoclusters which possess excellent fluorescence properties. Finally, a summary is given about how these materials have led to recent advances in the field of nanobiotechnology. Address Department of Chemical and Biomolecular Engineering (BK 21+ program), KAIST, Daehak-ro 291, Yuseong-gu, Daejeon 305-701, Republic of Korea Corresponding author: Park, Hyun Gyu ([email protected]) Current Opinion in Biotechnology 2014, 28:17–24 This review comes from a themed issue on Nanobiotechnology Edited by Jonathan S Dordick and Kelvin H Lee For a complete overview see the Issue and the Editorial Available online 24th November 2013 0958-1669/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. Moreover, a large number of nucleic acid sequences can be efficiently and cost-effectively generated by employing automated solid-phase synthetic processes. Finally, nucleic acids are highly stable entities that can be manipulated under a wide range of environmental conditions. Metal ions, which have unique chemical and physical properties related to electron conductivity, magnetism, and catalysis, have been incorporated into nucleic acids to create metal-mediated functioning materials [2]. A representative example is found in systems that use metal ions rather than hydrogen-bond driven base pairing to promote formation of base pairs between non-complementary DNA bases. This non-natural base pairing is stabilized by coordination of metal ions to DNA bases. Inorganic fluorescent nanomaterials such as silver nanoclusters have attracted special interest as replacements for conventional organic fluorophores owing to their desirable photophysical properties and high photostabilities. Among a number of synthetic routes developed to prepare the inorganic fluorescent nanomaterials, those that use nucleic acids, especially DNA, have been found to be highly versatile [3]. As a result, DNA-mediated fluorescent nanomaterials have been fabricated and extensively applied in the development of novel bio-sensing strategies, molecular logic gates, and nanomachines. http://dx.doi.org/10.1016/j.copbio.2013.10.013 Introduction Nucleic acids, polymers comprised of phosphodiester linked chains of purine (adenine (A) and guanine (G)) and pyrimidine (thymine (T) and cytosine (C)) bases, have long been recognized to be the carriers of genetic information in living systems. However, in recent years many attempts have been made to exploit the chemical properties of nucleic acids in non-biological contexts. Several desirable features of nucleic acids enable them to be used to produce the nanomaterials and nanostructures [1]. For example, the sizes of nucleic acids are in the nanometer range, with a duplex of ten nucleotides having an overall length of 3.4 nm and a width of 2 nm. In addition, nucleic acids undergo straightforward sequence-dependent hybridization with complementary strands and they capture specific target molecules with high specificities and affinities. This property enables information to be conveniently and specifically programmed into a variety of DNA nanostructures. www.sciencedirect.com The unique interactions of DNA bases with metal ions have been utilized as the basis for the generation of metalbase pairs and fluorescent nanomaterials. In this review, an overview of DNA-based metal-base pairs, especially those formed by naturally occurring nucleosides, is given along with a discussion of recent advances that have been made in applications of these substances, including their use to control enzymatic reactions. In addition, the synthesis, characterization and applications of DNA-templated fluorescent nanomaterials such as silver nanoclusters are reviewed. Metal ion-mediated base pairing The metal ion-linked duplexes have attracted recent interest because they possess metal-related properties such as electron conductivity and magnetism, and they can serve to govern interesting enzymatic reactions. The first example of incorporation of metal ions into the core of DNA double helixes was uncovered by Lee et al. In this study, Lee et al. observed that the divalent Current Opinion in Biotechnology 2014, 28:17–24 18 Nanobiotechnology Figure 1 (a) H (d) O O O N C NH HN T N T N O N N T HgII N O O O N N H N T H N O H O AgI C N N H N N G O O H N N H (b) (e) NH2 NH2 H2N H2N NH C N N C N C N O N N C AgI N O O N PdC O (c) O T H2N AgI T NH N N N N O C AgI N O O O H2N N N C N O C H2N N N N N N O O H2N O T HgII N O C N O Current Opinion in Biotechnology Schematic illustration of metal ion-mediated base pairing. (a) Hg2+ ion-mediated T–Hg2+–T pair formation [5]. (b) Ag+ ion-mediated C–Ag+–C pair formation [6]. (c) Predicted structures of T–Ag+–C and T–Hg2+–C pairs [7,8]. (d) Ag+ ion-mediated base triplet CG.CAg+ [9]. (e) Predicted structure of Ag+ ion-mediated PdC–Ag+–C pair [10,11]. PdC: pyrrolo-dC. metal ions such as Zn2+, Co2+ and Ni2+ stabilize unmodified DNA duplexes at elevated pH [4]. The more direct observation of binding of metal ions in the interiors of DNA duplexes was made in studies with the naturally occurring thymine (T)–thymine (T) mismatched base pair, which demonstrated that Hg2+ ions participate in formation of a stable T–Hg2+–T base pair (Figure 1a) [5]. In a similar way, the natural cytosine (C)–cytosine (C) mismatched base pair was found to be stabilized by Ag+ ions through formation of a C–Ag+–C base pair (Figure 1b) [6]. Also, Urata et al. and Ono et al. independently discovered that a duplex DNA containing a T–C mismatched base pair is moderately stabilized in the presence of Ag+ and Hg2+ ions, possibly as a result of the formation of respective T–Ag+–C and T–Hg2+–C base pairs (Figure 1c) [7,8]. Recently, Jyo et al. observed that Current Opinion in Biotechnology 2014, 28:17–24 Ag+ ions mediate the formation of a DNA triplex (CG.CAg+) by replacing Hoogsteen base pairing with the metal-base pairing (Figure 1d) [9]. A fluorescent base analog, which is structurally similar to the corresponding natural nucleobase and exhibits environment-dependent fluorescence properties, was also shown to form a metal ion-mediated base pair [10]. In this effort, Park et al. found that when pyrrolo-dC (PdC), a fluorescent analog of the cytosine nucleobase, is paired with cytosine within a duplex DNA, the fluorescence efficiency of PdC is significantly diminished in the presence of Ag+ ions (Figure 1e) [11]. Park et al. assumed that Ag+ ions stabilize the PdC–C mismatched base pair through simultaneous coordination with the N3 nitrogen of PdC and C. Formation of this complex leads to more www.sciencedirect.com Interactions of DNA bases with metal ions Park and Park 19 efficient pi-stacking of the mismatched nucleobases and collisional quenching of the intrinsic fluorescence of PdC. Application of metal ion-mediated base pairing The novel techniques have been devised to detect Hg2+ and Ag+ ions by taking advantage of specific interactions of these metal ions with the respective thymine–thymine (T–T) and cytosine–cytosine (C–C) mismatched base pairs (Figure 2a) [12,13]. In addition, new versions of molecular beacon probes, consisting of hairpin-structured DNA possessing fluorophore-quencher pairs at both ends, have been constructed to monitor DNA hybridization and to detect small molecules. The hydrogen bonding pattern in the DNA stem region is replaced by metal-base pairing (Figure 2b) [14,15]. The new molecular beacon was demonstrated to be superior because it exhibits a lower background signal, higher thermal stability, and more flexible stem structures, all of which enable it to be more applicable in complex biological environments. However, the new molecular beacon probes still require double labeling of DNA with a fluorophore and a quencher, a requirement that leads to the need for more difficult synthetic routes and a significant loss in the affinity and specificity of the probe. To overcome this limitation, several approaches have been used to design label-free probes. In these strategies, the molecular recognition events and signal generation steps occur separately (Figure 2c) [16–18]. Representative of this approach is the design of a molecular beacon probe that employs the complexes T–Hg2+–T or C–Ag+– C to release Hg2+ or Ag+ from the DNA stem region upon interaction with the target molecules. The released metal ions then are employed to generate a signal, which identifies the presence of the target molecules. By rationally designing the base sequence in the probes, this simple label-free strategy can be employed to detect various target molecules, such as complementary DNA, proteins, and small molecules. Metal ion-mediated base pairing has also been utilized to construct DNA-based nanomolecular machines that are capable of undergoing reversible and repeatable mechanical motion. Seminal studies by Sen et al. led to the development of a new three-way junction-based DNA nanomachine, fueled by binding of Hg2+ ions to T–T mismatches present in one of the three-way junction stems, that exhibits reversible, mechanical and electrical switching (Figure 2d). Interestingly, this type of switching can be employed to couple mechanical motion with changes in hole transport efficiency, a feature which could facilitate electrical monitoring of structural changes in DNA [19]. Finally, metal ion-mediated base pairing has also been utilized to induce enzymatic reactions. The first example www.sciencedirect.com of this feature was uncovered by Urata et al., in a study which demonstrated that, DNA polymerases in the presence of Hg2+ ions incorporate 20 -deoxy-thymidine 50 triphosphate (dTTP) at site opposite to thymine in a template DNA. This process involves formation of a stable T–Hg2+–T base pair and leads to elongation of the primer and synthesis of a full-length product (Figure 3a) [20]. At nearly the same time, Park et al. described a Hg2+ and Ag+ promoted ‘illusionary’ polymerase activity that accomplishes an unnatural extension reaction even at mismatched sites (T–T and C–C) of a primer with template DNA (Figure 3b) [21]. These workers utilized this unnatural polymerase activity induced by metal ions as the basis for a novel strategy to construct a molecular scale logic gate. Recently, Urata et al. found that in the presence of Ag+ ions, adenine is mis-incorporated into the site opposite a C residue in template DNA. This phenomenon probably takes place through formation of a C–Ag+–A base pair [22]. However, why a C–Ag+–A base pair instead of the more thermodynamically stable C–Ag+–C base pair is formed and, consequently, what is the molecular basis of the Ag+-mediated incorporation of adenine remain questions that do not have clear answers. In addition to the induction of DNA polymerase activity, metal ion-coordinated base pairing has been utilized to trigger other enzymatic activities such as those of nicking endonucleases (Figure 3c) [23], ligases (Figure 3d) [24], and exonucleases [25]. The catalytic activities of some deoxyribozymes, which are rationally modified to contain T–T or C–C mismatched base pairs, have also been modulated through formation of T-Hg2+-T or C-Ag+-C base pairs [26,27]. In the same manner, Shangguan et al. showed that the binding affinity of a DNA aptamer, which is rationally modified to contain T–T mismatched base pairs, can be controlled by Hg2+ ions through the formation of T–Hg2+–T [28]. DNA-templated fluorescent nanomaterials Owing to the unique properties, which include high fluorescence quantum yields, high photostabilities and facile syntheses, fluorescent nanomaterials have emerged as promising alternatives to conventional organic fluorophores [3]. A representative example of a fluorescent nanomaterial of this type is a nucleic acid-templated silver nanocluster consisting of a few to ten atoms of silver (<2 nm). Nucleic acids, especially DNA, serve as initial nucleation sites for fluorescent silver nanocluster formation and they stabilize the clusters by serving as the capping agents. The most common form of a DNA template used to produce fluorescent silver nanoclusters is the cytosine-rich single-stranded oligonucleotides [29], but other nucleobases such as guanine and adenine have also been reported to promote the formation of these nanoclusters [30]. Current Opinion in Biotechnology 2014, 28:17–24 20 Nanobiotechnology Figure 2 (a) T-HgII-T HgII Electron transfer Graphene oxide hv C-AgI-C AgI Electron transfer Graphene oxide hv (b) T-HgII-T HgII Target DNA F Q (c) T-HgII-T HgII Target DNA QD QD HgII (d) h+ h+ X HgII FRET FRET T-HgII-T Current Opinion in Biotechnology Application of metal ion-mediated base pairing to the development of target-detecting strategies and DNA-based nanomachines. (a) Schematic representation of the detection of heavy metal ions on the graphene oxide arrays [13]. (b) The use of stem flexibility to design metal ion-mediated Current Opinion in Biotechnology 2014, 28:17–24 www.sciencedirect.com Interactions of DNA bases with metal ions Park and Park 21 Figure 3 (a) dTTP w/o Hg2+ Klenow fragment T T T T w/ Hg2+ T (b) DNA primer HgII T DNA primer T T C C Template DNA X X Template DNA : Taq DNA polymerase T HgII : Taq DNA polymerase C T : Hg2+ AgI C : Ag+ C C T T (c) Recognition sequence T T T C T A G G w/ Hg2+ G G A T C Nt. AlwI T T T T T T C C T A G T T T (d) C C T A G Cleavage site T T w/ Hg2+ T T C C T A G G G A T C T T T T T T + Cleaved DNA product DNA ligase T T DNA polymerase dNTPs C C w/ Ag+ DNA ligase C C C C Rolling circle amplification (RCA) Current Opinion in Biotechnology Application of metal ion-mediated base pairing to induce enzymatic activities. (a) Incorporation of thymine nucleotides by DNA polymerase through T– Hg2+–T base pairing [20]. (b) ‘Illusionary’ polymerase activity triggered by Hg2+ and Ag+ ions via their interaction with T–T and C–C mismatched base pair [21]. (c) Cleavage activity of nicking endonuclease triggered by Hg2+ ions, which form the stable T–Hg2+–T base pair [23]. (d) DNA ligase activity induced by Hg2+ and Ag+ ions, which form corresponding stable T–Hg2+–T and C–Ag+–C base pairs [24]. Fluorescent silver nanoclusters have been also demonstrated to be efficiently generated from metal ion complexation with several secondary structures of DNA including hairpin, G-quadruplex, triplex, and i-motif (Table 1) [30–33]. Interestingly, double-stranded (ds) DNAs, which are not regarded as good templates for silver nanocluster generation [30], have been modified so that they produce fluorescent silver nanoclusters. Modified dsDNAs, which incorporate a loop moiety, mismatched site, abasic site, and gap-site [34–37], have been utilized as a synthetic template for fluorescent silver nanocluster formation (Table 1). (Figure 2 Legend Continued) molecular beacon probes that signal molecular interactions [14]. (c) Label-free metal ion-mediated molecular beacon probe that operates by separating the molecular recognition and signal reporting steps [18]. (d) A three-way junction-based DNA nanomachine exhibiting reversible, mechanical and electrical switching that is fueled by Hg2+ binding to T–T mismatched base pair [19]. www.sciencedirect.com Current Opinion in Biotechnology 2014, 28:17–24 22 Nanobiotechnology Table 1 Various types of DNA templates to promote formation of fluorescent silver nanoclusters DNA structures DNA sequences 0 0 lex/lem Reference Single-stranded linear DNA 5 -CCC TTT AAC CCC-3 50 -CCC TCT TAA CCC-30 50 -CCC TTA ATC CCC-30 50 -CCT CCT TCC TCC-30 50 -CCC TAA CTC CCC-30 350/485 420/520 470/572 530/620 650/705 [29] DNA hairpin 50 -TATC CGT CCCCC ACG GATA-30 a 50 -TATC CGT GGGGG ACG GATA-30 a 50 -GGG TTA GGG T CCC CCC ACCC TTA CCC-30 a 581.7/646.3 544.6/614.6 494/570 581/646 [30] G-quadruplex 50 -GGT GGT GGT GGT TGT GGT GGT GGT GG-30 325/420 510/680 [31] DNA triplex 50 -GAG AGG AGA GAG AAG AGG AAG-30 30 -CTC TCC TCT CTC TTC TCC TTC-50 50 -CTC TCC TCT CTC TTC TCC TTC-30 480/534 [32] i-motif 50 -(TAACCCC)4-30 50 -(CCCCAA)3CCCC-30 460/560 500/570 [33] Duplex DNA with a six-cytosine loop 30 -CACG TGGA CTGA GG CCCCCC ACA CCT CTTC-50 b 50 -GTGC ACCT GACT CC TGT GGA GAAG-30 520/572 [34] Duplex DNA with a mismatched site 30 -GGG ATT GGG X TTG GGA TTG GGA-50 c 50 -CCC TAA CCC T AAC CCT AAC CCT-30 520/570 [35] Duplex DNA with an abasic site 30 -TAC CAC CCC CGT CGC-50 50 -ATG GTG GXG GCA GCG-30 d 588/670 [36] Duplex DNA with a gap site 30 -CGA GTA CCA CCC CCG TCG CGG AG-50 e 50 -GCT CAT GGT GG-30 50 -GGC AGC GCC TC-30 560/643 [37] a b c d e The The The The The [46] underlined sequence is the stem region of hairpin structured DNA. underlined sequence is the six-cytosine loop bulged from the duplex DNA. underlined X is thymine, guanine, and cytosine nucleobases that is mismatched base paired with thymine in the opposite strand. underlined X is the abasic site (dSpacer, tetrahydrofuran residue). underlined C is the cytosine nucleobase that is positioned opposite the gap site within duplex DNA. Application of DNA-templated fluorescent nanomaterials Various strategies that take advantage of the high fluorescence properties of DNA-silver nanoclusters have been utilized to detect target DNA, microRNA, proteins, small molecules, and metal ions [3]. A representative example is found in the discovery by Werner et al. that the fluorescence of DNA-silver nanoclusters is dramatically enhanced when particular nucleotides such as guanine or thymine are placed in close proximity [38]. Based on this observation, these workers developed a novel DNA detection probe, named a ‘Nano-Cluster Beacon,’ that displays turn-on fluorescence upon target binding. The same group also developed another type of DNA probe, termed a ‘chameleon NanoCluster Beacon’ for the rapid and precise screening of single-nucleotide polymorphisms (SNPs) [39]. This new molecular probe consists of a silver nanocluster-containing strand and a guanine-rich enhancer strand, which are brought into close proximity when it binds to SNP targets. This phenomenon brings about fluorescence emission at various wavelengths (colors) depending on the alignment Current Opinion in Biotechnology 2014, 28:17–24 between silver nanoclusters and the enhancer. By following this approach, aptamer-based sensors have been designed. These sensors rely on a target-induced conformational change of the DNA aptamer probe, containing silver nanoclusters, and guanine-rich enhancer sequences at both ends. This change brings the guanine-rich enhancer sequence close to the silver nanoclusters and causes a resulting fluorescence enhancement [40]. In addition, novel strategies for sensing biological thiols such as cysteine, homocysteine, and glutathione and metal ions such as Cu2+ and Hg2+ ions have been devised based on the fact that these substances strongly interact with and change the fluorescence of silver nanoclusters [41–43]. DNA-silver nanoclusters have also been employed as labeling systems for biomolecule imaging. Dickson et al. initiated this area by designing DNA-protected silver nanoclusters that are covalently conjugated with avidin or a primary antibody for cell surface labeling [44]. Recently, it was also reported that the silver nanoclusters, which specifically mark the nucleus of live cells, can be www.sciencedirect.com Interactions of DNA bases with metal ions Park and Park 23 prepared by using aptamers against target cells as the nucleation template [45]. DNA-templated silver nanoclusters have been also utilized to construct molecular logic gates, switches, and fluorescent hydrogels that are applicable in the area of nanobiotechnology. For example, Wang et al. devised a molecular logic device, which employs K+ or H+ ions as inputs and modulates the fluorescence properties of DNA-silver nanoclusters by inducing the structural changes in template DNA [46]. The same group also designed a new type of fluorescent molecular switch, which operates through a DNA strand exchange reaction that leads to fluorescence on-off switching of DNA-silver nanoclusters [47]. In addition, fluorescent hydrogels, which are comprised of a Y-shaped DNA structure modified with silver nanoclusters as a functional component, have been prepared and shown by Willner et al. to exhibit thermally reversible solution– hydrogel transitions without loss of fluorescence intensities [48]. Acknowledgements This work was supported by the grant from the Basic Science and Public Welfare & Safety Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (No. 2009-0080602) (No. 2012M3A2A1051683) and the Industrial Source Technology Development Program of the Ministry of Knowledge Economy (MKE) (No. 2010-10038683). References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1. Berti L, Burley GA: Nucleic acid and nucleotide-mediated synthesis of inorganic nanoparticles. Nat Nanotechnol 2008, 3:81-87. 2. Takezawa Y, Shionoya M: Metal-mediated DNA base pairing: alternatives to hydrogen-bonded Watson–Crick base pairs. Acc Chem Res 2012, 45:2066-2076. 3. Latorre A, Somoza A: DNA-mediated silver nanoclusters: synthesis, properties and applications. Chembiochem 2012, 13:951-958. 4. Lee JS, Latimer LJ, Reid RS: A cooperative conformational change in duplex DNA induced by Zn2+ and other divalent metal-ions. Biochem Cell Biol 1993, 71:162-168. 5. Miyake Y, Togashi H, Tashiro M, Yamaguchi H, Oda S, Kudo M, Tanaka Y, Kondo Y, Sawa R, Fujimoto T et al.: MercuryIImediated formation of thymine-HgII-thymine base pairs in DNA duplexes. J Am Chem Soc 2006, 128:2172-2173. 6. Ono A, Cao S, Togashi H, Tashiro M, Fujimoto T, Machinami T, Oda S, Miyake Y, Okamoto I, Tanaka Y: Specific interactions between silver(I) ions and cytosine–cytosine pairs in DNA duplexes. Chem Commun 2008:4825-4827. 7. Urata H, Yamaguchi E, Nakamura Y, Wada S: Pyrimidine– pyrimidine base pairs stabilized by silver(I) ions. Chem Commun 2011, 47:941-943. 8. Ono A, Torigoe H, Tanaka Y, Okamoto I: Binding of metal ions by pyrimidine base pairs in DNA duplexes. Chem Soc Rev 2011, 40:5855-5866. 9. Ihara T, Ishii T, Araki N, Wilson AW, Jyo A: Silver ion unusually stabilizes the structure of a parallel-motif DNA triplex. J Am Chem Soc 2009, 131:3826-3827. Conclusions Unique interactions between DNA bases and metal ions, which lead to the formation of metal ion-mediated base pairs and the generation of fluorescent nanomaterials, have been used to construct novel sensing, imaging and computing systems, and to build nanostructures and nanomachines. Moreover, metal ion-mediated base pairing along with well-established sequence-specific hybridization also has the potential of being employed to produce complex DNA nanoarchitectures. In spite of the substantial progress that has been made in exploring the potential of specific interactions that take place between DNA bases and metal ions, many challenges remain. For example, the high specificities seen in metal ion interactions with DNA bases have not been explained fully because the formations of metal-base pairs in most cases have only been indirectly confirmed by observation of the enhanced thermal stabilities of the corresponding duplexes. Owing to this gap, in-depth investigations are required to fully understand the mechanism of metal ion binding to DNA bases. Furthermore, the exact structures of silver nanoclusters have not been unambiguously elucidated and, as a result, it is not possible to generate a molecular basis to predict precisely the emitting fluorescence, which has been observed to depend on several variables, such as DNA sequence, buffer, pH, and reagent concentration. Thus, additional basic investigations are needed in order to determine how the fluorescence properties of DNA-templated nanomaterials can be more concisely controlled. 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