Imidazolo‐dC Metal‐Mediated Base Pairs: Purine Nucleosides Capture Two Ag+ Ions and Form a Duplex with the Stability of a Covalent DNA Cross‐Link

8‐Phenylimidazolo‐dC (^phImidC, 2 ) forms metal‐mediated DNA base pairs by entrapping two silver ions. To this end, the fluorescent “purine” 2′‐deoxyribonucleoside 2 has been synthesised and converted into the phosphoramidite 6 . Owing to the ease of nucleobase deprotonation, the new Ag⁺‐mediated base pair containing a “purine” skeleton is much stronger than that derived from the pyrrolo‐ [3,4‐d]pyrimidine system (^phPyrdC, 1 ). The silver‐mediated ^phImidC–^phImidC base pair fits well into the DNA double helix and has the stability of a covalent cross‐link. The formation of such artificial metal base pairs might not be limited to DNA but may be applicable to other nucleic acids such as RNA, PNA and GNA as well as other biopolymers.     

Contiguous metal-mediated base pairs comprising two Ag(I) ions

The incorporation of transition‐metal ions into nucleic acids by using metal‐mediated base pairs has proved to be a promising strategy for the site‐specific functionalization of these biomolecules. We report herein the formation of Ag⁺‐mediated Hoogsteen‐type base pairs comprising 1,3‐dideaza‐2′‐deoxyadenosine and thymidine. By defunctionalizing the Watson–Crick edge of adenine, the formation of regular base pairs is prohibited. The additional substitution of the N3 nitrogen atom of adenine by a methine moiety increases the basicity of the exocyclic amino group. Hence, 1,3‐dideazaadenine and thymine are able to incorporate two Ag⁺ ions into their Hoogsteen‐type base pair (as compared with one Ag⁺ ion in base pairs with 1‐deazaadenine and thymine). We show by using a combination of experimental techniques (UV and circular dichroism (CD) spectroscopies, dynamic light scattering, and mass spectrometry) that this type of base pair is compatible with different sequence contexts and can be used contiguously in DNA double helices. The most stable duplexes were observed when using a sequence containing alternating purine and pyrimidine nucleosides. Dispersion‐corrected density functional theory calculations have been performed to provide insight into the structure, formation and stabilization of the twofold metalated base pair. They revealed that the metal ions within a base pair are separated by an Ag???Ag distance of about 2.88 Å. The Ag–Ag interaction contributes some 16 kcal mol^?1 to the overall stability of the doubly metal‐mediated base pair, with the dominant contribution to the Ag–Ag bonding resulting from a donor–acceptor interaction between silver 4d‐type and 4s orbitals. These Hoogsteen‐type base pairs enable a higher functionalization of nucleic acids with metal ions than previously reported metal‐mediated base pairs, thereby increasing the potential of DNA‐based nanotechnology.     

A Dinuclear Mercury(II)‐Mediated Base Pair in DNA

The first dinuclear metal‐mediated base pair containing divalent metal ions has been prepared. A combination of the neutral bis(monodentate) purine derivative 1,N⁶‐ethenoadenine (ϵA), which preferentially binds two metal ions with a parallel alignment of the N−M bonds, and the canonical nucleobase thymine (T), which readily deprotonates in the presence of Hg^II and thereby partially compensates the charge accumulation due to the two closely spaced divalent metal ions, yields the dinuclear T‐Hg^II₂‐ϵA base pair. This metal‐mediated base pair stabilizes the DNA oligonucleotide duplex as shown by an increase of 8 °C in its melting temperature. Formation of the base pair was demonstrated by temperature‐dependent UV spectroscopy as well as by titration experiments monitored by UV and CD spectroscopy.     

Highly Stable Double‐Stranded DNA Containing Sequential Silver(I)‐Mediated 7‐Deazaadenine/Thymine Watson–Crick Base Pairs

The oligonucleotide d(TX)₉, which consists of an octadecamer sequence with alternating non‐canonical 7‐deazaadenine (X) and canonical thymine (T) as the nucleobases, was synthesized and shown to hybridize into double‐stranded DNA through the formation of hydrogen‐bonded Watson–Crick base pairs. dsDNA with metal‐mediated base pairs was then obtained by selectively replacing W‐C hydrogen bonds by coordination bonds to central silver(I) ions. The oligonucleotide I adopts a duplex structure in the absence of Ag⁺ ions, and its stability is significantly enhanced in the presence of Ag⁺ ions while its double‐helix structure is retained. Temperature‐dependent UV spectroscopy, circular dichroism spectroscopy, and ESI mass spectrometry were used to confirm the selective formation of the silver(I)‐mediated base pairs. This strategy could become useful for preparing stable metallo‐DNA‐based nanostructures.     

Bifacial Base‐Pairing Behaviors of 5‐Hydroxyuracil DNA Bases through Hydrogen Bonding and Metal Coordination

A novel bifacial ligand‐bearing nucleobase, 5‐hydroxyuracil ( U^OH ), which forms both a hydrogen‐bonded base pair ( U^OH –A) and a metal‐mediated base pair ( U^OH –M– U^OH ) has been developed. The U^OH –M– U^OH base pairs were quantitatively formed in the presence of lanthanide ions such as Gd^III when U^OH – U^OH pairs were consecutively incorporated into DNA duplexes. This result established metal‐assisted duplex stabilization as well as DNA‐templated assembly of lanthanide ions. Notably, a duplex possessing U^OH –A base pairs was destabilized by addition of Gd^III ions. This observation suggests that the hybridization behaviors of the U^OH ‐containing DNA strands are altered by metal complexation. Thus, the U^OH nucleobase with a bifacial base‐pairing property holds great promise as a component for metal‐responsive DNA materials.     

A Reusable DNA Single‐Walled Carbon‐Nanotube‐Based Fluorescent Sensor for Highly Sensitive and Selective Detection of Ag+ and Cysteine in Aqueous Solutions

Here we report a reusable DNA single‐walled carbon nanotube (SWNT)‐based fluorescent sensor for highly sensitive and selective detection of Ag⁺ and cysteine (Cys) in aqueous solution. SWNTs can effectively quench the fluorescence of dye‐labeled single‐stranded DNA due to their strong π–π stacking interactions. However, upon incubation with Ag⁺, Ag⁺ can induce stable duplex formation mediated by C–Ag⁺–C (C=cytosine) coordination chemistry, which has been further confirmed by DNA melting studies. This weakens the interactions between DNA and SWNTs, and thus activates the sensor fluorescence. On the other hand, because Cys is a strong Ag⁺ binder, it can remove Ag⁺ from C–Ag⁺–C base pairs and deactivates the sensor fluorescence by rewrapping the dye‐labeled oligonucleotides around the SWNT. In this way, the fluorescence signal‐on and signal‐off of a DNA/SWNT sensor can be used to detect aqueous Ag⁺ and Cys, respectively. This sensing platform exhibits high sensitivity and selectivity toward Ag⁺ and Cys versus other metal ions and the other 19 natural amino acids, with a limit of detection of 1 nM for Ag⁺ and 9.5 nM for Cys. Based on these results, we have constructed a reusable fluorescent sensor by using the covalent‐linked SWNT–DNA conjugates according to the same sensing mechanism. There is no report on the use of SWNT–DNA assays for the detection of Ag⁺ and Cys. This assay is simple, effective, and reusable, and can in principle be used to detect other metal ions by substituting C–C base pairs with other native or artificial bases that selectively bind to other metal ions.     

7‐Iodo‐5‐aza‐7‐deazaguanine ribonucleoside: crystal structure,physical properties,base‐pair stability and functionalization

The positional change of nitrogen‐7 of the RNA constituent guanosine to the bridgehead position‐5 leads to the base‐modified nucleoside 5‐aza‐7‐deazaguanosine. Contrary to guanosine, this molecule cannot form Hoogsteen base pairs and the Watson–Crick proton donor site N3—H becomes a proton‐acceptor site. This causes changes in nucleobase recognition in nucleic acids and has been used to construct stable `all‐purine' DNA and DNA with silver‐mediated base pairs. The present work reports the single‐crystal X‐ray structure of 7‐iodo‐5‐aza‐7‐deazaguanosine, C<sub>10</sub>H<sub>12</sub>IN<sub>5</sub>O<sub>5</sub> ( 1 ). The iodinated nucleoside shows an anti conformation at the glycosylic bond and an N conformation (O4′‐endo) for the ribose moiety, with an antiperiplanar orientation of the 5′‐hydroxy group. Crystal packing is controlled by interactions between nucleobase and sugar moieties. The 7‐iodo substituent forms a contact to oxygen‐2′ of the ribose moiety. Self‐pairing of the nucleobases does not take place. A Hirshfeld surface analysis of 1 highlights the contacts of the nucleobase and sugar moiety (O—H…O and N—H…O). The concept of pK‐value differences to evaluate base‐pair stability was applied to purine–purine base pairing and stable base pairs were predicted for the construction of `all‐purine' RNA. Furthermore, the 7‐iodo substituent of 1 was functionalized with benzofuran to detect motional constraints by fluorescence spectroscopy.     

Sequence‐Dependent Duplex Stabilization upon Formation of a Metal‐Mediated Base Pair

An artificial nucleoside surrogate with 1H‐imidazo[4,5‐f][1,10]phenanthroline ( P ) acting as an aglycone has been introduced into DNA oligonucleotide duplexes. This nucleoside surrogate can act as a bidentate ligand, and so is useful in the context of metal‐mediated base pairs. Several duplexes involving a hetero base pair with an imidazole nucleoside have been investigated. The stability of DNA duplexes incorporating the respective Ag^I‐mediated base pairs strongly depends on the sequence context. Quantum mechanical/molecular mechanical (QM/MM) calculations have been performed in order to gain insight into the factors determining this sequence dependence. The results indicated that, in addition to the stabilizing effect that results from the formation of coordinative bonds, destabilizing effects may occur when the artificial base pair does not fit optimally into the surrounding B‐DNA duplex.     

Stable Copper(I)‐Mediated Base Pairing in DNA

A GNA (glycol nucleic acid) functionalized nucleoside analogue containing the artificial nucleobase 1H‐imidazo[4,5‐f][1,10]phenanthroline (P) was used to form a copper(I)‐mediated base pair within a DNA duplex. The geometrical constraints imposed by the artificial nucleobase play a pivotal role in this unprecedented stabilization of copper(I) in aqueous medium via metal‐mediated base pairing. The formation of the copper(I)‐mediated base pair was investigated by temperature‐dependent UV spectroscopy and CD spectroscopy. The metal‐mediated base pair stabilizes the DNA oligonucleotide duplex by 23 °C. A redox chemistry approach confirmed that this base pair formation was due to the incorporation of copper(I) into the duplex. This first report of a copper(I)‐mediated base pair adds metal‐based diversity to the field and consequently opens up the range of possible applications of metal‐modified nucleic acids.     

N‐Methyl‐2, 2'‐Dipicolylamine Complexes as Potential Models for Metal‐Mediated Base Pairs

Metal‐mediated base pairs can be used to insert metal ions into nucleic acids at precisely defined positions. As structural data on the resulting metal‐modified DNA are scarce, appropriate model complexes need to be synthesized and structurally characterized. Accordingly, the molecular structures of nine transition metal complexes of N‐methyl‐2, 2'‐dipicolylamine (dipic) are reported. In combination with an azole‐containing artificial nucleoside, this tridentate ligand had recently been used to generate metal‐mediated base pairs (Chem. Commun. 2011 , 47, 11041–11043). The Pd^II and Pt^II complexes reported here confirm that the formation of planar complexes (as required for a metal‐mediated base pair) comprising N‐methyl‐2, 2'‐dipicolylamine is possible. Two Hg^II complexes with differing stoichiometry indicate that a planar structure might also be formed with this metal ion, even though it is not favored. In the complex [Ag₂(dipic)₂](ClO₄)₂, the two Ag^I ions are located close to one another with an Ag ··· Ag distance of 2.9152(3) Å, suggesting the presence of a strong argentophilic interaction.     

Pyrrolo‐dC Metal‐Mediated Base Pairs in the Reverse Watson–Crick Double Helix: Enhanced Stability of Parallel DNA and Impact of 6‐Pyridinyl Residues on Fluorescence and Silver‐Ion Binding

Reverse Watson–Crick DNA with parallel‐strand orientation (ps DNA) has been constructed. Pyrrolo‐dC (PyrdC) nucleosides with phenyl and pyridinyl residues linked to the 6 position of the pyrrolo[2,3‐d]pyrimidine base have been incorporated in 12‐ and 25‐mer oligonucleotide duplexes and utilized as silver‐ion binding sites. Thermal‐stability studies on the parallel DNA strands demonstrated extremely strong silver‐ion binding and strongly enhanced duplex stability. Stoichiometric UV and fluorescence titration experiments verified that a single ^2pyPyrdC–^2pyPyrdC pair captures two silver ions in ps DNA. A structure for the PyrdC silver‐ion base pair that aligns 7‐deazapurine bases head‐to‐tail instead of head‐to‐head, as suggested for canonical DNA, is proposed. The silver DNA double helix represents the first example of a ps DNA structure built up of bidentate and tridentate reverse Watson–Crick base pairs stabilized by a dinuclear silver‐mediated PyrdC pair.     

A Family of “Click” Nucleosides for Metal‐Mediated Base Pairing: Unravelling the Principles of Highly Stabilizing Metal‐Mediated Base Pairs

A family of artificial nucleosides has been developed by applying the Cu^I‐catalyzed Huisgen 1,3‐dipolar cycloaddition. Starting from 2‐deoxy‐β‐D ‐glycosyl azide as a common precursor, three bidentate nucleosides have been synthesized. The 1,2,3‐triazole involved in all three nucleobases is complemented by 1,2,4‐triazole ( TriTri ), pyrazole ( TriPyr ), or pyridine ( TriPy ). Molecular structures of two metal complexes indicate that metal‐mediated base pairs of TriPyr may not be fully planar. An investigation of DNA oligonucleotide duplexes comprising the new “click” nucleosides showed that they can bind Ag^I to form metal‐mediated base pairs. In particular the mispair formed from TriPy and the previously established imidazole nucleoside is significantly stabilized in the presence of Ag^I. A comparison of different oligonucleotide sequences allowed the determination of general factors involved in the stabilization of nucleic acids duplexes with metal‐mediated base pairs.     

Infrared Spectroscopic Observation of a G–C+ Hoogsteen Base Pair in the DNA:TATA‐Box Binding Protein Complex Under Solution Conditions

Hoogsteen DNA base pairs (bps) are an alternative base pairing to canonical Watson–Crick bps and are thought to play important biochemical roles. Hoogsteen bps have been reported in a handful of X‐ray structures of protein–DNA complexes. However, there are several examples of Hoogsteen bps in crystal structures that form Watson–Crick bps when examined under solution conditions. Furthermore, Hoogsteen bps can sometimes be difficult to resolve in DNA:protein complexes by X‐ray crystallography due to ambiguous electron density and by solution‐state NMR spectroscopy due to size limitations. Here, using infrared spectroscopy, we report the first direct solution‐state observation of a Hoogsteen (G–C⁺) bp in a DNA:protein complex under solution conditions with specific application to DNA‐bound TATA‐box binding protein. These results support a previous assignment of a G–C⁺ Hoogsteen bp in the complex, and indicate that Hoogsteen bps do indeed exist under solution conditions in DNA:protein complexes.     

Nucleotide–amino acid interactions in the l‐His–IMP·MeOH·H2O complex

In the crystal structure of the methanol‐solvated monohydrated complex of l ‐histidine (His) with inosine 5′‐monophosphate (IMP), namely l ‐histidinium inosine‐5′‐phosphate methanol solvate monohydrate, C₆H₁₀N₃O₂⁺·C₁₀H₁₂N₄O₈P⁻·CH₃OH·H₂O, most of the interactions between IMP anions (anti/C3′‐endo/gauche–gauche conformers) are realized between the riboses and hypoxanthine bases in a trans sugar‐edge/sugar‐edge geometry, and between the phosphate groups. The base Watson–Crick edge is involved in additional methanol‐mediated IMP...MeOH...IMP contacts. Specific and nonspecific nucleotide–amino acid (IMP...His) interactions engage the Hoogsteen edges of the base and phosphate group, respectively. Additional stabilization of His...IMP contacts is provided by π–π stacking between the imidazolium ring of His and the hypoxanthine base of IMP. The results may indicate the possible recognition mechanism between His and IMP.     

A DNA Structure Containing AgI‐Mediated G:G and C:C Base Pairs

Metal‐mediated base pairs have been extensively utilized in many research fields, including genetic‐code extension, novel therapeutics development, and nanodevice design. Compared to other cations, Ag^I is more flexible in pairing with natural base pairs. Herein, we present a DNA structure containing two C‐Ag^I‐C pairs and the first reported G‐Ag^I‐G pair in a short 8mer DNA strand. This structure not only provides detailed insight into these Ag^I‐mediated base‐pairing patterns in DNA, but also represents the first nonhelical DNA structure driven by heavy‐metal ions, thus further contributing to the structural diversity of DNA. This unique complex structure is highly sequence‐dependent, thus implying functional potentials as a new DNA aptamer that can bind and recognize silver ions. These results not only advance our understanding of the interactions between Ag^I and nucleobases, but also provide a unique structural component for the rational design of new DNA nanodevices.     

Impedimetric DNA‐Based Biosensor for Silver Ions Detection with Hemin/G‐Quadruplex Nanowire as Enhancer

A DNA‐based biosensor was reported for detection of silver ions (Ag⁺) by electrochemical impedance spectroscopy (EIS) with [Fe(CN)₆]^4?/3? as redox probe and hybridization chain reaction (HCR) induced hemin/G‐quadruplex nanowire as enhanced label. In the present of target Ag⁺, Ag⁺ interacted with cytosine‐cytosine (C? C) mismatch to form the stable C? Ag⁺? C complex with the aim of immobilizing the primer DNA on electrode, which thus triggered the HCR to form inert hemin/G‐quadruplex nanowire with an amplified EIS signal. As a result, the DNA biosensor showed a high sensitivity with the concentration range spanning from 0.1 nM to 100 µM and a detection limit of 0.05 nM.     

Structure Determination of an AgI‐Mediated Cytosine–Cytosine Base Pair within DNA Duplex in Solution with 1H/15N/109Ag NMR Spectroscopy

The structure of an Ag^I‐mediated cytosine–cytosine base pair, C–Ag^I–C, was determined with NMR spectroscopy in solution. The observation of 1‐bond ¹⁵N‐¹⁰⁹Ag J‐coupling (¹J(¹⁵N,¹⁰⁹Ag): 83 and 84 Hz) recorded within the C–Ag^I–C base pair evidenced the N3–Ag^I–N3 linkage in C–Ag^I–C. The triplet resonances of the N4 atoms in C–Ag^I–C demonstrated that each exocyclic N4 atom exists as an amino group (?NH₂), and any isomerization and/or N4–Ag^I bonding can be excluded. The 3D structure of Ag^I–DNA complex determined with NOEs was classified as a B‐form conformation with a notable propeller twist of C–Ag^I–C (?18.3±3.0°). The ¹⁰⁹Ag NMR chemical shift of C‐Ag^I‐C was recorded for cytidine/Ag^I complex (δ(¹⁰⁹Ag): 442 ppm) to completed full NMR characterization of the metal linkage. The structural interpretation of NMR data with quantum mechanical calculations corroborated the structure of the C–Ag^I–C base pair.     

Structural,Electronic, and Optical Properties of Metallo Base Pairs in Duplex DNA: A Theoretical Insight

Using density functional theory calculations, we investigated the structural, energetic, electronic, and optical properties of recently synthesized duplex DNA containing metal‐mediated base pairs. The studied duplex DNA consists of three imidazole (Im) units linked through metal (Im‐M‐Im, M=metal) and four flanking A:T base pairs (two on each side). We examined the role of artificial base pairing in the presence of two distinctive metal ions, diamagnetic Ag⁺ and magnetic Cu²⁺ ions, on the stability of duplex DNA. We found that metal‐mediated base pairs form stable duplex DNA by direct metal ion coordination to the Im bases. Our results suggest a higher binding stability of base pairing mediated by Cu²⁺ ions than by Ag⁺ ions, which is attributed to a larger extent of orbital hybridization. We furthermore found that DNA modified with Im‐Ag⁺‐Im shows the low‐energy optical absorption characteristic of π–π*orbital transition of WC A:T base pairs. On the other hand, we found that the low‐energy optical absorption peaks for DNA modified with Im‐Cu²⁺‐Im originate from spin–spin interactions. Additionally, this complex exhibits weak ferromagnetic coupling between Cu²⁺ ions and strong spin polarization, which could be used for memory devices. Moreover, analyzing the role of counter ions (Na⁺) and the presence of explicit water molecules on the structural stability and electronic properties of the DNA duplex modified with Im‐Ag⁺‐Im, we found that the impact of these two factors is negligible. Our results are fruitful for understanding the experimental data and suggest a potential route for constructing effective metal‐mediated base pairs in duplex DNA for optoelectronic applications.     

Regulated Incorporation of Two Different Metal Ions into Programmed Sites in a Duplex by DNA Polymerase Catalyzed Primer Extension

Metal‐mediated base pairs formed by the coordination of metal ions to natural or artificial bases impart unique chemical and physical properties to nucleic acids and have attracted considerable interest in the field of nanodevices. Ag^I ions were found to mediate DNA polymerase catalyzed primer extension through the formation of a C–Ag^I–T base pair, as well as the previously reported C–Ag^I–A base pair. The comparative susceptibility of dNTPs to Ag^I‐mediated enzymatic incorporation into the site opposite cytosine in the template was shown to be dATP>dTTP?dCTP. Furthermore, two kinds of metal ions, Ag^I and Hg^II, selectively mediate the incorporation of thymidine 5′‐triphosphate into sites opposite cytosine and thymine in the template, respectively. In other words, the regulated incorporation of different metal ions into programmed sites in the duplex by DNA polymerase was successfully achieved.     

High‐Resolution Crystal Structure of a Silver(I)–RNA Hybrid Duplex Containing Watson–Crick‐like CSilver(I)C Metallo‐Base Pairs

Metallo‐base pairs have been extensively studied for applications in nucleic acid‐based nanodevices and genetic code expansion. Metallo‐base pairs composed of natural nucleobases are attractive because nanodevices containing natural metallo‐base pairs can be easily prepared from commercially available sources. Previously, we have reported a crystal structure of a DNA duplex containing T? Hg^II? T base pairs. Herein, we have determined a high‐resolution crystal structure of the second natural metallo‐base pair between pyrimidine bases C? Ag^I? C formed in an RNA duplex. One Ag^I occupies the center between two cytosines and forms a C? Ag^I? C base pair through N3? Ag^I? N3 linear coordination. The C? Ag^I? C base pair formation does not disturb the standard A‐form conformation of RNA. Since the C? Ag^I? C base pair is structurally similar to the canonical Watson–Crick base pairs, it can be a useful building block for structure‐based design and fabrication of nucleic acid‐based nanodevices.