Development of a Metal‐Ion‐Mediated Base Pair for Electron Transfer in DNA

A new C‐nucleoside structurally based on the hydroxyquinoline ligand was synthesized that is able to form stable pairs in DNA in both the absence and the presence of metal ions. The interactions between the metal centers in adjacent Cu^II‐mediated base pairs in DNA were probed by electron paramagnetic resonance (EPR) spectroscopy. The metal–metal distance falls into the range of previously reported values. Fluorescence studies with a donor–DNA–acceptor system indicate that photoinduced charge‐transfer processes across these metal‐ion‐mediated base pairs in DNA occur more efficiently than over natural base pairs.     

Conformationally Restricted Isoindoline‐Derived Spin Labels in Duplex DNA: Distances and Rotational Flexibility by Pulsed Electron–Electron Double Resonance Spectroscopy

Three structurally related isoindoline‐derived spin labels that have different mobilities were incorporated into duplex DNA to systematically study the effect of motion on orientation‐dependent pulsed electron–electron double resonance (PELDOR) measurements. To that end, a new nitroxide spin label, ^ExIm U , was synthesized and incorporated into DNA oligonucleotides. ^ExIm U is the first example of a conformationally unambiguous spin label for nucleic acids, in which the nitroxide N?O bond lies on the same axis as the three single bonds used to attach the otherwise rigid isoindoline‐based spin label to a uridine base. Continuous‐wave (CW) EPR measurements of ^ExIm U confirm a very high rotational mobility of the spin label in duplex DNA relative to the structurally related spin label ^Im U , which has restricted mobility due to an intramolecular hydrogen bond. The X‐band CW‐EPR spectra of ^ExIm U can be used to identify mismatches in duplex DNA. PELDOR distance measurements between pairs of the spin labels ^Im U , ^Ox U , and ^ExIm U in duplex DNA showed a strong angular dependence for ^Im U , a medium dependence for ^Ox U , and no orientation effect for ^ExIm U . Thus, precise distances can be extracted from ^ExIm U without having to take orientational effects into account.     

The “Speedy” Synthesis of Atom‐Specific 15N Imino/Amido‐Labeled RNA

Although numerous reports on the synthesis of atom‐specific ¹⁵N‐labeled nucleosides exist, fast and facile access to the corresponding phosphoramidites for RNA solid‐phase synthesis is still lacking. This situation represents a severe bottleneck for NMR spectroscopic investigations on functional RNAs. Here, we present optimized procedures to speed up the synthesis of ¹⁵N(1) adenosine and ¹⁵N(1) guanosine amidites, which are the much needed counterparts of the more straightforward‐to‐achieve ¹⁵N(3) uridine and ¹⁵N(3) cytidine amidites in order to tap full potential of ¹H/¹⁵N/¹⁵N‐COSY experiments for directly monitoring individual Watson–Crick base pairs in RNA. Demonstrated for two preQ₁ riboswitch systems, we exemplify a versatile concept for individual base‐pair labeling in the analysis of conformationally flexible RNAs when competing structures and conformational dynamics are encountered.     

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.     

A Novel DNA Helical Wire Containing HgII‐Mediated T:T and T:G Pairs

Numerous applications of metal‐mediated base pairs (metallo‐base‐pairs) to nucleic acid based nanodevices and genetic code expansion have been extensively studied. Many of these metallo‐base‐pairs are formed in DNA and RNA duplexes containing Watson–Crick base pairs. Recently, a crystal structure of a metal–DNA nanowire with an uninterrupted one‐dimensional silver array was reported. We now report the crystal structure of a novel DNA helical wire containing Hg^II‐mediated T:T and T:G base pairs and water‐mediated C:C base pairs. The Hg‐DNA wire does not contain any Watson–Crick base pairs. Crystals of the Hg‐DNA wire, which is the first DNA wire structure driven by Hg^II ions, were obtained by mixing the short oligonucleotide d(TTTGC) and Hg^II ions. This study demonstrates the potential of metallo‐DNA to form various structural components that can be used for functional nanodevices.     

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.     

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.     

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.     

Reaction of an “Invisible” Frustrated N/B Lewis Pair with Dihydrogen

D ‐(+)‐Camphor forms the enamine 2 with piperidine. Compound 2 adds HB(C₆F₅)₂ at the enamine carbon atom C3 to form a Lewis acid/Lewis base adduct (exo‐/endo‐isomers of 3 ). Exposure of 3 to dihydrogen (2.5 bar, room temperature) leads to heterolytic splitting of H₂ to form the H⁺/H^? addition products ( 4 , two diastereoisomers) of the “invisible” frustrated Lewis pairs ( 5 , two diastereoisomers) that were apparently generated in situ by enamine hydroboration under equilibrium conditions.     

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.     

Site‐Directed Spin‐Labeling of DNA by the Azide–Alkyne ‘Click’ Reaction: Nanometer Distance Measurements on 7‐Deaza‐2′‐deoxyadenosine and 2′‐Deoxyuridine Nitroxide Conjugates Spatially Separated or Linked to a ‘dA‐dT’ Base Pair

Nucleobase‐directed spin‐labeling by the azide‐alkyne ‘click’ (CuAAC) reaction has been performed for the first time with oligonucleotides. 7‐Deaza‐7‐ethynyl‐2′‐deoxyadenosine ( 1 ) and 5‐ethynyl‐2′‐deoxyuridine ( 2 ) were chosen to incorporate terminal triple bonds into DNA. Oligonucleotides containing 1 or 2 were synthesized on a solid phase and spin labeling with 4‐azido‐2,2,6,6‐tetramethylpiperidine 1‐oxyl (4‐azido‐TEMPO, 3 ) was performed by post‐modification in solution. Two spin labels ( 3 ) were incorporated with high efficiency into the DNA duplex at spatially separated positions or into a ‘dA‐dT’ base pair. Modification at the 5‐position of the pyrimidine base or at the 7‐position of the 7‐deazapurine residue gave steric freedom to the spin label in the major groove of duplex DNA. By applying cw and pulse EPR spectroscopy, very accurate distances between spin labels, within the range of 1–2 nm, were measured. The spin–spin distance was 1.8±0.2 nm for DNA duplex 17 ( dA*^7,10 ) ?11 containing two spin labels that are separated by two nucleotides within one individual strand. A distance of 1.4±0.2 nm was found for the spin‐labeled ‘dA‐dT’ base pair 15 ( dA*⁷ ) ?16 ( dT*⁶ ). The ‘click’ approach has the potential to be applied to all four constituents of DNA, which indicates the universal applicability of the method. New insights into the structural changes of canonical or modified DNA are expected to provide additional information on novel DNA structures, protein interaction, DNA architecture, and synthetic biology.     

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.     

Metal‐Ion‐Triggered Exonuclease III Activity for the Construction of DNA Colorimetric Logic Gates

The logic system is obtained by using a series of double‐stranded (ds) DNA templates with mismatched base pairs (T–T or C–C) and ion‐modulated exonuclease III (Exo III) activity, in which the Exo III cofactors, Hg²⁺ and Ag⁺ ions, are used as inputs for the activation of the respective scission of Exo III based on the formation of T–Hg²⁺–T or C–Ag⁺–C base pairs. Additionally, two kinds of signal probes are utilized to transduce the logic operations. One is the two split G‐rich DNA strands that are used to design the OR, AND, INHIBIT, and XOR gates, whereas the other is the self‐assembled split G‐quadruplex structure to construct NOR, NAND, IMPLICATION, and XNOR operations based on DNA hybridization and strand displacement. In the presence of hemin, the split G‐quadruplex biocatalyzes the formation of a colored product, which is an output signal for the different logic gates. Thus, we have constructed a complete set of colorimetric DNA logic gates based on the Exo III and split G‐quadruplex for the first time. In addition, we are able to effortlessly recognize the logic output signals by the naked eye and their simplicity and cost‐effective design is the most apparent feature for the logic gates developed in this work.     

A Highly Stabilizing Silver(I)‐Mediated Base Pair in Parallel‐Stranded DNA

The first parallel‐stranded DNA duplex with Hoogsteen base pairing that readily incorporates an Ag⁺ ion into an internal mispair to form a metal‐mediated base pair has been created. Towards this end, the highly stabilizing 6 FP ‐Ag⁺‐ 6 FP base pair comprising the artificial nucleobase 6‐furylpurine ( 6 FP ) was devised. A combination of temperature‐dependent UV spectroscopy, CD spectroscopy, and DFT calculations was used to confirm the formation of this base pair. The nucleobase 6 FP is capable of forming metal‐mediated base pairs both by the Watson–Crick edge (i.e. in regular antiparallel‐stranded DNA) and by the Hoogsteen edge (i.e. in parallel‐stranded DNA), depending on the oligonucleotide sequence and the experimental conditions. The 6 FP ‐Ag⁺‐ 6 FP base pair within parallel‐stranded DNA is the most strongly stabilizing Ag⁺‐mediated base pair reported to date for any type of nucleic acid, with an increase in melting temperature of almost 15 °C upon the binding of one Ag⁺ ion.     

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.     

Fluorescence Quenching over Short Range in a Donor‐DNA‐Acceptor System

A new donor‐DNA‐acceptor system has been synthesized containing Nile red‐modified 2′‐deoxyuridine as charge donor and 6‐N,N‐dimethylaminopyrene‐modified 2′‐deoxyuridine as acceptor to investigate the charge transfer in DNA duplexes using fluorescence spectroscopy and time‐resolved femtosecond pump‐probe techniques. Fluorescence quenching experiments revealed that the quenching efficiency of Nile red depends on two components: 1) the presence of a charge acceptor and 2) the number of intervening CG and AT base pairs between donor and acceptor. Surprisingly, the quenching efficiency of two base pairs (73 % for CG and the same for AT) is higher than that for one base pair (68 % for CG and 37 % for AT), while at a separation of three base pairs less than 10 % quenching is observed. A comparison with the results of time‐resolved measurements revealed a correlation between quenching efficiency and the first ultrafast time constant suggesting that quenching proceeds via a charge transfer from the donor to the acceptor. All transients are satisfactorily described with two decays: a rapid charge transfer with 600 fs (～10¹² s^?1) that depends strongly and in a non‐linear fashion on the distance between donor and acceptor, and a slower time constant of a few picoseconds (～10¹¹ s^?1) with weak distance dependence. A third time constant on a nanosecond time scale represents the fluorescence lifetime of the donor molecule. According to these results and time‐dependent density functional theory (TDDFT) calculations a combination of single‐step superexchange and multistep hopping mechanisms can be proposed for this short‐range charge transfer. Furthermore, significantly less quenching efficiency and slower charge transfer rates at very short distances indicate that the direct interaction between donor and acceptor leads to a local structural distortion of DNA duplexes which may provide some uncertainty in identifying the charge transfer rates in short‐range systems.     

Replication Experiments with Nucleotide Base Analogues

The principles governing the replication fidelity of genomes are not fully understood yet. Watson and Crick's base-pairing principle for matched deoxyribonucleotide (DNA) bases can explain why the guanine–cytosine and adenine-thymine base pairs are approximately one hundred times more stable thermodynamically than mismatched combinations. In vitro, DNA polymerases reduce the number of mismatched base pairs to about 10^?6 per Watson–Crick base pair. Replication fidelity can further be enhanced to a mutation probability of 10^?10or less in vivo if optimal conditions for DNA synthesis are provided by polymerase–assisting proteins and DNA-repairing enzymes. The precise reasons for the formation of mismatched base pairs (mispairs), which are responsible for a substantial part of DNA mutations, are still in debate. Although it is agreed that a template-directed “reading” of the hydrogen-substitution pattern in the heterocyclic bases is crucial for proper base pairing during DNA synthesis, it is not clear which type of “misreading” leads to mispairs. Misreading may be due to a non-Watson–Crick base pairing as well as to a change in the hydrogen-substitution pattern, leading to Watson-Crick-like mispairs. The surprising discovery of the selective and quantitative DNA-polymerase-catalyzed formation of a pyridine-pyrimidine base pair (involving a nucleotide base analogue) indicated that rare tautomeric forms in template DNA strands can lead to Watson-Crick-like mispairings that are hardly recognized by the polymerase's proofreading activity. This reveals new pathways for substitution mutations (replication-dependent DNA point mutations) and suggests a new type of mutagen in vivo.     

Biocomplementary interaction behavior in DNA‐like and RNA‐like polymers

A series of nucleobased polymers and copolymers were synthesized through atom transfer radical polymerization (ATRP). Biocomplementary DNA‐ and RNA‐like supramolecular complexes are formed in dilute DMSO solution through nucleobase recognition. ¹H NMR titration studies of these complexes in CDCl₃ indicated that thymine‐adenine (T‐A) and uracil‐adenine (U‐A) complexes form rapidly on the NMR time scale with high association constants (up to 534 and 671 M^–1, respectively) and result in significant T_g increase. WAXD and differential scanning calorimetry analyzes in the bulk state indicate the presence of highly physical cross‐linked structures and provide further details into the nature of the self‐assembly of these systems. Furthermore, this study is of discussion on the difference in the hydrogen bond strength between T‐A and U‐A base pairs within polymer systems, indicating that the strength of hydrogen bonds in RNA U‐A pairs is stronger than that in DNA T‐A base pairs. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 6388–6395, 2009     

AFM‐Correlated CSM‐Coupled Raman/Fluorescence Studies on Selective Interactions of Water Soluble Porphyrins with DNA

The Raman and fluorescence spectroscopic properties of water‐soluble oxo‐titanium(IV) mesotetrakis (1‐methyl pyridium‐4‐yl) porphyrin (O=Ti(TMPyP)⁴⁺) bound with calf thymus DNA and artificial DNAs such as double stranded poly[d(A‐T)₂] and poly[d(G‐C)₂] have been investigated on the single DNA molecule basis by AFM‐correlated confocal scanning microscope (CSM)‐coupled Raman and fluorescence spectroscopic techniques as well as the ensemble‐averaged spectroscopy. The ensemble‐averaged spectroscopic studies imply that the porphyrin interacts with DNA in different groove binding patterns depending on the base pairs. AFM‐images of the different DNAs bound with O=Ti(TMPyP)⁴⁺ were measured, and their morphologies are found to depend on kind of base pairs interacting with O=Ti(TMPyP)⁴⁺. Being correlated with the AFM images, the CSM‐coupled Raman and fluorescence spectral properties of the three different single O=Ti(TMPyP)⁴⁺‐DNA complexes were observed to be highly resolved and sensitive to base pair‐dependent axial ligation of Ti‐O bond as compared to the corresponding ensemble‐averaged spectral properties, which affect the groove binding and its strength of the O=Ti(TMPyP)⁴⁺ with DNA. The axial ligation was found to be accompanied by vibration structural change of the porphyrin ring, leading to keep the shape of double stranded poly[d(A‐T)₂] rigid while poly‐[d(G‐C)₂] and calf thymus DNA flexible after binding with the oxo‐titanyl porphyrin. The base pair dependence of the fluorescence decay times of the DNA‐bound porphyrins was also observed, implying that an excited‐state charge transfer takes place in the G‐C rich major groove in calf thymus DNA. These results suggest that binding of O=Ti(TMPyP)⁴⁺ is more preferential with the G‐C rich major groove than with the A‐T rich minor groove in calf thymus DNA so that the morphology of DNA is changed.     

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.