Metal Base Pairs Research Articles

Towards polymerase-mediated synthesis of artificial RNA–DNA metal base pairs

Polymerase-mediated synthesis of RNA-DNA metal base pairs.

Enzymatic construction of metal-mediated nucleic acid base pairs.

Artificial metal base pairs have become increasingly important in nucleic acids chemistry due to their high thermal stability, water solubility, orthogonality to natural base pairs, and low cost of production. These interesting properties combined with ease of chemical and enzymatic synthesis have prompted their use in several practical applications, including the construction of nanomolecular devices, ions sensors, and metal nanowires. Chemical synthesis of metal base pairs is highly efficient and enables the rapid screening of novel metal base pair candidates. However, chemical synthesis is limited to rather short oligonucleotides and requires rather important synthetic efforts. Herein, we discuss recent progress made for the enzymatic construction of metal base pairs that can alleviate some of these limitations. First, we highlight the possibility of generating metal base pairs using canonical nucleotides and then describe how modified nucleotides can be used in this context. We also provide a description of the main analytical techniques used for the analysis of the nature and the formation of metal base pairs together with relevant examples of their applications.

Enzymatic Formation of an Artificial Base Pair Using a Modified Purine Nucleoside Triphosphate.

The expansion of the genetic alphabet with additional, unnatural base pairs (UBPs) is an important and long-standing goal in synthetic biology. Nucleotides acting as ligands for the coordination of metal cations have advanced as promising candidates for such an expansion of the genetic alphabet. However, the inclusion of artificial metal base pairs in nucleic acids mainly relies on solid-phase synthesis approaches, and very little is known about polymerase-mediated synthesis. Herein, we report the selective and high yielding enzymatic construction of a silver-mediated base pair (dImC-AgI-dPurP) as well as a two-step protocol for the synthesis of DNA duplexes containing such an artificial metal base pair. Guided by DFT calculations, we also shed light into the mechanism of formation of this artificial base pair as well as into the structural and energetic preferences. The enzymatic synthesis of the dImC-AgI-dPurP artificial metal base pair provides valuable insights for the design of future, more potent systems aiming at expanding the genetic alphabet.

Enzymatic Construction of Artificial Base Pairs: The Effect of Metal Shielding.

Th formation of metal base pairs is a versatile method for the introduction of metal cations into nucleic acids that has been used in numerous applications including the construction of metal nanowires, development of energy, charge-transfer devices and expansion of the genetic alphabet. As an alternative, enzymatic construction of metal base pairs is an alluring strategy that grants access to longer sequences and offers the possibility of using such unnatural base pairs (UBPs) in SELEX experiments for the identification of functional nucleic acids. This method remains rather underexplored, and a better understanding of the key parameters in the design of efficient nucleotides is required. We have investigated the effect of methylation of the imidazole nucleoside (dImnMe TP) on the efficiency of the enzymatic construction of metal base pairs. The presence of methyl substituents on dImTP facilitates the polymerase-driven formation of dIm4Me -AgI -dIm and dIm2Me TP-CrIII -dIm base pairs. Steric factors rather than the basicity of the imidazole nucleobase appear to govern the enzymatic formation of such metal base pairs. We also demonstrate the compatibility of other metal cations rarely considered in the construction of artificial metal bases by enzymatic DNA synthesis under both primer extension reaction and PCR conditions. These findings open up new directions for the design of nucleotide analogues for the development of metal base pairs.

On the Enzymatic Formation of Metal Base Pairs with Thiolated and pKa -Perturbed Nucleotides.

The formation of artificial metal base pairs is an alluring and versatile method for the functionalization of nucleic acids. Access to DNA functionalized with metal base pairs is granted mainly by solid-phase synthesis. An alternative, yet underexplored method, envisions the installation of metal base pairs through the polymerization of modified nucleoside triphosphates. Herein, we have explored the possibility of using thiolated and pKa -perturbed nucleotides for the enzymatic construction of artificial metal base pairs. The thiolated nucleotides S2C, S6G, and S4T as well as the fluorinated analogue 5FU are readily incorporated opposite a templating S4T nucleotide through the guidance of metal cations. Multiple incorporation of the modified nucleotides along with polymerase bypass of the unnatural base pairs are also possible under certain conditions. The thiolated nucleotides S4T, S4T, S2C, and S6G were also shown to be compatible with the synthesis of modified, high molecular weight single-stranded (ss)DNA products through TdT-mediated tailing reactions. Thus, sulfur-substitution and pKa perturbation represent alternative strategies for the design of modified nucleotides compatible with the enzymatic construction of metal base pairs.

Methods of Synthesis and Characterization of Conductive DNA Nanowires Based on Metal Ion-Mediated Base Pairing for Single-Molecule Electronics

Advances in the field of molecular electronics have made possible the directmeasurement of charge transport across single molecules. In particular, workon DNA oligomers has demonstrated that this weakly-conducting biomoleculecan be functionalized through metal-mediated nucleobase pairing in order tosignificantly increase electron mobility across the molecule. The introductionof interacting stacks of single metal ions inside the DNA helix is an attractiveplatform for assay and optimization; for this reason we present a protocolfor the production and processing of nanowires with a metal base pair forsingle-molecule applications. In particular, we describe the construction of DNA duplex wires with a cytosine-Ag+-cytosine base pair (dC:Ag+:dC).A thorough investigation of buffer components suggests the use of divalentmagnesium counterions to stabilize highly mismatched oligonucleotides insolution. We further analyse cleaning and processing of thin gold films forbatch-fabrication of conductive imaging substrates for use in conductivescanning probe assays of single-molecule conductivity. With a clear path toelectrical assays, we suggest that the C:Ag+:C orthogonal nucleotide pair andother similar chemistries may provide a foundation for molecular electroniccomponents in integrated devices

Towards the enzymatic formation of artificial metal base pairs with a carboxy-imidazole-modified nucleotide

The identification of synthetic nucleotides that sustain the formation of orthogonal, unnatural base pairs is an important goal in synthetic biology. Such artificial synthons have been used for the generation of semi-synthetic organisms as well as functional nucleic acids with enhanced binding properties. The enzymatic formation of artificial metal-base pairs is a vastly underexplored and alluring alternative to existing systems. Here, we report the synthesis and biochemical characterization of 1‑(2-deoxy‑β‑d‑ribofuranosyl) imidazole‑4‑carboxylate nucleoside triphosphate (dImCTP) which is equipped with a carboxylic acid moiety on the imidazole moiety in order to increase the coordination environment to [2 + 2] and [2 + 1]. A clear metal dependence was observed for the single incorporation of the modified nucleotide into DNA by the DNA polymerase from Thermus aquaticus (Taq). The presence of AgI in primer extension reactions conducted with combinations of 1‑(2‑deoxy‑β‑d‑ribofuranosyl) imidazole nucleoside triphosphate (dImTP) and dImCTP supported the unusual [2 + 1] coordination pattern. The efficiency of the tailing reactions mediated by the terminal deoxynucleotidyl transferase (TdT) was markedly improved when using dImCTP instead of dImTP. Even though products with multiple modified nucleotides were not observed, the appendage of additional metal binding ligands on the imidazole nucleobase appears to be a valid approach to improve the biochemical properties of modified triphosphates in the context of an expansion of the genetic alphabet with metal base pairs.

A dinuclear Cu(i)-mediated complex: Theoretical studies of the G2Cu2 4+ cluster ion.

Recently, the T-Hg(ii)2-A base pair containing two equivalents of Hg(ii) has been prepared and characterized experimentally, which implies that there might exist considerable stable metal-mediated base pairs holding two neighbouring metal centers. Here we report a quantum chemical study on geometries, electronic structures, and bonding of various G2Cu2 4+ (G = guanine) isomers including one di-copper(i) unit. Different density functional methods [Becke 3-parameter-Lee-Yang-Parr, Perdew-Becke-Ernzerhof, Becke-Perdew, Density Functional Theory with Dispersion Corrections (DFT-D)] assign ambiguous relative energies to these isomers with the singlet and triplet states. High-level ab initio [domain-based local pair natural orbital (DLPNO) coupled-cluster with single and double excitations and DLPNO-coupled-cluster with single, double, and perturbative triple excitations] calculations confirm that the lowest-lying isomer of the G2Cu2 4+ ion has C 2h symmetry with the singlet state and is comparable to the singly and doubly charged homologues (G2Cu2 + and G2Cu2 2+). The extended transition state (ETS)-natural orbitals for the chemical valence (ETS-NOCV) calculations point out that it has larger instantaneous interaction energy and bond dissociation energy than the corresponding singly and doubly charged complexes due to its relatively stronger attractive energies and weaker Pauli repulsion. The orbital interactions in the quadruply charged cluster chiefly come from Cu2 4+ ← G⋯G π donations. The results may help the understanding of the bonding properties of other potential metal-base pair complexes with the electron transfer.

Silver(I)-Induced Conformation Change of DNA: Gold Nanocluster as a Spectroscopic Probe

DNA-based materials are promising avenues for designing optical sensors, light-harvesting devices, energy conversion devices, and other potential applications. Therefore, the understanding of DNA conformation is important for designing such materials. Here, we investigate the conformation of DNA by a photoexcited energy transfer process where the Au cluster is covalently attached with AlexaFluor 488 (A488) dye tagged DNA. Fluorescence resonance energy transfer (FRET) is a well-known method to determine the donor–acceptor distance where A488 acts as donor and the gold nanoclusters (Au NCs) act as acceptor. Spectroscopic studies reveal that Ag+ ions are incorporated into dsDNA to produce a Ag+-C (cytosine) metal base pair bond as the used DNA contains 55.6% G-C base pairs. Steady-state and time-resolved spectroscopic studies reveal that the distances between donor and acceptor are found to be 98 and 83 Å in the absence and presence of Ag+ ion, respectively, indicating the rigid conformation of dsDNA becomes flexible in the presence of a Ag+ ion. The efficiency of energy transfer increases from 63% to 82% in the presence of the Ag+ ion because the conformation of DNA changes with the formation of DNA-metal base pairs. Analysis suggests that the Au cluster is a useful spectroscopic probe to understand the conformation of DNA and others. Furthermore, this energy transfer from dye to Au NCs using the DNA scaffold may open up new opportunities in designing an artificial light-harvesting system.

On the enzymatic incorporation of an imidazole nucleotide into DNA.

The expansion of the genetic alphabet with an additional, artificial base pair is of high relevance for numerous applications in synthetic biology. The enzymatic construction of metal base pairs is an alluring strategy that would ensure orthogonality to canonical nucleic acids. So far, very little is known on the enzymatic fabrication of metal base pairs. Here, we report on the synthesis and the enzymatic incorporation of an imidazole nucleotide into DNA. The imidazole nucleotide dIm is known to form highly stable dIm-Ag+-dIm artificial base pairs that cause minimal structural perturbation of DNA duplexes and was considered to be an ideal candidate for the enzymatic construction of metal base pairs. We demonstrate that dImTP is incorporated with high efficiency and selectivity opposite a templating dIm nucleotide by the Kf exo-. The presence of Mn2+, and to a smaller extent Ag+, enhances the efficiency of this polymerization reaction, however, without being strictly required. In addition, multiple incorporation events could be observed, albeit with modest efficiency. We demonstrate that the dIm-Mn+-dIm cannot be constructed by DNA polymerases and suggest that parameters other than stability of a metal base pair and its impact on the structure of DNA duplexes govern the enzymatic formation of artificial metal base pairs.

Self-pairing of 1-methylthymine mediated by two and three Ag(I) ions: a gas phase study using infrared dissociation spectroscopy and density functional theory.

Metal base pairs of Ag(I) cations and 1-methylthymine (1MT) or deprotonated 1-methylthymine (1MT-H) are produced and analyzed by electrospray ionization mass spectrometry (ESI-MS). Mass-selected ions of type [Ag2(1MT)(1MT-H)](+) and [Ag3(1MT-H)2](+) are interrogated by infrared multiple-photon dissociation (IRMPD) in an ion trap in the range of 1200-3700 cm(-1). Supporting spectroscopic data were obtained from the investigation of the analogous 2'-deoxy-thymidine complexes which exhibit advantageously high fragment yields. By comparison with calculated linear IR spectra (obtained by density functional theory, DFT) we assign the structures and the possible isomeric forms of these metal base pairs and their dependence on the number of mediating Ag(I) ions. Based on the observed Ag(+)/1MT complexes and related polarizable continuum model DFT calculations we describe the probable formation pathways in aqueous solution. The present findings pave the way for subsequent UV investigations of the multi-metal mediated base pairs.

Metal-Triggered DNA Folding by Different Mechanisms

Metal-mediated base pairs by the interaction between metal ions and artificial bases in oligonucleotides has been widely used in DNA nanotechnology and biosensing technique. Using isothermal titration calorimetry, circular dichroism spectroscopy and fluorescence spectroscopy, the folding process of T-C-rich oligonucleotides (TCO) induced by Hg2+ and Ag+ with the synthetic sequence d(T6C6T6C6T6C6T6) was studied and analyzed. Although thermodynamic data predict that TCO should initially fold into a relatively stable hairpin through two possible pathways of conformational transitions whether Hg2+ or Ag+ were added at first, the mechanisms and final products between the two are entirely different from isothermal titration calorimetry outcomes. When Hg2+ were added first, the haipin was formed through T-Hg-T structure with further stabilization by C-Ag-C after Ag+ addition. However, it is proposed that an unusual metal-base pair for Ag+ binding is generated instead classical C-Ag-C when Ag+ was injected first. Moreover, further confirmation of this unconventional metal-base pair T-Ag-C was verified by circular dichroism and fluorescence spectroscopy.

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 ((ph) ImidC, 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 ((ph) PyrdC, 1). The silver-mediated (ph) ImidC-(ph) ImidC 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.

Synthesis and properties of a Cu(ii) complexing pyrazole ligandoside in DNA

The development of metal base pairs is of immense importance for the construction of DNA nanostructures. Here we report the synthesis of a biaryl pyrazole-phenol nucleoside that forms in DNA a stable self-pair upon complexation of a Cu(II) ion. A sequence with five consecutive pyrazole nucleotides allows the complexation of five Cu(II) ions in a row.

Rational design of Hg2+controlled streptavidin-binding aptamer

Through substituting two canonical base pairs of a streptavidin-binding aptamer with T-T mismatched base pairs, a new aptamer was constructed. Its binding ability could be controlled by Hg(2+) through the formation of T-Hg-T metal-base pairs.

Reversible bond formation enables the replication and amplification of a crosslinking salen complex as an orthogonal base pair

The universal genetic code relies on two hydrogen-bonded Watson-Crick base pairs that can form 64 triplet codons. This places a limit on the number of amino acids that can be encoded, which has motivated efforts to create synthetic base pairs that are orthogonal to the natural ones. An additional base pair would result in another 61 triplet codons. Artificial organic base pairs have been described in enzymatic incorporation studies, and inorganic T-Hg-T and C-Ag-C base pairs have been reported to form in primer extension studies. Here, we demonstrate a metal base pair that is fully orthogonal and can be replicated, and can even be amplified by polymerase chain reaction in the presence of the canonical pairs dA:dT and dG:dC. Crystal structures of a dS-Cu-dS base pair inside a polymerase show that reversible chemistry is possible directly inside the polymerase, which enables the efficient copying of the inorganic crosslink. The results open up the possibility of replicating and amplifying artificial inorganic DNA nanostructures by extending the genetic alphabet.

Metal–base pairing in DNA

The use of DNA as a molecular wire in nanoscale electronic architectures would greatly benefit from its capability of sequence-specific self-assembly. Although single electrons and positive charges have been shown to be transmitted by natural DNA over a distance of several base pairs, the high ohmic resistance of unmodified oligonucleotides imposes a serious obstacle. Exchanging some or all of the Watson–Crick base pairs in DNA by metal complexes may solve this problem and evolve DNA-like materials with superior conductivity for future nano-electronic applications. The so-called metal–base pairs are formed from suitable transition metal ions and ligand-like nucleosides which are introduced into both of the two pairing strands by automated DNA synthesis. This review illustrates the basic concepts of metal–base pairing and highlights recent developments in the field.

‘Turn-on’ detection of Hg2+ ion using a peroxidase-like split G-quadruplex–hemin DNAzyme

A highly sensitive and selective Hg(2+) detection method was developed based on the Hg(2+)-mediated formation of split G-quadruplex-hemin DNAzymes. In this method, two label-free oligonucleotides are used. In the presence of Hg(2+), the two oligonucleotides hybridize to each other to form a duplex, in which T-T mismatches are stabilized by T-Hg(2+)-T base pair. As a result, the G-rich sequences of the two oligonucleotides can associate to form a split G-quadruplex, which is able to bind hemin to form the catalytically active G-quadruplex-hemin DNAzymes. This can be reflected by an absorbance increase when monitored in the H(2)O(2)-ABTS (2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid) reaction system by using UV-vis absorption spectroscopy. This 'turn-on' process allows the detection of aqueous Hg(2+) at concentrations as low as 19 nM using a simple colorimetric technique. With the development of the studies on metal-base pairs, this Hg(2+)-sensing method can be easily extended to the analysis of other metal ions.

DNA—Metal Base Pairs

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DNA–Metal Base Pairs

Recent developments show encouraging results for the use of DNA as a construction material for nanometer-sized objects. Today, however, DNA-based molecular nanoarchitectures are constructed with mainly unmodified or at best end-modified oligonucleotides, thus shifting the development of functionalized DNA structures into the limelight. One of most recent developments in this direction is the substitution of the canonical Watson-Crick base pairs by metal complexes. In this way "metal-base pairs" are created, which could potentially impart magnetic or conductive properties to DNA-based nanostructures. This review summarizes research which started almost 45 years ago with the investigation of how metal ions interact with unmodified DNA and which recently culminated in the development of artificial ligand-like nucleobases so far able to coordinate up to ten metal ions inside a single DNA duplex in a programmable fashion.