Binding of metal ions by pyrimidine base pairs in DNA duplexes

Pyrimidine base pairs in DNA duplexes selectively capture metal ions to form metal ion-mediated base pairs, which can be evaluated by thermal denaturation, isothermal titration calorimetry, and nuclear magnetic resonance spectroscopy. In this critical review, we discuss the metal ion binding of pyrimidine bases (thymine, cytosine, 4-thiothymine, 2-thiothymine, 5-fluorouracil) in DNA duplexes. Thymine-thymine (T-T) and cytosine-cytosine (C-C) base pairs selectively capture Hg(II) and Ag(I) ions, respectively, and the metallo-base pairs, T-Hg(II)-T and C-Ag(I)-C, are formed in DNA duplexes. The metal ion binding properties of the pyrimidine-pyrimidine pairs can be changed by small chemical modifications. The binding selectivity of a metal ion to a 5-fluorouracil-5-fluorouracil pair in a DNA duplex can be switched by changing the pH of the solution. Two silver ions bind to each thiopyrimidine-thiopyrimidine pair in the duplexes, and the duplexes are largely stabilized. Oligonucleotides containing these bases are commercially available and can readily be applied in many scientific fields (86 references).     

Anomeric 5-Aza-7-deaza-2′-deoxyguanosines in Silver-Ion-Mediated Homo and Hybrid Base Pairs: Impact of Mismatch Structure,Helical Environment,and Nucleobase Substituents on DNA Stability

Nucleoside configuration (α-d vs. β-d ), nucleobase substituents, and the helical DNA environment of silver-mediated 5-aza-7-deazaguanine-cytosine base pairs have a strong impact on DNA stability. This has been demonstrated by investigations on oligonucleotide duplexes with silver-mediated base pairs of α-d and β-d anomeric 5-aza-7-deaza-2′-deoxyguanosines and anomeric 2′-deoxycytidines incorporated in 12-mer duplexes. To this end, a new synthetic protocol has been developed to access the pure anomers of 5-aza-7-deaza-2′-deoxyguanosine by glycosylation of either the protected nucleobase or its salt followed by separation of the glycosylation products by crystallization and chromatography. Thermal stability measurements were performed on duplexes with α-d /α-d and β-d /β-d homo base pairs or α-d /β-d and β-d /α-d hybrid pairs within two sequence environments, positions 6 or 7, of oligonucleotide duplexes. The respective T_m stability increases observed after silver ion addition differ significantly. Homo base pairs with β-d /β-d or α-d /α-d nucleoside combinations are more stable than α-d /β-d hybrid base pairs. The positional switch of silver-ion-mediated base pairs has a significant impact on stability. Nucleobase substituents introduced at the 5-position of the dC site of silver-mediated base pairs affect base pair stability to a minor extent. Our investigation might lead to applications in the construction of bioinspired nanodevices, in DNA diagnostics, or metal-DNA hybrid materials.     

Heterochiral DNA with Complementary Strands with α-d and β-d Configurations: Hydrogen-Bonded and Silver-Mediated Base Pairs with Impact of 7-Deazapurines Replacing Purines

Heterochiral DNA with hydrogen-bonded and silver-mediated base pairs have been constructed using complementary strands with nucleosides with α-d or β-d configuration. Anomeric phosphoramidites were employed to assemble the oligonucleotides. According to the T_m values and thermodynamic data, the duplex stability of the heterochiral duplexes was similar to that of homochiral DNA, but mismatch discrimination was better in heterochiral DNA. Replacement of purines by 7-deazapurines resulted in stable parallel duplexes, thereby confirming Watson–Crick-type base pairing. When cytosine was facing cytosine, thymine or adenine residues, duplex DNA formed silver-mediated base pairs in the presence of silver ions. Although the CD spectra of single strands with α-d configuration display mirror-like shapes to those with the β-d configuration, the CD spectra of the hydrogen-bonded duplexes and those with a limited number of silver pairs show a B-type double helix almost indistinguishable from natural DNA. Nonmelting silver ion–DNA complexes with entirely different CD spectra were generated when the number of silver ions was equal to the number of base pairs.     

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.     

Cyclohexyl "base pairs" stabilize duplexes and intensify pyrene fluorescence by shielding it from natural base pairs

In this study, we investigated the stability and structure of artificial base pairs that contain cyclohexyl rings. The introduction of a single pair of isopropylcyclohexanes into the middle of DNA slightly destabilized the duplex. Interestingly, as the number of the "base pairs" increased, the duplex was remarkably stabilized. A duplex with six base pairs was even more stable than one containing six A-T pairs. Thermodynamic analysis revealed that changes in entropy and not enthalpy contributed to duplex stability, demonstrating that hydrophobic interactions between isopropyl groups facilitated the base pairing, and thus stabilized the duplex. NOESY of a duplex containing an isopropylcyclohexane-methylcyclohexane pair unambiguously demonstrated its "pairing" in the duplex because distinct NOEs between the protons of cyclohexyl moieties and imino protons of both of the neighboring natural base pairs were observed. CD spectra of duplexes tethering cyclohexyl moieties also showed a positive-negative couplet that is characteristic of the B-form DNA duplex. Taken together, these results showed that cyclohexyl moieties formed base pairs in the DNA duplex without severely disturbing the helical structure of natural DNA. Next, we introduced cyclohexyl base pairs between pyrene and nucleobases as an "insulator" that suppresses electron transfer between them. We found a massive increase in the quantum yield of pyrene due to the efficient shielding of pyrene from nucleobases. The cyclohexyl base pairs reported here have the potential to prepare highly fluorescent labeling agents by multiplying fluorophores and insulators alternately into DNA duplexes.     

Structures and energetics of base flipping of the thymine dimer depend on DNA sequence

The cis,syn-cyclobutane pyrimidine dimer (CPD) is a photoinduced DNA lesion leading to a significant distortion of the DNA structure. Its repair by DNA photolyase requires a flip of the damaged base into an extrahelical position. This base flip is expected to be sequence-dependent, but the structures and energetics as a function of the bases 3' and 5' to the CPD lesion are unknown. Eight-nanosecond MD simulations of four different hexadecamer duplexes with the CPD were performed for the flipped-in and flipped-out structures. Analysis of these results indicates clear sequence-dependent differences. Significant disruptions of the base pairs to the 3' side of the CPD are observed for the flipped-out structures with adjacent A-T pairs, whereas those with G-C pairs adjacent show no such distortions. The conformational spaces occupied by these two duplexes are significantly different. The structural differences correlate well with the free energy differences for base flipping calculated using the previously established 2D potential of mean force (PMF) method. The energy differences for base flipping in duplexes containing A, T, G, and C pairs adjacent to the CPD were found to be 6.25-6.5, 5.25-5.5, 7.25-7.5, and 6.5-6.75 kcal/mol, respectively. These energy differences of up to 2 kcal/mol should be large enough to be detected experimentally using sensitive probes.     

N1...N3 hydrogen bonds of A:U base pairs of RNA are stronger than those of A:T base pairs of DNA

Trans-hydrogen-bond deuterium isotope effects of Watson-Crick A:U and A:T base pairs of 10 homologous RNA and DNA duplexes are compared. The isotope effect at 13C2 of adenosine residues due to deuterium/protium substitution at the imino H3 site, 2hDelta13C2, is larger in RNA than in DNA. The virtually consistent larger isotope effects in RNA suggest that the N1...N3 hydrogen bonds of A:U base pairs of RNA are stronger than those of the A:T base pairs of DNA.     

A novel silver(i)-mediated DNA base pair

We have generated a novel silver(I)-mediated unnatural DNA base pair consisting of two 2,6-bis(ethylthiomethyl)pyridine nucleobases SPy. This metallo-base pair has a remarkably high pairing stability and selectivity which rivals that of the natural base pairs dA:dT and dC:dG. UV-melting experiments revealed that the dSPy:dSPy self-pair can replace natural base pairs at multiple sites and still form stable DNA duplexes.     

Structural motifs of DNA complexes in the gas phase

DNA duplexes are known to be quite stable in the condensed phase but recent mass spectrometry results have shown that DNA complexes are also stable (at least for a limited time) in the gas phase. However, very little is known about the overall shape of the complexes in a solvent-free environment and what factors influence that shape. In this article, we present recent ion mobility and molecular modeling results that address some issues concerning the gas-phase conformations of DNA duplexes. Examples include the effect of metal ions on Watson–Crick base pairing, investigating the onset of helicity in duplexes as a function of strand length, comparison of the stability of C·G and A·T base pairs, and examining the formation of quadruplex structures.     

Efforts toward expansion of the genetic alphabet: structure and replication of unnatural base pairs

Expansion of the genetic alphabet has been a long-time goal of chemical biology. A third DNA base pair that is stable and replicable would have a great number of practical applications and would also lay the foundation for a semisynthetic organism. We have reported that DNA base pairs formed between deoxyribonucleotides with large aromatic, predominantly hydrophobic nucleobase analogues, such as propynylisocarbostyril (dPICS), are stable and efficiently synthesized by DNA polymerases. However, once incorporated into the primer, these analogues inhibit continued primer elongation. More recently, we have found that DNA base pairs formed between nucleobase analogues that have minimal aromatic surface area in addition to little or no hydrogen-bonding potential, such as 3-fluorobenzene (d3FB), are synthesized and extended by DNA polymerases with greatly increased efficiency. Here we show that the rate of synthesis and extension of the self-pair formed between two d3FB analogues is sufficient for in vitro DNA replication. To better understand the origins of efficient replication, we examined the structure of DNA duplexes containing either the d3FB or dPICS self-pairs. We find that the large aromatic rings of dPICS pair in an intercalative manner within duplex DNA, while the d3FB nucleobases interact in an edge-on manner, much closer in structure to natural base pairs. We also synthesized duplexes containing the 5-methyl-substituted derivatives of d3FB (d5Me3FB) paired opposite d3FB or the unsubstituted analogue (dBEN). In all, the data suggest that the structure, electrostatics, and dynamics can all contribute to the extension of unnatural primer termini. The results also help explain the replication properties of many previously examined unnatural base pairs and should help design unnatural base pairs that are better replicated.     

Apolar carbohydrates as DNA capping agents

Mono- and disaccharides have been shown to stack on top of DNA duplexes stabilizing sequences with terminal C-G base pairs. Here we present an apolar version of glucose and cellobiose as new capping agents that stack on DNA increasing considerably its stability with respect to their natural polyhydroxylated mono- and disaccharide DNA conjugates.     

The structure of LNA:DNA hybrids from molecular dynamics simulations: the effect of locked nucleotides

Locked nucleic acids (LNAs) exhibit a modified sugar fragment that is restrained to the C3'-endo conformation. LNA-containing duplexes are rather stable and have a more rigid structure than DNA duplexes, with a propensity for A-conformation of the double helix. To gain detailed insight into the local structure of LNA-modified DNA oligomers (as a foundation for subsequent exploration of the electron-transfer capabilities of such modified duplexes), we carried out molecular dynamics simulations on a set of LNA:DNA 9-mer duplexes and analyzed the resulting structures in terms of base step parameters and the conformations of the sugar residues. The perturbation introduced by a single locked nucleotide was found to be fairly localized, extending mostly to the first neighboring base pairs; such duplexes featured a B-type helix. With increasing degree of LNA modification the structure gradually changed; the duplex with one complete LNA strand assumed a typical A-DNA structure. The relative populations of the sugar conformations agreed very well with NMR data, lending credibility to the validity of the computational protocol.     

Light-Induced Formation of Thymine-Containing Mercury(II)-Mediated Base Pairs

By applying caged thymidine residues, DNA duplexes were created in which Hg^II-mediated base pair formation can be triggered by irradiation with light. When a bidentate ligand was used as the complementary nucleobase, an unprecedented stepwise formation of different metal-mediated base pairs was achieved.     

Metal ion-binding properties of DNA duplexes containing thiopyrimidine base pairs

Thiopyrimidine pairs in DNA duplexes were unexpectedly largely stabilized by complexation with two equivalents of Ag(I) ions and their binding properties were evaluated. The metal ion-binding properties of the thiopyrimidine base pairs differed significantly from those of unpaired bases.     

Recognition properties of donor- and acceptor-modified biphenyl-DNA

The recognition properties of DNA duplexes containing single or triple incorporations of eight different donor-modified (OMe, NH(2)) and acceptor-modified (NO(2)) biphenyl residues as base replacements in opposite positions were probed by UV-melting and by CD and fluorescence spectroscopy. We found a remarkable dependence of duplex stability on the natures of the substituents (donor vs. acceptor). The stabilities of duplexes with one biphenyl pair increase in the order donor/donor < acceptor/donor < acceptor/acceptor substitution. The most stable biphenyl pairs stabilize duplexes by up to 6 degrees C in T(m). In duplexes with three consecutive biphenyl pairs the stability increases in the inverse order (acceptor/acceptor < donor/acceptor < donor/donor) with increases in T(m), relative to an unmodified duplex, of up to 10 degrees C. A thermodynamic analysis, combined with theoretical calculations of the physical properties of the biphenyl substituents, suggests that in duplexes with single biphenyl pairs the affinity is dominated by electrostatic forces between the biphenyl/nearest neighbor natural base pairs, whereas in the triple-modified duplexes the increase in thermal stability is predominantly determined by hydrophobic interactions of the biphenyl residues with each other. Oligonucleotides containing amino biphenyl residues are fluorescent. Their fluorescence is largely quenched when they are paired with themselves or with nitrobiphenyl-containing duplex partners.     

Selective pairing of polyfluorinated DNA bases

Novel selective non-hydrogen-bonding DNA base pairs utilizing fluorinated nucleoside analogues have been investigated. Melting studies of DNA duplexes containing 2,3,4,5-tetrafluorobenzene and 4,5,6,7-tetrafluoroindole bases on opposite strands show greater stabilization of the duplex compared with nonfluorinated hydrocarbon controls. Overall, these hydrophobic analogues are destabilizing compared with natural base pairs but are stabilizing compared with natural base mismatches. Such selective pairing may be due to solvent avoidance of these hydrophobic structures, burying their surfaces within the duplex. Our findings suggest that polyfluoroaromatic bases might be employed as a new, selective base-pairing system orthogonal to the natural genetic system.     

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.     

DNA duplexes and triplex-forming oligodeoxynucleotides incorporating modified nucleosides forming stable and selective triplexes

We have previously reported DNA triplexes containing the unnatural base triad G-PPI·C3, in which PPI is an indole-fused cytosine derivative incorporated into DNA duplexes and C3 is an abasic site in triplex-forming oligonucleotides (TFOs) introduced by a propylene linker. In this study, we developed a new unnatural base triad A-ψ·C(R1) where ψ and C(R1) are base moieties 2'-deoxypseudouridine and 5-substituted deoxycytidine, respectively. We examined several electron-withdrawing substituents for R1 and found that 5-bromocytosine (C(Br)) could selectively recognize ψ. In addition, we developed a new PPI derivative, PPI(Me), having a methyl group on the indole ring in order to achieve selective triplex formation between DNA duplexes incorporating various Watson-Crick base pairs, such as T-A, C-G, A-ψ, and G-PPI(Me), and TFOs containing T, C, C(Br), and C3. We studied the selective triplex formation between these duplexes and TFOs using UV-melting and gel mobility shift assays.     

Are radical cation states delocalized over GG and GGG hole traps in DNA?

Recently, Kurnikov et al. (J. Phys. Chem. B 2002, 106, 7) have shown that solvation of DNA duplexes destabilizes holes of sizes larger than three base pairs. In this paper, we consider the effects of solvation and internal reorganization on the hole charge distribution in sequences 5'-X-GG-Y-3'. Radical cation states in DNA are found to be localized to a single guanine site independent of the nature of adjacent base pairs.     

Preparation of supramolecular chromophoric assemblies using a DNA duplex

Organization of supramolecular assemblies of chromophores with precisely-controlled orientation and sequence remains challenging. Nucleic acids with complementary base sequences spontaneously form double-helical structures. Therefore, covalent attachment of chromophores to DNA or RNA can be used to control assembly and orientation of chromophores. In this perspective, we first review our recent work on the assemblies of fluorophores (pyrene and perylene) by using natural base pairs. The interaction between dyes can be strictly controlled by means of cluster and interstrand wedge motifs. We then discuss novel artificial base pairs that can suppress the interaction between fluorophores and nucleobases. We incorporated a cyclohexane moiety into DNA, and showed that these artificial base pairs suppressed the electron-hole transfer between fluorophores and nucleobases and enhanced the quantum yields of fluorophores. These base pairs can potentially be used to accumulate fluorophores inside DNA duplexes without decreasing quantum yields.