Crystal Structure of Metallo DNA Duplex Containing Consecutive Watson–Crick‐like T–HgII–T Base Pairs

The metallo DNA duplex containing mercury‐mediated T–T base pairs is an attractive biomacromolecular nanomaterial which can be applied to nanodevices such as ion sensors. Reported herein is the first crystal structure of a B‐form DNA duplex containing two consecutive T–Hg^II–T base pairs. The Hg^II ion occupies the center between two T residues. The N3‐Hg^II bond distance is 2.0 Å. The relatively short Hg^II‐Hg^II distance (3.3 Å) observed in consecutive T–Hg^II–T base pairs suggests that the metallophilic attraction could exist between them and may stabilize the B‐form double helix. To support this, the DNA duplex is largely distorted and adopts an unusual nonhelical conformation in the absence of Hg^II. The structure of the metallo DNA duplex itself and the Hg^II‐induced structural switching from the nonhelical form to the B‐form provide the basis for structure‐based design of metal‐conjugated nucleic acid nanomaterials.     

Base Pairing of 8‐Aza‐7‐deazapurine‐2,6‐diamine Linked via the N(8)‐Position to the DNA Backbone: Universal Base‐Pairing Properties and Formation of Highly Stable Duplexes when Alternating with dT

The unusually N⁸‐glycosylated pyrazolo[3,4‐d]pyrimidine‐4,6‐diamine 2′‐deoxyribonucleoside ( 3 ) was synthesized and converted to the phosphoramidite 11 . Oligonucleotides were prepared by solid‐phase synthesis, and the base pairing of compound 3 was studied. In non‐self‐complementary duplexes containing compound 3 located opposite to the four canonical DNA constituents, strong base pairs are formed that show ambiguous pairing properties. The self‐complementary duplex d( 3 ‐T)₆ ( 34 ⋅ 34 ) is significantly more stable than d(A‐T)₆.     

Evaluation of Novel Third‐Strand Bases for the Recognition of a C⋅G Base Pair in the Parallel DNA Triple‐Helical Binding Motif

We describe the synthesis and the incorporation into oligonucleotides of the novel nucleoside building blocks 9, 10 , and 16 , carrying purine‐like double H‐bond‐acceptor bases. These base‐modified nucleosides were conceived to recognize selectively a cytosine⋅guanine (C⋅G) inversion site within a homopurine⋅homopyrimidine DNA duplex, when constituent of a DNA third strand designed to bind in the parallel binding motif. While building block 16 turned out to be incompatible with standard oligonucleotide‐synthesis conditions, UV/triplex melting experiments with third‐strand 15‐mers containing β‐D ‐nucleoside 6 (from 9 ) showed that recognition of the four natural Watson‐Crick base pairs follows the order G⋅C≈C⋅G>A⋅T>T⋅A. The recognition is sequence‐context sensitive, and G⋅C or C⋅G recognition does not involve protonated species of β‐D ‐nucleoside 6 . The data obtained fit (but do not prove) a structural model for C⋅G recognition via one conventional and one C−H⋅⋅⋅O H‐bond. The unexpected G⋅C recognition is best explained by third‐strand base intercalation. A comparison of the triplex binding properties of these new bases with those of 4‐deoxothymine (5‐methylpyrimidine‐2(1H)‐one, ^{4 H}T), previously shown to be C⋅G selective but energetically weak, is also described.     

Synthesis of Fluorobenzene and Benzimidazole Nucleic‐Acid Analogues and Their Influence on Stability of RNA Duplexes

Six different ribonucleoside phosphoramidites with fluorobenzenes or fluorobenzimidazoles as base analogues, one abasic site, and inosine were synthesized and incorporated into oligoribonucleotides. The oligomers were investigated by means of UV and CD spectroscopy to assess the contribution of H‐bonding, base stacking, and solvation to the stability of the RNA duplex. CD Spectra show that the incorporation of modified nucleosides does not lead to changes in the structure of RNA. The T_m differences determined are based on changes in base stacking and solvation. Individual contributions of base stacking and solvation of the modified nucleosides could be determined. In fluorobenzene⋅fluorobenzimidazole‐modified base pairs, a duplex‐stabilizing force was found that points to a weak F⋅⋅⋅H H‐bond.     

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.     

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.     

On RNA Triplet Interactions: NMR Study of the Short Intramolecular Duplex Formed by r[GCAm1G‐p‐O(CH2CH2O)6‐p‐UGCC], Preliminary Communication

The flexible hexaethylene‐glycol linker enhances the stability of the duplex between the two tetranucleotides in compound 1 sufficiently to allow determination of the solution structure by NMR. At 4.8°, two of the three possible imino NH protons are detected as sharp signals and establish the presence of two G⋅C Watson‐Crick base pairs. Through assignment of all but one of the non‐labile protons and measurement of ¹H,¹H and ¹H,³¹P coupling constants, as well as NOEs of labile and non‐labile protons, it was possible for the first time to derive detailed structural information on such a short duplex. It forms an A‐type double helix over the full length, including the dangling nucleotides. Small variations of coupling constants and a broadening of the H−C(8) signal of m¹G4 indicate that the two nucleotides connected to the linker are conformationally slightly distorted and/or more flexible than the unlinked end of the duplex.     

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.     

The Crystal Structure of Non‐Modified and Bipyridine‐Modified PNA Duplexes

Peptide nucleic acid (PNA) is a synthetic analogue of DNA that commonly has an N‐aminoethyl glycine backbone. The crystal structures of two PNA duplexes, one containing eight standard nucleobase pairs (GGCATGCC)₂, and the other containing the same nucleobase pairs and a central pair of bipyridine ligands, have been solved with a resolution of 1.22 and 1.10 Å, respectively. The non‐modified PNA duplex adopts a P‐type helical structure similar to that of previously characterized PNAs. The atomic‐level resolution of the structures allowed us to observe for the first time specific modes of interaction between the terminal lysines of the PNA and the backbone and the nucleobases situated in the vicinity of the lysines, which are considered an important factor in the induction of a preferred handedness in PNA duplexes. Our results support the notion that whereas PNA typically adopts a P‐type helical structure, its flexibility is relatively high. For example, the base‐pair rise in the bipyridine‐containing PNA is the largest measured to date in a PNA homoduplex. The two bipyridines bulge out of the duplex and are aligned parallel to the major groove of the PNA. In addition, two bipyridines from adjacent PNA duplexes form a π‐stacked pair that relates the duplexes within the crystal. The bulging out of the bipyridines causes bending of the PNA duplex, which is in contrast to the structure previously reported for biphenyl‐modified DNA duplexes in solution, where the biphenyls are π stacked with adjacent nucleobase pairs and adopt an intrahelical geometry. This difference shows that relatively small perturbations can significantly impact the relative position of nucleobase analogues in nucleic acid duplexes.     

A Pyrimidopyrimidine Janus‐AT Nucleoside with Improved Base‐Pairing Properties to both A and T within a DNA Duplex: The Stabilizing Effect of a Second Endocyclic Ring Nitrogen

Janus bases are heterocyclic nucleic acid base analogs that present two different faces able to simultaneously hydrogen bond to nucleosides that form Watson–Crick base pairs. The synthesis of a Janus‐AT nucleotide analogue, ^N J_AT , that has an additional endocyclic ring nitrogen and is thus more capable of efficiently discriminating T/A over G/C bases when base‐pairing in a standard duplex‐DNA context is described. Conversion to a phosphoramidite ultimately afforded incorporation into an oligonucleotide. In contrast to the first generation of carbocyclic Janus heterocycles, it remains in its unprotonated state at physiological pH and, therefore, forms very stable Watson–Crick base pairs with either A or T bases. Biophysical and computational methods indicate that ^N J_AT is an improved candidate for sequence‐specific genome targeting.     

Duplex structure of a minimal nucleic acid

The crystal structure of an 8-mer (S)-GNA duplex is presented. As a tool for phasing, the anomalous diffraction of two copper(II) ions within two artificial metallo-base pairs was employed. The duplex structure confirms a canonical Watson-Crick base pairing scheme of GNA with antiparallel strands. The duplex secondary structure is distinct from canonical A- and B-form nucleic acids and can be described as a right-handed helical ribbon wrapped around the helix axis, resulting in a large hollow core. Most intriguingly, neighboring base pairs slide strongly against each other, resulting in extensive interstrand base-base hydrophobic interactions along with unusual hydrophobic intrastrand interactions of nucleobases with their backbone. These results reveal how a minimal nucleic acid backbone can support highly stable Watson-Crick-like duplex formation.     

Oligonucleotide Analogues with Integrated Bases and Backbone. Part 22

The tritylated and silylated self‐complementary A*[s]U*[s]A*[s]U* and U*[s]A*[s]U*[s]A* tetramers 18 and 24 , linked by thiomethylene groups (abbreviated as [s]) between a nucleobase and C(5′) of the neighbouring nucleoside unit were prepared by a linear synthesis based on S‐alkylation of 5′‐thionucleosides by 6‐(chloromethyl)uridines, 7 or 10 , or 8‐(chloromethyl)adenosines, 12 or 15 . The tetramers 18 and 24 were detritylated to the monoalcohols 19 and 25 , and these were desilylated to the diols 20 and 26 , respectively. The association of the tetramers 18 – 21 and 24 – 26 in CDCl₃ or in CDCl₃/(D₆)DMSO 95 : 5 was investigated by the concentration dependence of the chemical shifts for H? N(3) or H₂N? C(6). The formation of cyclic duplexes connected by four base pairs is favoured by the presence of one and especially of two OH groups. The diol 20 with the AUAU sequence prefers reverse‐Hoogsteen, and diol 26 with the UAUA sequence Watson–Crick base pairing. The structure of the cyclic duplex of 26 in CDCl₃ at 2° was derived by a combination of AMBER* modeling and simulated annealing with NMR‐derived distance and torsion‐angle restraints resulting in a Watson–Crick base‐paired right‐handed antiparallel helix showing large roll angles, especially between the centre base pairs, leading to a bent helix axis.     

Rational Design for Building Blocks of DNA‐Based Conductive Nanowires through Multi‐Copper Incorporation into Mismatched Base Pairs

Metal‐modified DNA base pairs, which possess potential electrical conductivity and can serve as conductive nanomaterials, have recently attracted much attention. Inspired by our recent finding that multicopper incorporation into natural DNA base pairs could improve the electronic properties of base pairs, herein, we designed two novel multi‐copper‐mediated mismatched base pairs (G_3CuT and A_2CuC), and examined their structural and electronic properties by means of density functional theory calculations. The results reveal that these multi‐Cu‐mediated mismatched base pairs still have planar geometries that are thermodynamically favorable to stability, and their binding energies are close to those of multi‐Cu‐mediated normal base pairs (G_3CuC and A_2CuT). Their HOMO–LUMO gaps and ionization potentials decrease significantly compared to the corresponding natural base pairs. As evidenced by the charge transfer excitation transitions, transverse electronic communication of G_3CuT and A_2CuC is remarkably enhanced, suggesting that they facilitate electron migration along the DNA wires upon incorporation. Further examinations also clarify the possibility to build promising DNA helices using the G_3CuT and/or A_2CuC base pairs. The calculated electronic properties of the three‐layer‐stacked multi‐Cu‐mediated mismatched base pairs illustrate that the Cu_m‐DNA have better conductivity. This work provides perspectives for the development and application of DNA nanowires.     

Oligonucleotide Analogues with a Nucleobase‐Including Backbone,Part 5, 2′‐Deoxy‐8‐(hydroxymethyl)adenosine‐ and 2′‐Deoxy‐6‐(hydroxymethyl)uridine‐Derived Phosphoramidites: Synthesis and Incorporation into 14‐Mer DNA Strands

The pairing propensity of new DNA analogues with a phosphinato group between O−C(3′) and a newly introduced OCH₂ group at C(8) and C(6) of 2′‐deoxyadenosine and 2′‐deoxyuridine, respectively, was evaluated by force‐field calculations and Maruzen model studies. These studies suggest that these analogues may form autonomous pairing systems, and that the incorporation of single modified units into DNA 14mers is compatible with duplex formation. To evaluate the incorporation, we prepared the required phosphoramidites 3 and 4 from 2′‐deoxyadenosine and 2′‐deoxyuridine, respectively. The phosphoramidite 5 was similarly prepared to estimate the influence of a CH₂OH group at C(8) on the duplex stability. The modified 14‐mers 6 – 9 were prepared by solid‐phase synthesis. Pairing studies show a decrease of the melting temperature by 2.5° for the duplex 13 ⋅ 9 , and of 6 – 8° for the duplexes 10 ⋅ 6 , 11 ⋅ 6 , 13 ⋅ 7 , and 14 ⋅ 8 , as compared to the unmodified duplexes.     

Spontaneous Assembly of an Organic–Inorganic Nucleic Acid Z‐DNA Double‐Helix Structure

Herein, we report a hybrid polyoxometalate organic–inorganic compound, Na₂[(HGMP)₂Mo₅O₁₅]⋅7 H₂O ( 1 ; where GMP=guanosine monophosphate), which spontaneously assembles into a structure with dimensions that are strikingly similar to those of the naturally occurring left‐handed Z‐form of DNA. The helical parameters in the crystal structure of the new compound, such as rise per turn and helical twist per dimer, are nearly identical to this DNA conformation, allowing a close comparison of the two structures. Solution circular dichroism studies show that compound 1 also forms extended secondary structures in solution. Gel electrophoresis studies demonstrate the formation of non‐covalent adducts with natural plasmids. Thus we show a route by which simple hybrid inorganic–organic monomers, such as compound 1 , can spontaneously assemble into a double helix without the need for a covalently connected linear sequence of nucleic acid base pairs.     

The crystal structure of the CeNA:RNA hybrid ce(GCGTAGCG):r(CGCUACGC)

Cyclohexenyl nucleic acids (CeNA) are characterised by the carbon–carbon double bond replacing the O4′‐oxygen atom of the natural D ‐2′‐deoxyribose sugar ring in DNA. CeNAs exhibit a high conformational flexibility, are stable against nuclease activity and their hybridisation is RNA selective. Additionally, CeNA has been shown to induce an enhanced biological activity when incorporated in siRNA. This makes CeNA a good candidate for siRNA and synthetic aptamer applications. The crystal structure of the synthetic CeNA:RNA hybrid ce(GCGTAGCG):r(CGCUACGC) has been solved with a resolution of 2.50 Å. The CeNA:RNA duplex adopts an anti‐parallel, right‐handed double helix with standard Watson–Crick base pairing. Analyses of the helical parameters revealed the octamer to form an A‐like double helix. The cyclohexenyl rings mainly adopt the ³H₂ conformation, which resembles the C3′‐endo conformation of RNA ribose ring. This C3′‐endo ring puckering was found in most of the RNA residues and is typical for A‐family helices. The crystal structure is stabilised by the presence of hexahydrated magnesium ions. The fact that the CeNA:RNA hybrid adopts an A‐type double helical conformation confirms the high potential of CeNAs for the construction of efficient siRNAs which can be used for therapeutical applications.     

Molecular‐Dynamics Studies of Single‐Stranded Hexitol,Altritol, Mannitol,and Ribose Nucleic Acids (HNA,MNA, ANA,and RNA,Resp.) and of the Stability of HNA⋅RNA,ANA⋅RNA,and MNA⋅RNA Duplexes

The influence of the orientation of a 3′‐OH group on the conformation and stability of hexitol oligonucleotides in complexes with RNA and as single strands in aqueous solution was investigated by molecular‐dynamics (MD) simulations with AMBER 4.1. The particle mesh Ewald (PME) method was used for the treatment of long‐range electrostatic interactions. An equatorial orientation of the 3′‐OH group in the single‐stranded D ‐mannitol nucleic acid (MNA) m(GCGTAGCG) and in the complex with the RNA r(CGCAUCGC) has an unfavorable influence on the helical stability. Frequent H‐bonds between the 3′‐OH group and the O−C(6′) of the phosphate backbone of the following nucleotide explain the distorted conformation of the MNA⋅RNA complex as well as that of the single MNA strand. This is consistent with experimental results that show lowered hybridization potentials for MNA⋅RNA complexes. An axial orientation of the 3′‐OH group in the D ‐altritol nucleic acid (ANA) a(GCGTAGCG) leads to a stable complex with the complementary RNA r(CGCAUCGC), as well as to a more highly preorganized single‐stranded ANA chain. The averaged conformation of the ANA⋅RNA complex is similar to that of A‐RNA, with only minor changes in groove width, helical curvature, and H‐bonding pattern. The relative stabilities of ANA⋅RNA vs. HNA⋅RNA (HNA=D ‐hexitol nucleic acid without 3′‐OH group) can be explained by differences in restricted movements, H‐bonds, and solvation effects.     

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.     

Oligonucleotide Analogues with Integrated Bases and Backbone. Part 30

The protected G*[s ]C*[s ]U*[s ]A*[s ]U*[s ]A*[s ]G*[s ]C* octanucleoside 24 was prepared by S‐alkylation of the thiolate derived from tetranucleoside 23 with the methanesulfonate 22 , and transformed to the silylated and isopropylidenated 25 , and further into the fully deprotected octanucleoside 26 . Compound 22 was derived from the methoxytrityl‐protected tetranucleoside 21 , and 21 was obtained by S‐alkylation of the thiolate derived from the dinucleoside 19 with methanesulfonate 17 derived from 16 by detritylation and mesylation. Similarly, tetranucleoside 23 resulted from S‐alkylation of the thiolate derived from 18 with the methanesulfonate 20 derived from 19 . Dinucleosides 16 and 18 resulted from S‐alkylation of the thiolate derived from the known cytidine‐derived thioacetate 15 with the C(8)‐substituted guanosine‐derived methanesulfonates 12 and 14 , respectively, that were synthesized from the protected precursors 4 and 7 by formylation, reduction, protection, and mesylation. The structures of the duplexes of 25 and 26 were calculated using AMBER* modelling and based on the known structure of the core tetranucleoside U*[s ]A*[s ]U*[s ]A*. The former shows a helix with a bent helix axis and strong buckle and propeller twists, whereas the latter is a regular, right‐handed, and apparently strain‐free helix. In agreement with modelling, the silylated and isopropylidenated octanucleoside 25 in (CDCl₂)₂ solution led to a mixture of associated species possessing at most four Watson? Crick base pairs, while the fully deprotected octanucleoside 26 in aqueous medium forms a duplex, as evidenced by a decreasing CD absorption upon increasing the temperature and by a UV‐melting curve with a melting temperature of ca. 10° below the one of the corresponding RNA octamer, indicating cooperativity between base pairing and base‐pair stacking.