Solution Structure of a Hexitol Nucleic Acid Duplex with Four Consecutive T⋅T Base Pairs

The structure of the hexitol nucleic acid (HNA) h(GCGCTTTTGCGC) was determined by NMR spectroscopy. This unnatural nucleic acid was developed as a mimic for A‐RNA. In solution, the studied sequence is forming a symmetric double‐stranded structure with four central consecutive T⋅T wobble pairs flanked by G⋅C Watson‐Crick base pairs. The stem regions adopt an A‐type helical structure. Discrete changes in backbone angles are altering the course of the helix axis in the internal loop region. Two H‐bonds are formed in each wobble pair, and base stacking is preserved in the duplex, explaining the stability of the duplex. This structure elucidation provides information about the influence of a (T)₄ fragment on local helix geometries as well as on the nature of the T⋅T mismatch base pairing in a TTTT tract.     

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.     

Effects of Six‐Membered Carbohydrate Rings on Structure,Stability, and Kinetics of G‐Quadruplexes

We have evaluated the conformational, thermal, and kinetic properties of d(TGGGGT) analogues with one or five of the ribose nucleotides replaced with the carbohydrate residues hexitol nucleic acid (HNA), cyclohexenyl nucleic acid (CeNA), or altritol nucleic acid (ANA). All of the modified oligonucleotides formed G‐quadruplexes, but substitution with the six‐membered rings resulted in a mixture of G‐quadruplex structures. UV and CD melting analyses showed that the structure formed by d(TGGGGT) modified with HNA was stabilized whereas that modified with CeNA was destabilized, relative to the structure formed by the unmodified oligonucleotide. Substitution at the fourth base of the G‐tract with ANA resulted in a greater stabilization effect than substitution at the first G residue; substitution with five ANA residues resulted in significant stabilization of the G‐quadruplex. A single substitution with CeNA at the first base of the G‐tract or five substitutions with HNA resulted in striking deceleration or acceleration of G‐quadruplex formation, respectively. Our results shed light on the effect of the sugar moiety on the properties of G‐quadruplex structures.     

On the structure and dynamics of duplex GNA

Glycol nucleic acid (GNA), with a nucleotide backbone comprising of just three carbons and the stereocenter derived from propylene glycol (1,2-propanediol), is a structural analog of nucleic acids with intriguing biophysical properties, such as formation of highly stable antiparallel duplexes with high Watson-Crick base pairing fidelity. Previous crystallographic studies of double stranded GNA (dsGNA) indicated two forms of backbone conformations, an elongated M-type (containing metallo-base pairs) and the condensed N-type (containing brominated base pairs). A herein presented new crystal structure of a GNA duplex at 1.8 ? resolution from self-complementary 3'-CTC(Br)UAGAG-2' GNA oligonucleotides reveals an N-type conformation with alternating gauche-anti torsions along its (O3'-C3'-C2'-O2') backbone. To elucidate the conformational state of dsGNA in solution, molecular dynamic simulations over a period of 20 ns were performed with the now available repertoire of structural information. Interestingly, dsGNA adopts conformational states in solution intermediate between experimentally observed backbone conformations: simulated dsGNA shows the all-gauche conformation characteristic of M-type GNA with the higher helical twist common to N-type GNA structures. The so far counterintuitive, smaller loss of entropy upon duplex formation as compared to DNA can be traced back to the conformational flexibility inherent to dsGNA but missing in dsDNA. Besides extensive interstrand base stacking and conformational preorganization of single strands, this flexibility contributes to the extraordinary thermal stability of GNA.     

Solution structure of a HNA-RNA hybrid

BACKGROUND: Synthetic nucleic acid analogues with a conformationally restricted sugar-phosphate backbone are widely used in antisense strategies for biomedical and biochemical applications. The modified backbone protects the oligonucleotides against degradation within the living cell, which allows them to form stable duplexes with sequences in target mRNAs with the aim of arresting their translation. The biologically most active antisense oligonucleotides also trigger cleavage of the target RNA through activation of endogenous RNase H. Systematic studies of synthetic oligonucleotides have also been conducted to delineate the origin of the chirality of DNA and RNA that are both composed of D-nucleosides. RESULTS: Hexitol nucleic acids (HNA) are the first example of oligonucleotides with a six-membered carbohydrate moiety that can bind strongly and selectively to complementary RNA oligomers. We present the first high resolution nuclear magnetic resonance structure of a HNA oligomer bound to a complementary RNA strand. The HNA-RNA complex forms an anti-parallel heteroduplex and adopts a helical conformation that belongs to the A-type family. Possibly, due to the rigidity of the rigid chair conformation of the six-membered ring both the HNA and RNA strand in the duplex are well defined. The observed absence of end-fraying effects also indicate a reduced conformational flexibility of the HNA-RNA duplex compared to canonical dsRNA or an RNA-DNA duplex. CONCLUSIONS: The P-P distance across the minor groove, which is close to A-form, and the rigid conformation of the HNA-RNA complex, explain its resistance towards degradation by Rnase H. The A-form character of the HNA-RNA duplex and the reduced flexibility of the HNA strand is possibly responsible for the stereoselectivity of HNA templates in non-enzymatic replication of oligonucleotides, supporting the theory that nucleosides with six-membered rings could have existed at some stage in molecular evolution.     

Sequence-dependent effects in A-DNA double helices

To date, more than ten different A-DNA structures have been analyzed at near atomic resolution by X-ray methods. This structural information enables us for the first time, to begin to discover the rules which govern the conformation of the double helix in the A form.A detailed analysis of the various helical conformations show that local structural changes are induced by base-pair stacking interactions. Variations in stacking geometry are sequence dependent. For pyrimidine-purine sites, the stacking mode is determined by the nature of the base-pair doublet only, whereas for purine-pyrimidine and homopolymer sites the identity of neighboring base pairs is important as well.     

Enantioselective Assembly of a Ruthenium(II) Polypyridyl Complex into a Double Helix

Evolution can increase the complexity of matter by self‐organization into helical architectures, the best example being the DNA double helix. One common aspect, apparently shared by most of these architectures, is the presence of covalent bonds within the helix backbone. Here, we report the unprecedented crystal structures of a metal complex that self‐organizes into a continuous double helical structure, assembled by non‐covalent building blocks. Built up solely by weak stacking interactions, this alternating tread stairs‐like double helical assembly mimics the DNA double helix structure. Starting from a racemic mixture in aqueous solution, the ruthenium(II) polypyridyl complex forms two polymorphic structures of a left‐handed double helical assembly of only the Λ‐enantiomer. The stacking of the helices is different in both polymorphs: a crossed woodpile structure versus a parallel columnar stacking.     

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.     

Crystal structure of a partly self-complementary peptide nucleic acid (PNA) oligomer showing a duplex-triplex network

The X-ray structure of a partly self-complementary peptide nucleic acid (PNA) decamer (H-GTAGATCACT-l-Lys-NH(2)) to 2.60 A resolution is reported. The structure is mainly controlled by the canonical Watson-Crick base pairs formed by the self-complementary stretch of four bases in the middle of the decamer (G(4)A(5)T(6)C(7)). One right- and one left-handed Watson-Crick duplex are formed. The two PNA units C(9)T(10) change helical handedness, so that each PNA strand contains both a right- and a left-handed section. The changed handedness in C(9)T(10) allows formation of Hoogsteen hydrogen bonding between C(9)T(10) and G(4)A(5) of a PNA strand in an adjacent Watson-Crick double helix of the same handedness. Thereby, a PNA-PNA-PNA triplex is formed. The PNA unit A(3) forms a noncanonical base pair with A(8) in a symmetry-related strand of opposite handedness; the base pair is of the A-A reverse Hoogsteen type. The structural diversity of this PNA demonstrates how the PNA backbone is able to adapt to structures governed by the stacking and hydrogen-bonding interactions between the nucleobases. The crystal structure further shows how PNA oligomers containing limited sequence complementarity may form complex hydrogen-bonding networks.     

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.     

SCF CI MO calculations on the base–base interactions in dinucleoside monophosphates

In order to elucidate further details of RNA conformations we have studied base stacking in dinucleoside monophosphates (DNP's) using UV difference spectra and the hypochromic effect. SCF CI MO calculations according to PPP and MIM approximations were carried out for six pyrimidine-containing DNP's in each of several stacking geometries. The calculated and plotted difference spectra were fitted to the experimental spectra. Different DNP's showed distinct geometries in the range of the helix angle of 35°. From our results we conclude that there is a microstructure in the helix. This would imply an additional content of information in this macromolecule, a higher precision in nucleic acid interactions, and make possible the prediction of the conformation of polynucleotides.     

Dynamic chemical devices: generation of reversible extension/contraction molecular motion by ion-triggered single/double helix interconversion

The polyheterocyclic strands 1-H and 2-H adopt a helical shape enforced by the pyridine-pyrimidine helicity codon. The crystal structure of 2-H shows the formation of stacks of dimers of right- and left-handed individual helices. Treatment of 1-H and 2-H with silver triflate results in the generation of double-helical entities 1-DH and 2-DH, containing two strands and two silver ions. NMR studies and determination of the crystal structure of 2-DH indicate that the duplex is stabilized by coordination of each Ag(+) ion to two terminal bipyridine units, one from each strand, and by pronounced pi-pi stacking interactions between the internal heterocycles of the strands, yielding a very robust double helical structure. Reversible interconversion of the single and double helix may be achieved by addition of a cryptand capable of sequestering Ag(+) and releasing it by protonation. Thus, successive addition of acid and base leads to reversible interconversion between the shorter ( approximately 3.6 A) single helix and the longer ( approximately 10.3 A) double helix, resulting in the generation of pronounced extension/contraction motion. The system 1,2-H/1,2-DH represents a dynamic chemical device undergoing ionic modulation of reversible molecular mechanical motion fueled by acid/base neutralization.     

Nonenzymatic template-directed reactions on altritol oligomers, preorganized analogues of oligonucleotides

Altritol nucleic acids (ANAs) are RNA analogues with a phosphorylated D-altritol backbone. The nucleobase is attached at the 2-(S)-position of the carbohydrate moiety. We report that ANA oligomers are superior to the corresponding DNA, RNA, and HNA (hexitol nucleic acid) in supporting efficient nonenzymatic template-directed synthesis of complementary RNAs from nucleoside-5'-phosphoro-2-methyl imidazolides. Activated ANA and HNA monomers do not oligomerize efficiently on DNA, RNA, HNA, or ANA templates.     

Functional monomers and polymers. LXXI. Conformation and interaction studies of poly-L-lysine derivatives with pendant nucleic acid bases

Copolymers of L -lysine and L -lysine derivatives which contained nucleic acid bases substituted on the N^ε position were synthesized by grafting nucleic acid derivatives onto poly-L -lysine. The conformation and interaction of these copolymers in solution were studied by using spectroscopic measurements. They existed in helical conformation at neutral pH values, and the polymer complex formation among them was examined by ultraviolet (UV) measurements in organic solvents. A decrease in the nucleic acid base content of the copolymers resulted in a decrease in helical structures and also in interactions with the polymer-containing complementary bases.     

Solution structure of a DNA duplex containing a biphenyl pair

Hydrogen-bonding and stacking interactions between nucleobases are considered to be the major noncovalent interactions that stabilize the DNA and RNA double helices. In recent work we found that one or multiple biphenyl pairs, devoid of any potential for hydrogen bond formation, can be introduced into a DNA double helix without loss of duplex stability. We hypothesized that interstrand stacking interactions of the biphenyl residues maintain duplex stability. Here we present an NMR structure of the decamer duplex d(GTGACXGCAG) d(CTGCYGTCAC) that contains one such X/Y biaryl pair. X represents a 3',5'-dinitrobiphenyl- and Y a 3',4'-dimethoxybiphenyl C-nucleoside unit. The experimentally determined solution structure shows a B-DNA duplex with a slight kink at the site of modification. The biphenyl groups are intercalated side by side as a pair between the natural base pairs and are stacked head to tail in van der Waals contact with each other. The first phenyl rings of the biphenyl units each show tight intrastrand stacking to their natural base neighbors on the 3'-side, thus strongly favoring one of two possible interstrand intercalation structures. In order to accommodate the biphenyl units in the duplex the helical pitch is widened while the helical twist at the site of modification is reduced. Interestingly, the biphenyl rings are not static in the duplex but are in dynamic motion even at 294 K.     

Probing possible left- and right-handed poly(dinucleotide) helical conformations from (n–h) plots. models for polysequential nucleotides

Introducing the concept of the “dinucleotide” as the helical repeat, theoretical attempts have been made to determine possible single and double stranded helical structures by using helical parameter calculations and model building investigations. By virtue of its flexible framework, the dinucleotide repeat offers a much greater scope of finding new secondary structural forms for nucleic acids. Considering only those conformations which show tendency for at least partial base overlap as does the dinucleotide helical repeat, it has been possible to predict poly(dinucleotide) helical models in which successive phosphodiesters as well as nucleotide conformations alternate. More important, the recently found left-handed Z-type polynucleotide helix is characterized rather uniquely on the helical parameter plot. The results further suggest the possibility of other Z-type helices obtainable by alternative conformations for the exocyclic C4'–C5' bond and sugar pucker. Near neighbor long range conformational correlations between the dinucleotide repeat and the phosphodiester linking them have been established similar to poly(mononucleotide) helices. Need for considering higher repeats such as trinucleotide has been suggested to obtain models for looped out helical conformations.     

Locked nucleic acid (LNA) recognition of RNA: NMR solution structures of LNA:RNA hybrids

Locked nucleic acids (LNAs) containing one or more 2'-O,4'-C-methylene-linked bicyclic ribonucleoside monomers possess a number of the prerequisites of an effective antisense oligonucleotide, e.g. unprecedented helical thermostability when hybridized with cognate RNA and DNA. To acquire a detailed understanding of the structural features of LNA giving rise to its remarkable properties, we have conducted structural studies by use of NMR spectroscopy and now report high-resolution structures of two LNA:RNA hybrids, the LNA strands being d(5'-CTGAT(L)ATGC-3') and d(5'-CT(L)GAT(L)AT(L)GC-3'), respectively, T(L) denoting a modified LNA monomer with a thymine base, along with the unmodified DNA:RNA hybrid. In the structures, the LNA nucleotides are positioned as to partake in base stacking and Watson-Crick base pairing, and with the inclusion of LNA nucleotides, we observe a progressive change in duplex geometry toward an A-like duplex structure. As such, with the inclusion of three LNA nucleotides, the hybrid adopts an almost canonical A-type duplex geometry, and thus it appears that the number of modifications has reached a saturation level with respect to structural changes, and that further incorporations would furnish only minute changes in the duplex structure. We attempt to rationalize the conformational steering induced by the LNA nucleotides by suggesting that the change in electronic density at the brim of the minor groove, introduced by the LNA modification, is causing an alteration of the pseudorotational profile of the 3'-flanking nucleotide, thus shifting this sugar equilibrium toward N-type conformation.     

Structure of the fully modified left-handed cyclohexene nucleic acid sequence GTGTACAC

CeNA oligonucleotides consist of a phosphorylated backbone where the deoxyribose sugars are replaced by cyclohexene moieties. The X-ray structure determination and analysis of a fully modified octamer sequence GTGTACAC, which is the first crystal structure of a carbocyclic-based nucleic acid, is presented. This particular sequence was built with left-handed building blocks and crystallizes as a left-handed double helix. The helix can be characterized as belonging to the (mirrored) A-type family. Crystallographic data were processed up to 1.53 A, and the octamer sequence crystallizes in the space group R32. The sugar puckering is found to adopt the 3H2 half-chair conformation which mimics the C3'-endo conformation of the ribose sugar. The double helices stack on top of each other to form continuous helices, and static disorder is observed due to this end-to-end stacking.     

Visualisation of inter loop tertiary base pairing and stacking interactions in an unusual slipped loop DNA structure

The conformation of an unusual slipped loop DNA structure exhibited by the sequence d(GAATTCCCGAATTC)₂ is determined using a combination of geometrical and molecular mechanics methods. This sequence is known to form a B-DNA-like duplex with the central non-complementary cytosines extruded into single stranded loop regions. The unusual feature is that the interior guanine does not pair with the cytosine across, instead, it pairs with the cytosine upstream by skipping two cytosines, leading to a slipped loop DNA structure with the loops staggered by two base pairs. The two loops, despite being very small, can fold across minor or major groove symmetrically or asymmetrically disposed, with one of the loop bases partially blocking the major or minor groove. Most interestingly, for certain conformations, the loop bases approach one another at close proximity so as to engage even in base pairing as well as base stacking interactions across the major groove. While such pairing and stacking are common in the tertiary folds of RNA, this is the first time that such an interaction is visualized in a DNA. This observation demonstrates that a W-C pair can readily be accomplished in a typical slipped loop structure postulated for DNA. Such tertiary loop interaction may prevent access to regulatory proteins across the major groove of the duplex DNA, thus providing a structure-function relation for the occurrence of slipped loop structure in DNA. Contribution no. 839 from this department     

The structure of a TNA-TNA complex in solution: NMR study of the octamer duplex derived from alpha-(L)-threofuranosyl-(3'-2')-CGAATTCG

TNA (alpha-( l)-threofuranosyl-(3'-2') nucleic acid) is a nucleic acid in which the ribofuranose building block of the natural nucleic acid RNA is replaced by the tetrofuranose alpha-( l)-threose. This shortens the repetitive unit of the backbone by one bond as compared to the natural systems. Among the alternative nucleic acid structures studied so far in our laboratories in the etiological context, TNA is the only one that exhibits Watson-Crick pairing not only with itself but also with DNA and, even more strongly, with RNA. Using NMR spectroscopy, we have determined the structure of a duplex consisting entirely of TNA nucleotides. The TNA octamer (3'-2')-CGAATTCG forms a right-handed double helix with antiparallel strands paired according to the Watson-Crick mode. The dominant conformation of the sugar units has the 2'- and 3'-phosphodiester substituents in quasi-diaxial position and corresponds to a 4'-exo puckering. With 5.85 A, the average sequential P i -P i+1 distances of TNA are shorter than for A-type DNA (6.2 A). The helix parameters, in particular the slide and x-displacement, as well as the shallow and wide minor groove, place the TNA duplex in the structural vicinity of A-type DNA and RNA.