Base-pairing Scheme Research Articles

Unusual Paradigm for DNA-DNA Recognition and Binding: "Socket-Plug" Complementarity.

DNA is the key informational polymer in biology by virtue of its precisely defined self-assembling properties. Watson-Crick complementarity, which underlies DNA's self-assembly, is required not only in biology but has also proved powerful in the field of nanoscience, where it has been utilized to assemble complex 2D and 3D architectures and nanodevices built from the DNA double-helix. Aside from Watson-Crick base-pairing, however, DNA also participates in alternative base pairing schemes, giving rise to DNA triplexes and G-quadruplexes. Herein, we describe "sticky-ended" DNA triplex-quadruplex composites that specifically recognize and bind to each other using a wholly different logic, "socket-plug" complementarity, a shape-sensing fitting of guanine "prongs" into guanine-lacking "cavities." A remarkable property of this kind of complementarity is the key role played in it by specific counter-cations: thus, exclusive "self" socket-plug recognition occurs over "other" in sodium salt solutions while precisely the reverse occurs in potassium salt solutions. We have used gel electrophoresis, Förster resonance energy transfer, alkylation protection, and structural modeling to study this remarkable fundamental property of DNA, that we anticipate will find wide practical application.

Building blocks for recognition-encoded oligoesters that form H-bonded duplexes.

Competition from intramolecular folding is a major challenge in the design of synthetic oligomers that form intermolecular duplexes in a sequence-selective manner. One strategy is to use very rigid backbones that prevent folding, but this design can prejudice duplex formation if the geometry is not exactly right. The alternative approach found in nucleic acids is to use bases (or recognition units) that have different dimensions. A long-short base-pairing scheme makes folding geometrically difficult and is compatible with the flexible backbones that are required to guarantee duplex formation. A monomer building block equipped with a long hydrogen bond donor (phenol, D) recognition unit and a monomer building block equipped with a short hydrogen bond acceptor (phosphine oxide, A) recognition unit were prepared with differentially protected alcohol and carboxylic acid groups. These compounds were used to synthesise the homo and hetero-sequence 2-mers AA, DD and AD. 19F and 31P NMR experiments were used to characterize the assembly properties of these compounds in toluene solution. AA and DD form a stable doubly-hydrogen-bonded duplex with an effective molarity of 20 mM for formation of the second intramolecular hydrogen bond. AD forms a duplex of similar stability. There is no evidence of intramolecular folding in the monomeric state of this compound, which shows that the long-short base-pairing scheme is effective. The ester coupling chemistry used here is an attractive method for the synthesis of long oligomers, and the properties of the 2-mers indicate that this molecular architecture should give longer mixed sequence oligomers that show high fidelity sequence-selective duplex formation.

2-Methoxypyridine as a Thymidine Mimic in Watson-Crick Base Pairs of DNA and PNA: Synthesis, Thermal Stability, and NMR Structural Studies.

The development of nucleic acid base-pair analogues that use new modes of molecular recognition is important both for fundamental research and practical applications. The goal of this study was to evaluate 2-methoxypyridine as a cationic thymidine mimic in the A-T base pair. The hypothesis was that including protonation in the Watson-Crick base pairing scheme would enhance the thermal stability of the DNA double helix without compromising the sequence selectivity. DNA and peptide nucleic acid (PNA) sequences containing the new 2-methoxypyridine nucleobase (P) were synthesized and studied by using UV thermal melting and NMR spectroscopy. Introduction of P nucleobase caused a loss of thermal stability of ≈10 °C in DNA-DNA duplexes and ≈20 °C in PNA-DNA duplexes over a range of mildly acidic to neutral pH. Despite the decrease in thermal stability, the NMR structural studies showed that P-A formed the expected protonated base pair at pH 4.3. Our study demonstrates the feasibility of cationic unnatural base pairs; however, future optimization of such analogues will be required.

Where have all the inosines gone? Conflicting evidence for A-to-I editing of the anticodon of higher eukaryotic tRNAACGArg questions the dogma of a universal wobble-mediated decoding of CGN codons.

Codon-anticodon recognition between triplets of an mRNA and a specific tRNA is the key element in the translation of the genetic code. In general, the precision of this process is dominated by a strict Watson-Crick base-pairing scheme. However, the degeneracy of the genetic code led Crick to propose the Wobble Hypothesis, permitting a less restraining interaction with the third base of the codon and involving the participation of inosine for decoding C-ending codons. The concept that the anticodon base A34 of tRNAACGArg in all eukaryotes, eubacteria, and plant chloroplasts is converted to I34 is firmly anchored in the literature despite conflicting evidence for its existence in higher eukaryote cytoplasmic tRNAACGArg. Here, we provide additional data and summarize the arguments favoring and contradicting post-transcriptional deamination of this position. A hypothesis that resolves the apparent conflict is proposed. © 2016 IUBMB Life, 68(6):419-422, 2016.

DPT tautomerisation of the wobble guanine·thymine DNA base mispair is not mutagenic: QM and QTAIM arguments

We have shown for the first time, connecting QM methods with QTAIM analysis and using the methodology of the sweeps of the energetical, electron-topological and geometrical parameters, that the tautomerisation of the wobble guanine·thymine (wG·T) DNA base mispair into the wG*·T* base mispair induced by the double proton transfer (DPT), which undergoes a concerted asynchronous pathway, is not mutagenic. The wG·T → wG*·T* DPT tautomerisation does not result in the transition of the G base into its mutagenic tautomeric form G* able to mispair with the T base within the Watson–Crick base pairing scheme. This observation is explained by the so-called quantum protection of the wG·T DNA base mispair from its mutagenic tautomerisation – the dynamical non-stability of the tautomerised wG*·T* base mispair and significantly negative value of the Gibbs free energy of activation for the reverse reaction of the wG·T → wG*·T* DPT tautomerisation.

Targeting microRNAs involved in human diseases: A novel approach for modification of gene expression and drug development

The identification of all epigenetic modifications (i.e. DNA methylation, histone modifications and expression of noncoding RNAs such as microRNAs) involved in gene regulation is one of the major steps forward for understanding human biology in both normal and pathological conditions and for development of novel drugs. In this context, microRNAs play a pivotal role. This review article focuses on the involvement of microRNAs in the regulation of gene expression, on the possible role of microRNAs in the onset and development of human pathologies, and on the pharmacological alteration of the biological activity of microRNAs. RNA and DNA analogs, which can selectively target microRNAs using Watson–Crick base pairing schemes, provide a rational and efficient way to modulate gene expression. These compounds, termed antago-miR or anti-miR have been described in many examples in the recent literature and have proved to be able to perform regulatory as well as therapeutic functions. Among these, a still not fully exploited class is that of peptide nucleic acids (PNAs), promising tools for the inhibition of miRNA activity, with important applications in gene therapy and in drug development. PNAs targeting miR-122, miR-155 and miR-210 have already been developed and their biological effects studied both in vitro and in vivo.

Synthesis and Properties of the Simplified Nucleic Acid Glycol Nucleic Acid

The nucleosides of glycol nucleic acid (GNA), with the backbone comprising just the three carbons and one stereocenter of propylene glycol (1,2-propanediol), probably constitute the simplest possible building blocks for a chemically stable nucleic acid that contains phosphodiester bonds. However, it was not until 2005 that the astonishing duplex formation properties of GNA homoduplexes were discovered in our laboratory. The R- and S-enantiomers of GNA, (R)-GNA and (S)-GNA, pair in like-symmetric combinations to form highly stable antiparallel duplexes in a Watson-Crick fashion, with thermal and thermodynamic stabilities exceeding those of analogous duplexes of DNA and RNA. Interestingly, (R)-GNA and (S)-GNA do not significantly cross-pair with each other, either in a parallel or antiparallel fashion. GNA discriminates strongly in favor of the Watson-Crick base-pairing scheme, with only slightly lower fidelity than DNA. Two (S)-GNA homoduplex structures recently determined by X-ray crystallography, one a brominated 6-mer duplex and the other an 8-mer duplex containing two copper(II) ions, reveal that the overall GNA double helix is distinct from canonical A- and B-form nucleic acids. The structure is perhaps best described as a helical ribbon loosely wrapped around the helix axis. Within the backbone, the propylene glycol nucleotides adopt two different conformations, gauche and anti, with respect to the torsional angles between the vicinal C3'-O and C2'-O bonds. A strikingly large backbone-base inclination results in extensive zipper-like interstrand and reduced intrastrand base-base interactions. This strong backbone-base inclination might explain the observation that neither the R- nor S-enantiomer of GNA cross-pairs with DNA, whereas (S)-GNA can interact with RNA strands that are devoid of G:C base pairs. Given the combination of structural simplicity, straightforward synthetic accessibility, and high duplex stability of GNA duplexes, GNA affords a promising nucleic acid scaffold for biotechnology and nanotechnology. Along these lines, we describe the functionalization of GNA duplexes through the incorporation of metal-ion-mediated base pairs. Finally, the properties of GNA discussed here reinforce its candidacy as one of the initial genetic molecules formed during the origins of life on Earth.

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.

Base Pairing within the ψ32,ψ39-Modified Anticodon Arm of Escherichia coli tRNAPhe

The base-base hydrogen bond interactions of the psi32,psi39-modified anticodon arm of Escherichia coli tRNAPhe have been investigated using heteronuclear NMR spectroscopy. psi32 and psi39 were enzymatically introduced into a [13C,15N]-isotopically enriched RNA sequence corresponding to the tRNAPhe anticodon arm. Both the psi32-A38 and A31-psi39 nucleotide pairs form Watson-Crick base pairing schemes and the anticodon nucleotides adopt a triloop conformation. Similar effects were observed previously with D2-isopentenyl modification of the A37 N6 that also is native to the tRNAPhe anticodon arm. These results demonstrate that the individual modifications are not sufficient to produce the 32-38 bifurcated hydrogen bond or the U-turn motifs that are observed in crystal structures of tRNAs and tRNA-protein complexes. Thus the formation of these conserved structural features in solution likely require the synergistic interaction of multiple modifications.

Exploring the Limits of DNA Size: Naphtho-Homologated DNA Bases and Pairs

A new design for DNA bases and base pairs is described in which the pyrimidine bases are widened by naphtho-homologation. Two naphtho-homologated deoxyribosides, dyyT (1) and dyyC (2), were synthesized and could be incorporated into oligonucleotides as suitably protected phosphoramidite derivatives. The deoxyribosides were found to be fluorescent, with emission maxima at 446 and 433 nm, respectively. Studies with single substitutions of 1 and 2 in the natural DNA context revealed exceptionally strong base stacking propensity for both. Sequences containing multiple substitutions of 1 and 2 paired opposite adenine and guanine were subsequently mixed and studied by several analytical methods. Data from UV mixing experiments, FRET measurements, fluorescence quenching experiments, and hybridizations on beads suggest that complementary "doublewide DNA" (yyDNA) strands may self-assemble into helical complexes with 1:1 stoichiometry. Data from thermal denaturation plots and CD spectra were less conclusive. Control experiments in one sequence context gave evidence that yyDNA helices, if formed, are preferentially antiparallel and are sequence selective. Hypothesized base pairing schemes are analogous to Watson-Crick pairing, but with glycosidic C1'-C1' distances widened by over 45%, to ca. 15.2 A. The possible self-assembly of the double-wide DNA helix establishes a new limit for the size of information-encoding, DNA-like molecules, and the fluorescence of yyDNA bases suggests uses as reporters in monomeric and oligomeric forms.

Stable, Specific, and Reversible Base Pairing via Schiff Base

We here present a novel covalently linked base pair via Schiff base formation between 5-formyluracil (fU) and 5-aminocytosine (AmC). Formation of the Schiff base linkage proceeds reversibly and does not require any additives. The cross-linked DNA is very stable under denaturing conditions, whereas it completely dissociates upon heating at 90 degrees C. The pairing ability of AmC and fU is very specific and is applicable to the detection of fU, which is the major oxidative lesion of T in DNA. We propose the Schiff base linkage as a new artificial base pairing scheme to create functional DNAs.

Characterisation of hydrogen bonding networks in RNAs via magic angle spinning solid state NMR spectroscopy

It is demonstrated that the spatial proximity of (1)H nuclei in hydrogen bonded base-pairs in RNAs can be conveniently mapped via magic angle spinning solid state NMR experiments involving proton spin diffusion driven chemical shift correlation of low gamma nuclei such as the imino and amino nitrogens of nucleic acid bases. As different canonical and non-canonical base-pairing schemes encountered in nucleic acids are characterised by topologically different networks of proton dipolar couplings, different base-pairing schemes lead to characteristic cross-peak intensity patterns in such correlation spectra. The method was employed in a study of a 100 kDa RNA composed of 97 CUG repeats, or (CUG)(97) that has been implicated in the neuromuscular disease myotonic dystrophy. (15)N-(15)N chemical shift correlation studies confirm the presence of Watson-Crick GC base pairs in (CUG)(97).

Synthesis and incorporation into DNA fragments of the artificial nucleobase, 2-amino-8-oxopurine

The nucleoside 2-amino-9-(2-deoxy-β- d-ribofuranosyl)-7,8-dihydro-8-oxo-purine (dJ) was obtained in eight steps from 2′-deoxyguanosine. The appropriate protected phosphoramidite was synthesized and incorporated into DNA oligonucleotides. The thermal stability of heteroduplexes containing 2-amino-8-oxopurine (J) was investigated by UV-thermal denaturation experiments. The results obtained can be interpreted by the base pairing schemes involving the two edges of dJ depending on the anti and syn orientations.

Substrate specificity and kinetic framework of a DNAzyme with an expanded chemical repertoire: a putative RNaseA mimic that catalyzes RNA hydrolysis independent of a divalent metal cation.

This work addresses the binding, cleavage and dissociation rates for the substrate and products of a synthetic RNaseA mimic that was combinatorially selected using chemically modified nucleoside triphosphates. This trans-cleaving DNAzyme, 9(25)-11t, catalyzes sequence-specific ribophosphodiester hydrolysis in the total absence of a divalent metal cation, and in low ionic strength at pH 7.5 and in the presence of EDTA. It is the first such sequence capable of multiple turnover. 9(25)-11t consists of 31 bases, 18 of which form a catalytic domain containing 4 imidazole and 6 allylamino modified nucleotides. This sequence cleaves the 15 nt long substrate, S1, at one embedded ribocytosine at the eighth position to give a 5'-product terminating in a 2',3'-phosphodiester and a 3'-product terminating in a 5'-OH. Under single turnover conditions at 24 degrees C, 9(25)-11t displays a maximum first-order rate constant, k(cat), of 0.037 min(-1) and a catalytic efficiency, k(cat)/K(m), of 5.3 x 10(5) M(-1) min(-1). The measured value of k(cat) under catalyst excess conditions agrees with the value of k(cat) observed for steady-state multiple turnover, implying that slow product release is not rate limiting with respect to multiple turnover. The substrate specificity of 9(25)-11t was gauged in terms of k(cat) values for substrate sequence variants. Base substitutions on the scissile ribose and at the two bases immediately downstream decrease k(cat) values by a factor of 4 to 250, indicating that 9(25)-11t displays significant sequence specificity despite the lack of an apparent Watson-Crick base-pairing scheme for recognition.

An Extremely Stable and Orthogonal DNA Base Pair with a Simplified Three-Carbon Backbone

A nucleotide C3HQ with a minimal three-carbon backbone displays unprecedented pairing strength and orthogonality in a homopair C3HQ:C3HQ in the presence of one equivalent of Cu2+. The pairing stability in DNA even exceeds the related base pair having the regular 2'-deoxyribose backbone. This discovery of a synergy between an artificial backbone and base-pairing scheme opens new avenues for the economical design of modified oligonucleotides with tailored properties.

Parallel DNA double helices incorporating isoG or m 5isoC bases studied by FTIR, CD and molecular modeling

FTIR spectroscopy has been used to follow the formation of parallel stranded DNA duplexes incorporating isoG or m 5isoC bases and determine their base pairing scheme. The results are discussed in comparison with data concerning anti-parallel duplexes with comparable base composition and sequence. In duplexes containing A–T and isoG–C or m 5isoC–G base pairs shifts of the thymine C2 O2 and C4 O4 carbonyl stretching vibrations (to lower and higher wavenumbers, respectively, when compared to their positions in classical cis Watson–Crick (WC) base pairs) reflect the formation of trans Watson–Crick A–T base pairs. All carbonyl groups of cytosines, m 5isocytosines, guanines and isoguanines are found to be involved in hydrogen bonds, indicative of the formation of isoG–C and m 5isoC–G base pairs with three hydrogen bonds. Molecular modeling shows that both structures form regular right handed helices with C2′endo sugar puckers. The role of the water content on the helical conformation of the parallel duplexes has been studied by FTIR and CD. It is found that a conformational transition similar to the B → A transition observed for anti-parallel duplexes induced by a decrease of the water content of the samples can occur for these parallel duplexes. Their helical flexibility has been evidenced by FTIR studies on hydrated films by the emergence of absorption bands characteristic of A type geometry, in particular by an S-type → N-type repuckering of the deoxyribose. All sugars in the parallel duplex with alternating d(isoG–A)/d(C–T) sequence can adopt an N-type geometry in low water content conditions. The conformational transition of the parallel hairpin duplex with alternating d(isoG–A)/d(C–T) sequence was followed by circular dichroism in water/trifluoroethanol solutions and its free energy at 0 °C was estimated to be 6.6 ± 0.3 kcal mol −1.

The solution structure of the VS ribozyme active site loop reveals a dynamic "hot-spot".

The VS ribozyme is the largest ribozyme in its class and is also the least structurally characterized thus far. The current working model of the VS ribozyme locates the active site in stem-loop VI. The solution structure of this active site loop was determined using high resolution NMR spectroscopy. The structure reveals that the ground-state conformation of the active site differs significantly from that determined previously from chemical structure probing and mutational analysis of the ribozyme in its active conformation, which contains several looped out bases. In contrast, the base-pairing scheme found for the isolated loop contains three mismatched base-pairs: an A+-C, a G-U wobble, and a sheared G-A base-pair and no looped out bases. Dynamics observed within the active site loop provide insight into the mechanism by which the RNA can rearrange its secondary structure into an "activated" conformation prior to cleavage. These findings lend support to the idea that RNA secondary structure is more fluid than once believed and that a better understanding of structure and dynamic features of ribozymes is required to unravel the intricacies of their catalytic abilities.

A second-generation copper(II)-mediated metallo-DNA-base pair

Metal-dependent pairing of nucleobases represents an alternative DNA base pairing scheme. Our first-generation copper(II)-mediated pyridine-2,6-dicarboxylate ( Dipic) and pyridine ( Py) metallo-base pair has a stability comparable to the natural base pairs dA:dT and dC:dG but does not have the selectivity of the Watson Crick base pairs. In order to increase the selectivity of base pair formation, a second-generation metallo-base pair was generated consisting of a pyridine-2,6-dicarboxamide ( Dipam) and a pyridine ( Py) nucleobase. This new metallo-base pair is more stable than the natural base pairs dA:dT and dC:dG and highly selective against mispairing. In addition, incorporation of multiple metallo-base pairs into DNA results in the formation of stable duplexes demonstrating that hydrogen bonding base pairs can efficiently be replaced by metal-dependent base pairs at multiple sites in DNA.

Crystallographic Characterization of an Exocyclic DNA Adduct: 3,N4-etheno-2′-deoxycytidine in the Dodecamer 5′-CGCGAATTεCGCG-3′

Exocyclic DNA adducts are formed from metabolites of chemical carcinogens and have also been detected as endogenous lesions in human DNA. The exocyclic adduct 3,N4-etheno-2′-deoxycytidine (εdC), positioned opposite deoxyguanosine in the B-form duplex of the dodecanucleotide d(CGCGAATTεCGCG), has been crystallographically characterized at 1.8Å resolution. This self-complementary oligomer crystallizes in space group P3212, containing a single strand in the asymmetric unit. The crystal structure was solved by isomorphous replacement with the corresponding unmodified dodecamer structure. Exposure of both structures to identical crystal packing forces allows a detailed investigation of the influence of the exocyclic base adduct on the overall helical structure and local geometry. Structural changes are limited to the εC:G and adjacent T:A and G:C base-pairs. The standard Watson–Crick base-pairing scheme, retained in the T:A and G:C base-pairs, is blocked by the etheno bridge in the εC:G pair. In its place, a hydrogen bond involving O2 of εC and N1 of G is present. Comparison with an εdC-containing NMR structure confirms the general conformation reported for εC:G, including the hydrogen bonding features. Superposition with the crystal structure of a DNA duplex containing a T:G wobble pair shows similar structural changes imposed by both mismatches. Evaluation of the hydration shell of the duplex with bond valence calculations reveals two sodium ions in the crystal.

HYDRATE MODEL OF THE EQUILIBRIUM DNA–WATER SYSTEMS

A hydrate model for the DNA– H2O system is proposed. For the notions developed, available data on a tendency of H2O molecules to form structured hydrates containing atomic groups, molecules, or atoms housed within the structural cavities formed by H-bonded H2O molecules are used. It is shown that the large and small cavities of the hydrate structure II are in close geometric agreement with N-bases and deoxyribose and with other atomic groups of DNA molecules, respectively. On the basis of the model proposed and with the Watson–Crick base-pairing scheme, the number of pairs of N-bases per helix turn (11.25), the density (1.161 g/cm3), and the helix step (0.567 nm) for the quasi-equilibrium DNA (RNA)–water system are computed, the last value nearly coinciding with the well-known alpha-helix step in the protein secondary structure (5.44 nm). Assumptions on the phenomenology of some stages of mitosis are presented.