Crick Base Pairs Research Articles

6-Chloroisocytosine and 5-bromo-6-methylisocytosine: again, one or two tautomers present in the same crystal?

It is well known that pyrimidin-4-one derivatives are able to adopt either the 1H- or the 3H-tautomeric form in (co)crystals, depending on the coformer. As part of ongoing research to investigate the preferred hydrogen-bonding patterns of active pharmaceutical ingredients and their model systems, 2-amino-6-chloropyrimidin-4-one and 2-amino-5-bromo-6-methylpyrimidin-4-one have been cocrystallized with several coformers and with each other. Since Cl and Br atoms both have versatile possibilities to interact with the coformers, such as via hydrogen or halogen bonds, their behaviour within the crystal packing was also of interest. The experiments yielded five crystal structures, namely 2-aminopyridin-1-ium 2-amino-6-chloro-4-oxo-4H-pyrimidin-3-ide-2-amino-6-chloropyrimidin-4(3H)-one (1/3), C5H7N2(+)·C4H3ClN3O(-)·3C4H4ClN3O, (Ia), 2-aminopyridin-1-ium 2-amino-6-chloro-4-oxo-4H-pyrimidin-3-ide-2-amino-6-chloropyrimidin-4(3H)-one-2-aminopyridine (2/10/1), 2C5H7N2(+)·2C4H3ClN3O(-)·10C4H4ClN3O·C5H6N2, (Ib), the solvent-free cocrystal 2-amino-5-bromo-6-methylpyrimidin-4(3H)-one-2-amino-5-bromo-6-methylpyrimidin-4(1H)-one (1/1), C5H6BrN3O·C5H6BrN3O, (II), the solvate 2-amino-5-bromo-6-methylpyrimidin-4(3H)-one-2-amino-5-bromo-6-methylpyrimidin-4(1H)-one-N-methylpyrrolidin-2-one (1/1/1), C5H6BrN3O·C5H6BrN3O·C5H9NO, (III), and the partial cocrystal 2-amino-5-bromo-6-methylpyrimidin-4(3H)-one-2-amino-5-bromo-6-methylpyrimidin-4(1H)-one-2-amino-6-chloropyrimidin-4(3H)-one (0.635/1/0.365), C5H6BrN3O·C5H6BrN3O·C4H4ClN3O, (IV). All five structures show R2(2)(8) hydrogen-bond-based patterns, either by synthon 2 or by synthon 3, which are related to the Watson-Crick base pairs.

Formation of supramolecular assemblies and liquid crystals by purine nucleobases and cyanuric acid in water: implications for the possible origins of RNA.

The free nucleobases and mononucleotides of RNA do not form Watson-Crick base pairs in water, a fact that presents several challenges for the prebiotic synthesis of RNA. 2,6-Diaminopurine and adenosine-5'-monophosphate (AMP) are shown to form supramolecular assemblies with cyanuric acid in water. These assemblies and their propensity to form liquid crystals suggest a possible means by which non-covalent structures might have originally selected the shape of the Watson-Crick base pairs.

Metal-ion nucleic-acid interactions: A personal account

A brief personal account is given on how I observed and enjoyed studying metal-ion nucleic-acid binding over the last 40years, based on contributions from my own group and from our collaborators and competitors.Metal ions do bind to ligands, and the nucleic acids not only can act as metal-binding ligands, but also as hydrogen-bonding species, as typically known for Watson–Crick base pairs. In the study of metal-nucleic acid binding, coordination and hydrogen bonding do often occur together and may act synergistically. In this account I will highlight a selection of our most important findings within the context of this special issue.

Correlations of NBO energies of individual hydrogen bonds in nucleic acid base pairs with some QTAIM parameters

DNA and RNA base-pairs are the most important systems containing multiple hydrogen bonds. Characterizing the energy of individual intermolecular interactions in such systems is vital and still an open problem that has been tackled here within the framework of the natural bond orbital (NBO) and the quantum theory of atoms in molecules (QTAIM) theories. In the NBO language, energy of an individual H-bond depends on the interaction of the n Y, σ*XH, and σXH orbitals directly involved in H-bonding. A partial charge transfer between donor (n Y) and acceptor (σ*XH) orbitals provides a substantial bonding contribution to the energies of the H-bonds, and in the end, the H-bonded complexes. It is accompanied with a repulsive contribution due to the proximity of the n Y and σXH orbitals. Energies of the individual H-bonds, resulting from addition of the both terms, were correlated with several parameters, provided by the QTAIM analysis which has also been extensively used to characterize the hydrogen bond. The calculations were performed for the G–C and A–T Watson–Crick base pairs, their substituted derivatives (by one of two substituents, NH3 + or OH2 +), A–U occurring in RNA and a wobble pair G–U. The best correlations were found for the NBO energy with the electron density and the potential energy density at H-bond critical points. The correlations held for the heterogeneous samples of HBs of different types, i.e. N–H···O, N–H···N, and C–H···O, occurring simultaneously in DNA base pairs.

Biogenesis and growth phase-dependent alteration of 5-methoxycarbonylmethoxyuridine in tRNA anticodons.

Post-transcriptional modifications at the anticodon first (wobble) position of tRNA play critical roles in precise decoding of genetic codes. 5-carboxymethoxyuridine (cmo5U) and its methyl ester derivative 5-methoxycarbonylmethoxyuridine (mcmo5U) are modified nucleosides found at the anticodon wobble position in several tRNAs from Gram-negative bacteria. cmo5U and mcmo5U facilitate non-Watson–Crick base pairing with guanosine and pyrimidines at the third positions of codons, thereby expanding decoding capabilities. By mass spectrometric analyses of individual tRNAs and a shotgun approach of total RNA from Escherichia coli, we identified mcmo5U as a major modification in tRNAAla1, tRNASer1, tRNAPro3 and tRNAThr4; by contrast, cmo5U was present primarily in tRNALeu3 and tRNAVal1. In addition, we discovered 5-methoxycarbonylmethoxy-2′-O-methyluridine (mcmo5Um) as a novel but minor modification in tRNASer1. Terminal methylation frequency of mcmo5U in tRNAPro3 was low (≈30%) in the early log phase of cell growth, gradually increased as growth proceeded and reached nearly 100% in late log and stationary phases. We identified CmoM (previously known as SmtA), an AdoMet-dependent methyltransferase that methylates cmo5U to form mcmo5U. A luciferase reporter assay based on a +1 frameshift construct revealed that terminal methylation of mcmo5U contributes to the decoding ability of tRNAAla1.

Sequence-dependent structural changes in a self-assembling DNA oligonucleotide.

DNA has proved to be a remarkable molecule for the construction of sophisticated two-dimensional and three-dimensional architectures because of its programmability and structural predictability provided by complementary Watson-Crick base pairing. DNA oligonucleotides can, however, exhibit a great deal of local structural diversity. DNA conformation is strongly linked to both environmental conditions and the nucleobase identities inherent in the oligonucleotide sequence, but the exact relationship between sequence and local structure is not completely understood. This study examines how a single-nucleotide addition to a class of self-assembling DNA 13-mers leads to a significantly different overall structure under identical crystallization conditions. The DNA 13-mers self-assemble in the presence of Mg(2+) through a combination of Watson-Crick and noncanonical base-pairing interactions. The crystal structures described here show that all of the predicted Watson-Crick base pairs are present, with the major difference being a significant rearrangement of noncanonical base pairs. This includes the formation of a sheared A-G base pair, a junction of strands formed from base-triple interactions, and tertiary interactions that generate structural features similar to tandem sheared G-A base pairs. The adoption of this alternate noncanonical structure is dependent in part on the sequence in the Watson-Crick duplex region. These results provide important new insights into the sequence-structure relationship of short DNA oligonucleotides and demonstrate a unique interplay between Watson-Crick and noncanonical base pairs that is responsible for crystallization fate.

Electrochemical behavior of 7-deazaguanine- and 7-deazaadenine-modified DNA at the hanging mercury drop electrode

DNA modification with synthetic analogs of natural nucleotides and/or their conjugates with external redox active groups is applied in the development of electrochemical DNA sensors or assay for DNA hybridization, SNP typing, DNA damage and so forth. 7-Deazapurines (Pu*) are analogs of natural purine bases in which N7 atom is replaced by CH group. The Pu* bases retain Watson–Crick base pairing of their parent purines (and the ability to form duplex DNA) but are incapable of Hoogsteen pairing (and thus cannot be involved in triplex or quadruples DNA structures). Previously, we studied electrochemical oxidation of Pu* residues in DNA fragments (prepared by PCR in the presence of Pu* deoxynucleoside triphosphates) at a carbon electrode and reported on significantly lower potentials of oxidation of both 7-deazaguanine (G*) and 7-deazaadenine (A*), compared to natural guanine (G) and adenine (A), respectively. In this work, we studied faradaic and tensammetric responses of G*- or A*-modified DNA on the hanging mercury drop electrode (HMDE). While A* was reduced at the HMDE, giving rise to a similar irreversible cathodic peak as the natural A, G* did not yield any peak analogous to the peak G due to guanine, in agreement with a loss of corresponding redox site in G*. Responses of DNA modified with A* were relatively similar to those of unmodified DNA (albeit we observed certain differences in tensammetric peak currents). Effects of G substitution by G* were more pronounced, being reflected in diminution of peak due to guanine, decrease of the peak CA (due to cytosine and adenine reduction) and in significantly changed shape of tensammetric DNA signals, indicating altered adsorption/desorption processes. While substitution of A by A* resulted in certain destabilization of the DNA duplex at the negatively charged HMDE surface (in qualitative agreement with significantly decreased melting temperature of the same DNA duplexes in solution), G*-modified duplex DNA displayed apparently lower susceptibility to surface denaturation.

A model of compensatory molecular evolution involving multiple sites in RNA molecules

Consider two sites under compensatory fitness interaction, such as a Watson–Crick base pair in an RNA helix or two interacting residues in a protein. A mutation at any one of these two sites may reduce the fitness of an individual. However, fitness may be restored by the occurrence of a second mutation at the other site. Kimura modeled this process using a two-locus haploid fitness scheme with two alleles at each locus. He predicted that compensatory evolution following this model is very rare unless selection against the deleterious single mutations is weak and linkage between the interacting sites is tight. Here we investigate the question whether the rate of compensatory evolution increases if we take the context of the two directly interacting sites into account. By “context”, we mean the effect of neighboring sites in an RNA helix. Interaction between the focal pair of sites under consideration and the context may lead to so-called indirect compensation. Thus, extending Kimura׳s classical model of compensatory evolution, we study the effects of both direct and indirect compensation on the rate of compensatory evolution. It is shown that the effects of indirect compensation are very strong. We find that recombination does not slow down the rate of compensatory evolution as predicted by the classical model. Instead, compensatory substitutions may be relatively frequent, even if linkage between the focal interacting sites is loose, selection against deleterious mutations is strong, and mutation rate is low. We compare our theoretical results with data on RNA secondary structures from vertebrate introns.

Ultraviolet Absorption Induces Hydrogen‐Atom Transfer in G⋅C Watson–Crick DNA Base Pairs in Solution

AbstractUltrafast deactivation pathways bestow photostability on nucleobases and hence preserve the structural integrity of DNA following absorption of ultraviolet (UV) radiation. One controversial recovery mechanism proposed to account for this photostability involves electron‐driven proton transfer (EDPT) in Watson–Crick base pairs. The first direct observation is reported of the EDPT process after UV excitation of individual guanine–cytosine (G⋅C) Watson–Crick base pairs by ultrafast time‐resolved UV/visible and mid‐infrared spectroscopy. The formation of an intermediate biradical species (G[−H]⋅C[+H]) with a lifetime of 2.9 ps was tracked. The majority of these biradicals return to the original G⋅C Watson–Crick pairs, but up to 10 % of the initially excited molecules instead form a stable photoproduct G*⋅C* that has undergone double hydrogen‐atom transfer. The observation of these sequential EDPT mechanisms across intermolecular hydrogen bonds confirms an important and long debated pathway for the deactivation of photoexcited base pairs, with possible implications for the UV photochemistry of DNA.

Ultraviolet Absorption Induces Hydrogen-Atom Transfer in G⋅C Watson-Crick DNA Base Pairs in Solution.

Ultrafast deactivation pathways bestow photostability on nucleobases and hence preserve the structural integrity of DNA following absorption of ultraviolet (UV) radiation. One controversial recovery mechanism proposed to account for this photostability involves electron-driven proton transfer (EDPT) in Watson-Crick base pairs. The first direct observation is reported of the EDPT process after UV excitation of individual guanine-cytosine (G⋅C) Watson-Crick base pairs by ultrafast time-resolved UV/visible and mid-infrared spectroscopy. The formation of an intermediate biradical species (G[-H]⋅C[+H]) with a lifetime of 2.9 ps was tracked. The majority of these biradicals return to the original G⋅C Watson-Crick pairs, but up to 10% of the initially excited molecules instead form a stable photoproduct G*⋅C* that has undergone double hydrogen-atom transfer. The observation of these sequential EDPT mechanisms across intermolecular hydrogen bonds confirms an important and long debated pathway for the deactivation of photoexcited base pairs, with possible implications for the UV photochemistry of DNA.

High‐Resolution Crystal Structure of a Silver(I)–RNA Hybrid Duplex Containing Watson–Crick‐like CSilver(I)C Metallo‐Base Pairs

AbstractMetallo‐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HgIIT base pairs. Herein, we have determined a high‐resolution crystal structure of the second natural metallo‐base pair between pyrimidine bases CAgIC formed in an RNA duplex. One AgI occupies the center between two cytosines and forms a CAgIC base pair through N3AgIN3 linear coordination. The CAgIC base pair formation does not disturb the standard A‐form conformation of RNA. Since the CAgI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.

ChemInform Abstract: The “Speedy” Synthesis of Atom‐Specific 15N Imino/Amido‐Labeled RNA

AbstractReview: 82 refs.

Duplex formation and secondary structure of γ-PNA observed by NMR and CD

Peptide nucleic acids (PNAs) are non-natural oligonucleotides mimics, wherein the phosphoribose backbone has been replaced by a peptidic moiety (N-(2-aminoethyl)glycine). This peptidic backbone lends itself to substitution and the γ-position has proven to yield oligomers with enhanced hybridization properties. In this study, we use Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) to explore the properties of the supramolecular duplexes formed by these species. We show that standard Watson–Crick base pair as well as non-standard ones are formed in solution. The duplexes thus formed present marked melting transition temperatures substantially higher than their nucleic acid homologs. Moreover, the presence of a chiral group on the γ-peptidic backbone increases further this transition temperature, leading to very stable duplexes.PNA duplexes with a chiral backbone present a marked chiral secondary structure, observed by CD, and showing a common folding pattern for all studied structures. Nevertheless small differences are observed depending on the details of the nucleobase sequence.

N6-Methyladenosine Modification in a Long Noncoding RNA Hairpin Predisposes Its Conformation to Protein Binding

N6-Methyladenosine (m6A) is a reversible and abundant internal modification of messenger RNA (mRNA) and long noncoding RNA (lncRNA) with roles in RNA processing, transport, and stability. Although m6A does not preclude Watson–Crick base pairing, the N6-methyl group alters the stability of RNA secondary structure. Since changes in RNA structure can affect diverse cellular processes, the influence of m6A on mRNA and lncRNA structure has the potential to be an important mechanism for m6A function in the cell. Indeed, an m6A site in the lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) was recently shown to induce a local change in structure that increases the accessibility of a U5-tract for recognition and binding by heterogeneous nuclear ribonucleoprotein C (HNRNPC). This m6A-dependent regulation of protein binding through a change in RNA structure, termed “m6A-switch”, affects transcriptome-wide mRNA abundance and alternative splicing. To further characterize this first example of an m6A-switch in a cellular RNA, we used nuclear magnetic resonance and Förster resonance energy transfer to demonstrate the effect of m6A on a 32-nucleotide RNA hairpin derived from the m6A-switch in MALAT1. The observed imino proton nuclear magnetic resonance resonances and Förster resonance energy transfer efficiencies suggest that m6A selectively destabilizes the portion of the hairpin stem where the U5-tract is located, increasing the solvent accessibility of the neighboring bases while maintaining the overall hairpin structure. The m6A-modified hairpin has a predisposed conformation that resembles the hairpin conformation in the RNA–HNRNPC complex more closely than the unmodified hairpin. The m6A-induced structural changes in the MALAT1 hairpin can serve as a model for a large family of m6A-switches that mediate the influence of m6A on cellular processes.

The structural and dipole-structural peculiarities of the Hoogsteen base pairs that are formed from complementary nucleobases according to ab initio quantum-mechanical studies

The structural and dipole-structural peculiarities of the Hoogsteen base pairs that are formed from complementary nucleobases were investigated in comparison with those of the Watson–Crick type by the ab initio quantum-mechanical methods. Both the latter and the former base pairs were found to be formed depending on the mutual positions of adenine and thymine. Cytosine–guanine Hoogsteen pairs were formed only if cytosine was originally protonated. Both types of Hoogsteen dimers considerably differed from the Watson–Crick dimers by the bond distances and angles between the adjacent bonds both in the purine and pyrimidine parts of the dimers. The Hoogsteen pairs mainly had shorter lengths of hydrogen bonds and significantly larger angles of hydrogen bonds and those between the hydrogen bonds in comparison with the Watson–Crick base pairs. Considerable differences were also observed in the distribution of charges on atoms and dipole moments. In this paper, we propose a new method for the determination of the fine structure of dipole moments and we used this method for a quantitative characterization of the differences in the dipole moments. The values of local parameters (according to Cambridge classification of the parameters which determine the DNA properties) were shown to be greatly different for the Hoogsteen and the Watson–Crick base pairs.

Interaction between Small Gold Clusters and Nucleobases: A Density Functional Reactivity Theory Based Study

The thermodynamic and kinetic aspects associated with the interaction of small gold clusters (Aun, where n = 3–6) with nucleobases are assessed using a density functional reactivity theory based comprehensive decomposition analysis of stabilization energy scheme. It is observed that the trend of interaction between Aun clusters and nucleobases follows the order G > A > C > T > U. Also, the Watson–Crick base pair GC interacts with Aun clusters more preferably than that of the AT pair. The observed trend is further supported by conventional binding energy and transition-state calculations at B3PW91 and MP2 levels.

Synthetic biology: Six pack and stack.

A pair of artificial DNA bases have now been shown to adopt an edge-to-edge geometry in DNA which is similar that found in Watson–Crick base pairing. Aptamers containing these bases have also been shown to bind more strongly to a target than those developed using only the four naturally occurring bases.

G-quadruplex formation in double strand DNA probed by NMM and CV fluorescence.

G-quadruplexes (GQs) are alternative DNA secondary structures that can form throughout the human genome and control the replication and transcription of important regulatory genes. Here, we established an ensemble fluorescence assay by employing two GQ-interacting compounds, N-methyl mesoporphyrin IX (NMM) and Crystal Violet (CV). This enables quantitative measurement of the GQ folding propensity and conformation specificity in both single strand (ss) and double strand (ds) DNA. Our GQ mapping indicates that the likelihood of GQ formation is substantially diminished in dsDNA, likely due to the competition from the Watson–Crick base pairing. Unlike GQ folding sequence in ssDNA which forms both parallel and antiparallel GQs, dsDNA displays only parallel folding. Additionally, we employed single molecule FRET to obtain a direct quantitation of stably formed-, weakly folded and unfolded GQ conformations. The findings of this study and the method developed here will enable identifying and classifying potential GQ-forming sequences in human genome.

FDF-PAGE: a powerful technique revealing previously undetected small RNAs sequestered by complementary transcripts.

Small RNAs, between 18nt and 30nt in length, are a diverse class of non-coding RNAs that mediate a range of cellular processes, from gene regulation to pathogen defense. They guide ribonucleoprotein complexes to their target nucleic acids by Watson–Crick base pairing. We report here that current techniques for small RNA detection and library generation are biased by formation of RNA duplexes. To address this problem, we established FDF-PAGE (fully-denaturing formaldehyde polyacrylamide gel electrophoresis) to prevent annealing of sRNAs to their complement. By applying FDF-PAGE, we provide evidence that both strands of viral small RNA are present in near equimolar ratios, indicating that the predominant precursor is a long double-stranded RNA. Comparing non-denaturing conditions to FDF-PAGE uncovered extensive sequestration of miRNAs in model organisms and allowed us to identify candidate small RNAs under the control of competing endogenous RNAs (ceRNAs). By revealing the full repertoire of small RNAs, we can begin to create a better understanding of small RNA mediated interactions.

88 Interplay of water molecules and Mg2+ ions in stability of RNA containing non-Watson–Crick base pairs

The segments of RNA, containing non-Watson–Crick base pairs, are stabilized by external factors like other part of RNA, proteins or metal ions. Eubacterial 5S RNA Loop-E [pdb: 354D] is made up of 1...