Base-pairing Properties Research Articles

Bis(6-carboxypurine)-Cu2+: a possibly primitive metal-mediated nucleobase pair.

A metal-mediated self-pair is described that emulates Watson-Crick base pair properties in a DNA double helix.

A base-modified PNA–graphene oxide platform as a turn-on fluorescence sensor for the detection of human telomeric repeats

Given the biological and therapeutic significance of telomeres and other G-quadruplex forming sequences in human genome, it is highly desirable to develop simple methods to study these structures, which can also be implemented in screening formats for the discovery of G-quadruplex binders. The majority of telomere detection methods developed so far are laborious and use elaborate assay and instrumental setups, and hence, are not amenable to discovery platforms. Here, we describe the development of a simple homogeneous fluorescence turn-on method, which uses a unique combination of an environment-sensitive fluorescent nucleobase analogue, the superior base pairing property of PNA, and DNA-binding and fluorescence quenching properties of graphene oxide, to detect human telomeric DNA repeats of varying lengths. Our results demonstrate that this method, which does not involve a rigorous assay setup, would provide new opportunities to study G-quadruplex structures.

Surprising base pairing and structural properties of 2'-trifluoromethylthio-modified ribonucleic acids.

The chemical synthesis of ribonucleic acids (RNA) with novel chemical modifications is largely driven by the motivation to identify eligible functional probes for the various applications in life sciences. To this end, we have a strong focus on the development of novel fluorinated RNA derivatives that are powerful in NMR spectroscopic analysis of RNA folding and RNA ligand interactions. Here, we report on the synthesis of 2′-SCF3 pyrimidine nucleoside containing oligoribonucleotides and the comprehensive investigation of their structure and base pairing properties. While this modification has a modest impact on thermodynamic stability when it resides in single-stranded regions, it was found to be destabilizing to a surprisingly high extent when located in double helical regions. Our NMR spectroscopic investigations on short single-stranded RNA revealed a strong preference for C2′-endo conformation of the 2′-SCF3 ribose unit. Together with a recent computational study (L. Li, J. W. Szostak, J. Am. Chem. Soc. 2014, 136, 2858–2865) that estimated the extent of destabilization caused by a single C2′-endo nucleotide within a native RNA duplex to amount to 6 kcal mol−1 because of disruption of the planar base pair structure, these findings support the notion that the intrinsic preference for C2′-endo conformation of 2′-SCF3 nucleosides is most likely responsible for the pronounced destabilization of double helices. Importantly, we were able to crystallize 2′-SCF3 modified RNAs and solved their X-ray structures at atomic resolution. Interestingly, the 2′-SCF3 containing nucleosides that were engaged in distinct mismatch arrangements, but also in a standard Watson–Crick base pair, adopted the same C3′-endo ribose conformations as observed in the structure of the unmodified RNA. Likely, strong crystal packing interactions account for this observation. In all structures, the fluorine atoms made surprisingly close contacts to the oxygen atoms of the corresponding pyrimidine nucleobase (O2), and the 2′-SCF3 moieties participated in defined water-bridged hydrogen-bonding networks in the minor groove. All these features allow a rationalization of the structural determinants of the 2′-SCF3 nucleoside modification and correlate them to base pairing properties.

DFT investigation of the vibrational properties of GC Watson-Crick and Hoogsteen base pairs in the presence of Mg²⁺, Ca²⁺, and Cu²⁺ ions.

The binding effects of Mg²⁺, Ca²⁺, and Cu²⁺ ions on the vibrational properties of guanine-cytosine base pairs have been performed using density functional theory investigations. Both Watson-Crick and Hoogsteen configurations of the base pairs were investigated. In Watson-Crick configuration, the metal was coordinated at N7 atom of guanine, while in the case of Hoogsteen configuration, the coordination is at N3 atom of guanine. We have pointed out the geometric properties of the metal-GC base pairs structure, as well as the vibrational bands that can be used to detect the presence of metallic ions in the Watson-Crick and Hoogsteen GC structures. For the geometric models used by us, the vibrational amplitudes of metallic atoms were stronger for wavenumbers lower than 500 cm⁻¹. This suggests that in the experimental studies on DNA the presence of the three metallic atoms (Mg, Ca, and Cu) can be explicitly detected at low frequencies.

Pyrazolo[1,5-a]-1,3,5-triazine C-Nucleoside as Deoxyadenosine Analogue: Synthesis, Pairing, and Resistance to Hydrolysis

The synthesis of a pyrazolo[1,5-a]-1,3,5-triazine C-nucleoside (dA(PT)), designed to form two hydrogen bonds with a complementary dT residue, is reported. Oligonucleotides including this dA nucleoside analogue possess base-pairing properties similar to those of the parent oligonucleotide. This dA nucleoside analogue is more resistant to acid-catalyzed hydrolysis than dA.

Synthesis of 8-oxoguanosine phosphoramidite and its incorporation into Oligoribonucleotides..

The detailed synthetic protocol for preparation of the phosphoramidite of an oxidatively damaged ribonucleotide, 8-oxoguanosine (8-oxo-G), and its incorporation into RNA are described. The O(6)- and N(7)-bisdiphenylcarbamoyl-protected 8-oxoguanosine phosphoramidite was synthesized as a new phosphoramidite precursor unit for the synthesis of RNA. It was successfully incorporated into the RNA sequences, and the synthesized RNAs were completely deprotected with 28% aqueous ammonia solution at 55°C for 24 hr. After purification using HPLC, they were identified by MALDI-TOF mass measurement. The base-pairing properties showed that 8-oxo-G forms base pairs not only with rC or dC in anti-conformation, but also with rA in syn conformation within the RNA duplexes or RNA/DNA heteroduplexes.

Synthesis of 8-halogenated-7-deaza-2′-deoxyguanosine as an 8-oxo-2′-deoxyguanosine analogue and evaluation of its base pairing properties

8-Halogenated-7-deaza-2′-deoxyguanosines (8-halo-7-deaza-dG) were designed to structurally mimic 8-oxo-2′-deoxyguanosine (8-oxo-dG), which is representative of an oxidized nucleoside. It has been shown by NMR that the conformation around the N-glycosidic bond of (8-halo-7-deaza-dG) is preferably syn, similar to 8-oxo-dG. The base pairing properties of 8-halo-7-deaza-dG were studied by measuring the thermal denaturation temperature of the duplexes, showing that their base pair with dC is destabilized compared with natural dG. These results also support their preference for syn conformation. Unlike 8-oxo-dG, 8-halo-7-deaza-dG did not form a stable base pair with dA, most likely due to the lack of N7-H hydrogen bonding with dA. In conclusion, the newly-designed 8-halo-7-deaza-dG analogs resemble 8-oxo-dG in its shape and preference for syn conformation, but they do not form Hoogsteen base pair with the opposing dA.

Inside Cover: 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 (Chem. Eur. J. 6/2014)

Inside Cover: 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 (Chem. Eur. J. 6/2014)

Multistep DNA-Templated Synthesis Using a Universal Template

We report a DNA-templated synthesis method that allows construction of the entire DNA-encoded library with a single DNA template. Taking advantage of deoxyinosine's indiscriminate base-pairing property, we designed a "universal template" that is capable of directing chemical reactions with multiple reactant DNAs with different sequences. In combination with other design features including photocleavable linkers and direct encoding by the reactant DNA, we demonstrated the capabilities of the universal template in library synthesis, target selection, and hit decoding. Our method can be generally and straightforwardly applied to prepare a variety of chemically diverse DNA-encoded libraries.

RNA nanotechnology for computer design and in vivo computation.

Molecular-scale computing has been explored since 1989 owing to the foreseeable limitation of Moore's law for silicon-based computation devices. With the potential of massive parallelism, low energy consumption and capability of working in vivo, molecular-scale computing promises a new computational paradigm. Inspired by the concepts from the electronic computer, DNA computing has realized basic Boolean functions and has progressed into multi-layered circuits. Recently, RNA nanotechnology has emerged as an alternative approach. Owing to the newly discovered thermodynamic stability of a special RNA motif (Shu et al. 2011 Nat. Nanotechnol. 6, 658-667 (doi:10.1038/nnano.2011.105)), RNA nanoparticles are emerging as another promising medium for nanodevice and nanomedicine as well as molecular-scale computing. Like DNA, RNA sequences can be designed to form desired secondary structures in a straightforward manner, but RNA is structurally more versatile and more thermodynamically stable owing to its non-canonical base-pairing, tertiary interactions and base-stacking property. A 90-nucleotide RNA can exhibit 4⁹⁰ nanostructures, and its loops and tertiary architecture can serve as a mounting dovetail that eliminates the need for external linking dowels. Its enzymatic and fluorogenic activity creates diversity in computational design. Varieties of small RNA can work cooperatively, synergistically or antagonistically to carry out computational logic circuits. The riboswitch and enzymatic ribozyme activities and its special in vivo attributes offer a great potential for in vivo computation. Unique features in transcription, termination, self-assembly, self-processing and acid resistance enable in vivo production of RNA nanoparticles that harbour various regulators for intracellular manipulation. With all these advantages, RNA computation is promising, but it is still in its infancy. Many challenges still exist. Collaborations between RNA nanotechnologists and computer scientists are necessary to advance this nascent technology.

Radical–Radical Interactions among Oxidized Guanine Bases Including Guanine Radical Cation and Dehydrogenated Guanine Radicals

We present here a theoretical investigation of the structural and electronic properties of di-ionized GG base pairs (G(•+)G(•+),G(-H1)(•)G(•+), and G(-H1)(•)G(-H1)(•)) consisting of the guanine cation radical (G(•+)) and/or dehydrogenated guanine radical (G(-H1)(•)) using density functional theory calculations. Different coupling modes (Watson-Crick/WC, Hoogsteen/Hoog, and minor groove/min hydrogen bonding, and π-π stacking modes) are considered. We infer that a series of G(•+)G(•+) complexes can be formed by the high-energy radiation. On the basis of density functional theory and complete active space self-consistent (CASSCF) calculations, we reveal that in the H-bonded and N-N cross-linked modes, (G(•+)G(•+))WC, (G(-H1)(•)G(-H1)(•))WC, (G(-H1)(•)G(-H1)(•))minI, and (G(-H1)(•)G(-H1)(•))minIII have the triplet ground states; (G(•+)G(•+))HoogI, (G(-H1)(•)G(•+))WC, (G(-H1)(•)G(•+))HoogI, (G(-H1)(•)G(•+))minI, (G(-H1)(•)G(•+))minII, and (G(-H1)(•)G(-H1)(•))minII possess open-shell broken-symmetry diradical-characterized singlet ground states; and (G(•+)G(•+))HoogII, (G(•+)G(•+))minI, (G(•+)G(•+))minII, (G(•+)G(•+))minIII, (G(•+)G(•+))HoHo, (G(-H1)(•)G(•+))minIII, (G(-H1)(•)G(•+))HoHo, and (G(-H1)(•)G(-H1)(•))HoHo are the closed-shell systems. For these H-bonded diradical complexes, the magnetic interactions are weak, especially in the diradical G(•+)G(•+) series and G(-H1)(•)G(-H1)(•) series. The magnetic coupling interactions of the diradical systems are controlled by intermolecular interactions (H-bond, electrostatic repulsion, and radical coupling). The radical-radical interaction in the π-π stacked di-ionized GG base pairs ((G(•+)G(•+))ππ, (G(-H1)(•)G(•+))ππ, and (G(-H1)(•)G(-H1)(•))ππ) are also considered, and the magnetic coupling interactions in these π-π stacked base pairs are large. This is the first theoretical prediction that some di-ionized GG base pairs possess diradical characters with variable degrees of ferromagnetic and antiferromagnetic characteristics, depending on the dehydrogenation characters of the bases and their interaction modes. Hopefully, this work provides some helpful information for the understanding of different structures and properties of the di-ionized GG base pairs.

7-Aminobutynyl-8-Aza-7-Deazahypoxanthine as a Quasi-Universal Nucleobase

Several 7-(hydroxy, amino, methylureido, and guanidino)alkynyl-substituted 8-aza-7-deaza- hypoxanthine analogues were investigated as potential universal nucleobases. 7-Aminobutynyl-8-aza-7-deazahypoxanthine was found to be the most promising quasi-universal nucleobase with improved hybridization and polymerase chain reaction (PCR) enhancing properties as compared to commonly used hypoxanthine (the nucleobase of inosine). It demonstrated improved ambiguity for pairing with A, T, and C bases and its base pairing properties can be summarized as follows: X:C∼X:A∼X:T > X:G. The improvement in PCR performance directly correlated with primer's Tm. Primers containing multiple 7-aminobutynyl-8-aza-7-deazahypoxanthines were successfully used without noticeable inhibition of Taq polymerase activity provided the modifications are positioned more than two bases away from the 3′ end.

Base Pairing and Miscoding Properties of 1,N6-Ethenoadenine- and 3,N4-Ethenocytosine-Containing RNA Oligonucleotides

Two RNA phosphoramidites containing the bases 1,N(6)-ethenoadenine (εA) and 3,N(4)-ethenocytosine (εC) were synthesized. These building blocks were incorporated into two 12-mer oligoribonucleotides for evaluation of the base pairing properties of these base lesions by UV melting curve (Tm) and circular dichroism measurements. The Tm data of the resulting duplexes with the etheno modifications opposing all natural bases showed a substantial destabilization compared to the corresponding natural duplexes, confirming their inability to form base pairs. The coding properties of these lesions were further investigated by introducing them into 31-mer oligonucleotides and assessing their ability to serve as templates in primer extension reactions with HIV, AMV, and MMLV reverse transcriptases (RT). Primer extension reactions showed complete arrest of the incorporation process using MMLV RT and AMV RT, while HIV RT preferentially incorporates dAMP opposite εA and dAMP as well as dTMP opposite εC. The properties of these RNA lesions are discussed in the context of its putative biological role.

Specific recognition of cytosine by hypoxanthine in pyrrolidinyl peptide nucleic acid

Hypoxanthine is an unnatural base that can potentially pair with all natural nucleobases. While hypoxanthine in DNA exhibits marginal preference for pairing with cytosine (C), little is known about its pairing behavior in other DNA analogues. In this study, we synthesized a hypoxanthine-containing monomer and incorporated it into pyrrolidinyl peptide nucleic acid with α/β-peptide backbone derived from D-prolyl-(1S,2S)-2-aminocyclopentanecarboxylic acid (acpcPNA). DNA binding studies clearly revealed that hypoxanthine in acpcPNA behaves like G-analogue because it can specifically form a stable base pair with dC in DNA. The ability to replace G by hypoxanthine without affecting the base pairing properties of acpcPNA can solve a number of problems associated with G-rich acpcPNA including difficult synthesis, formation of secondary structures and fluorescence quenching.

Reciprocal regulation of A-to-I RNA editing and the vertebrate nervous system

The fine control of molecules mediating communication in the nervous system is key to adjusting neuronal signaling during development and in maintaining the stability of established networks in the face of altered sensory input. To prevent the culmination of pathological recurrent network excitation or debilitating periods of quiescence, adaptive alterations occur in the signaling molecules and ion channels that control membrane excitability and synaptic transmission. However, rather than encoding (and thus “hardwiring”) modified gene copies, the nervous systems of metazoa have opted for expanding on post-transcriptional pre-mRNA splicing by altering key encoded amino acids using a conserved mechanism of A-to-I RNA editing: the enzymatic deamination of adenosine to inosine. Inosine exhibits similar base-pairing properties to guanosine with respect to tRNA codon recognition, replication by polymerases, and RNA secondary structure (i.e.,: forming-capacity). In addition to recoding within the open reading frame, adenosine deamination also occurs with high frequency throughout the non-coding transcriptome, where it affects multiple aspects of RNA metabolism and gene expression. Here, we describe the recoding function of key RNA editing targets in the mammalian central nervous system and their potential to be regulated. We will then discuss how interactions of A-to-I editing with gene expression and alternative splicing could play a wider role in regulating the neuronal transcriptome. Finally, we will highlight the increasing complexity of this multifaceted control hub by summarizing new findings from high-throughput studies.

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(3Cu)T and A(2Cu)C), 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(3Cu)C and A(2Cu)T). 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(3Cu)T and A(2Cu)C 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(3Cu)T and/or A(2Cu) C 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.

Binding effects of Mn2+ and Zn2+ ions on the vibrational properties of guanine-cytosine base pairs in the Watson-Crick and Hoogsteen configurations

The binding effects of Mn²⁺ and Zn²⁺ ions on the vibrational properties of guanine-cytosine base pairs have been performed using density functional theory investigations. The calculations were carried out on Watson-Crick and Hoogsteen configurations of the base pairs. We have found, that in Watson-Crick configuration, the metal is coordinated to N7 atom of guanine while, in the case of Hoogsteen configuration, the coordination is at N3 atom of guanine. We have pointed out the vibrational bands that can be used to detect the presence of metallic ions in the Watson-Crick and Hoogsteen structures. Our results show that the vibrational amplitudes of metallic atoms are strong for wavenumbers lower than 600 cm⁻¹. Also, we predict that the distinction between Watson-Crick and Hoogsteen configurations can be seen around 85, 170 and 310 cm⁻¹.

Thermochemical properties of some vinyl chloride-induced DNA lesions: detailed view from NBO & AIM analysis

Etheno-damaged DNA adducts such as 3,N 4-ethenocytosine, N 2,3-ethenoguanine, and 1,N 2-ethenoguanine are associated with carcinogenesis and cell death. These inevitable damages are counteracted by glycosylase enzymes, which cleave damaged nucleobases from DNA. Escherichia coli alkyl purine DNA glycosylase is the enzyme responsible for excising damaged etheno adducts from DNA in humans. In an effort to understand the intrinsic properties of these molecules, we examined gas-phase acidity values and proton affinities (PA) of multiple sites of these molecules as well as equilibrium tautomerization and base pairing properties by quantum mechanical calculations. We also used calculations to compare the acidic and basic properties of these etheno adduct with those of the normal bases—cytosine and guanine nucleobases. We hypothesize that alkyl DNA glycosylase may cleave certain damaged nucleobases as anions and that the active site may take advantage of a nonpolar environment to favor deprotonated cytosine or guanine as a leaving group versus damaged nucleobases.

Role of pK(a) of nucleobases in the origins of chemical evolution.

The formation of canonical base pairs through Watson–Crick hydrogen bonding sits at the heart of the genetic apparatus. The specificity of the base pairing of adenine with thymine/uracil and guanine with cytosine preserves accurate information for the biochemical blueprint and replicates the instructions necessary for carrying out biological function. The chemical evolution question of how these five canonical nucleobases were selected over various other possibilities remains intriguing. Since these and alternative nucleobases would have been available for chemical evolution, the reasons for the emergence of this system appear to be primarily functional.While investigating the base-pairing properties of structural nucleic acid analogs, we encountered a relationship between the pKa of a series of nonstandard (and canonical) nucleobases and the pH of the aqueous medium. This relationship appeared to correspond with the propensity of these molecules to self-assemble via Watson–Crick-type base-pairing interactions. A simple correlation of the “magnitude of the difference between the pKa and pH” (pKa–pH correlation) enables a general prediction of which types of heterocyclic recognition elements form hydrogen-bonded base pairs in aqueous media. Using the pKa–pH relationship, we can rationalize why nature chose the canonical nucleobases in terms of hydrophobic and hydrophilic interactions, and further extrapolate its significance within the context of chemical evolution.The connection between the physicochemical properties of bioorganic compounds and the interactions with their aqueous environment directly affects structure and function, at both a molecular and a supramolecular level. A general structure–function pattern emerges in biomolecules and biopolymers in aqueous media near neutral pH. A pKa – pH < 2 generally prompts catalytic functions, central to metabolism, but a difference in pKa – pH > 2 seems to result in the emergence of structure, central to replication. While this general trend is observed throughout extant biology, it could have also been an important factor in chemical evolution.

Quadracyclic Adenine: A Non-Perturbing Fluorescent Adenine Analogue

Fluorescent-base analogues (FBAs) comprise a group of increasingly important molecules for the investigation of nucleic acid structure and dynamics as well as of interactions between nucleic acids and other molecules. Here, we report on the synthesis, detailed spectroscopic characterisation and base-pairing properties of a new environment-sensitive fluorescent adenine analogue, quadracyclic adenine (qA). After developing an efficient route of synthesis for the phosphoramidite of qA it was incorporated into DNA in high yield by using standard solid-phase synthesis procedures. In DNA qA serves as an adenine analogue that preserves the B-form and, in contrast to most currently available FBAs, maintains or even increases the stability of the duplex. We demonstrate that, unlike fluorescent adenine analogues, such as the most commonly used one, 2-aminopurine, and the recently developed triazole adenine, qA shows highly specific base-pairing with thymine. Moreover, qA has an absorption band outside the absorption of the natural nucleobases (>300 nm) and can thus be selectively excited. Upon excitation the qA monomer displays a fluorescence quantum yield of 6.8 % with an emission maximum at 456 nm. More importantly, upon incorporation into DNA the fluorescence of qA is significantly less quenched than most FBAs. This results in quantum yields that in some sequences reach values that are up to fourfold higher than maximum values reported for 2-aminopurine. To facilitate future utilisation of qA in biochemical and biophysical studies we investigated its fluorescence properties in greater detail and resolved its absorption band outside the DNA absorption region into distinct transition dipole moments. In conclusion, the unique combination of properties of qA make it a promising alternative to current fluorescent adenine analogues for future detailed studies of nucleic acid-containing systems.