Canonical Watson-Crick Base Pairs Research Articles

Hidden Hoogsteens in the Data

Although extremely rare in the crystallographic database, Hoogsteen (HG) base pairs have been modeled, especially at duplex termini, protein-DNA interfaces, damaged DNA and DNA mismatches. Formation of HG base pairs in duplex DNA requires a 180 degree rotation along the glycosidic bond of the purine relative to the canonical Watson-Crick (WC) base-pair. In a recent survey of the PDB we identified 75 HS base pairs in 2,910 x-ray derived models. Recent evidence suggests that the prevalence of HG base pairs in nature to be greater than previously thought. Due to electron density density ambiguity at mid to low resolution, it is possible for bases to be modeled incorrectly. We have developed a high-throughput method which examines the difference density around DNA to identify mis-modeled bases. Here we show where bases were incorrectly modeled and our subsequent fixes which show pairs modeled as WC that really are HG, vice versa, and evidence of WC-HG alternates. This research shows that base pairing conformations in DNA are more heterogeneous than previously thought.

Crystal structure of metallo-DNA duplex containing T-Hg(II)-T base pairs

The DNA duplex containing mercury-mediated base pairs (T-Hg(II)-T) is an attractive biomacromolecular nanomaterials. In a recent study, it was confirmed that the Hg(II) ion significantly stabilizes a DNA duplex by binding selectively to a T-T mispair [1]. Based on the phenomenon observed, a DNA-based sensing system that selectively and sensitively detects Hg(II) ions in aqueous solution was developed [2]. In the present study, we have solved the first crystal structure of a B-form DNA duplex containing two consecutive T-Hg(II)-T base pairs [3]. The Hg(II) ion occupies the center between two T residues. The geometry of the T-Hg(II)-T base pair is very similar to that of the canonical Watson-Crick base pairs. The distance of N3-Hg(II) bond is 2.0 Å, suggesting that the N3 nitrogen releases an imino-proton even at neutral pH (pKa of N3 position of T is 9.8) and directly bonds to Hg(II). In the B-form DNA, the helical axis runs through the center of base pairs, and the Hg(II) ions are therefore aligned along the helical axis. The distance between the two neighboring Hg(II) ions is 3.3 Å. The relatively short Hg(II)-Hg(II) distance indicates that the metallophilic attraction could exit between them and may stabilize the B-form duplex. To support this, the DNA duplex is largely distorted and adopts an unusual non-helical conformation in the absence of Hg(II). In conclusion, the Hg(II) ion is essential for maintaining the B-form conformation of the DNA duplex containing T-T mispairs. The structure of the Hg(II)-DNA hybrid duplex itself and the Hg(II)-induced structural switching from the non-helical form to the B-form provide the basis for the structure-based design of metal-conjugated nucleic acid nanomaterials.

Transition-state destabilization reveals how human DNA polymerase β proceeds across the chemically unstable lesion N7-methylguanine.

N7-Methyl-2′-deoxyguanosine (m7dG) is the predominant lesion formed by methylating agents. A systematic investigation on the effect of m7dG on DNA replication has been difficult due to the chemical instability of m7dG. To gain insights into the m7dG effect, we employed a 2′-fluorine-mediated transition-state destabilzation strategy. Specifically, we determined kinetic parameters for dCTP insertion opposite a chemically stable m7dG analogue, 2′-fluoro-m7dG (Fm7dG), by human DNA polymerase β (polβ) and solved three X-ray structures of polβ in complex with the templating Fm7dG paired with incoming dCTP or dTTP analogues. The kinetic studies reveal that the templating Fm7dG slows polβ catalysis ∼300-fold, suggesting that m7dG in genomic DNA may impede replication by some DNA polymerases. The structural analysis reveals that Fm7dG forms a canonical Watson–Crick base pair with dCTP, but metal ion coordination is suboptimal for catalysis in the polβ-Fm7dG:dCTP complex, which partially explains the slow insertion of dCTP opposite Fm7dG by polβ. In addition, the polβ-Fm7dG:dTTP structure shows open protein conformations and staggered base pair conformations, indicating that N7-methylation of dG does not promote a promutagenic replication. Overall, the first systematic studies on the effect of m7dG on DNA replication reveal that polβ catalysis across m7dG is slow, yet highly accurate.

Role of the LEXE Motif of Protein-primed DNA Polymerases in the Interaction with the Incoming Nucleotide

The LEXE motif, conserved in eukaryotic type DNA polymerases, is placed close to the polymerization active site. Previous studies suggested that the second Glu was involved in binding a third noncatalytic ion in bacteriophage RB69 DNA polymerase. In the protein-primed DNA polymerase subgroup, the LEXE motif lacks the first Glu in most cases, but it has a conserved Phe/Trp and a Gly preceding that position. To ascertain the role of those residues, we have analyzed the behavior of mutants at the corresponding ϕ29 DNA polymerase residues Gly-481, Trp-483, Ala-484, and Glu-486. We show that mutations at Gly-481 and Trp-483 hamper insertion of the incoming dNTP in the presence of Mg(2+) ions, a reaction highly improved when Mn(2+) was used as metal activator. These results, together with previous crystallographic resolution of ϕ29 DNA polymerase ternary complex, allow us to infer that Gly-481 and Trp-483 could form a pocket that orients Val-250 to interact with the dNTP. Mutants at Glu-486 are also defective in polymerization and, as mutants at Gly-481 and Trp-483, in the pyrophosphorolytic activity with Mg(2+). Recovery of both reactions with Mn(2+) supports a role for Glu-486 in the interaction with the pyrophosphate moiety of the dNTP.

Global and Local Conformation of Mismatched Duplex DNA Upon MSH2-MSH6 Binding Studied by Steady-State and Time-Resolved Fluorescence

The DNA mismatch repair (MMR) system guards the integrity of genetic material by scanning and correcting errors in a post-replicative manner. In eukaryotic cells, the initiation of MMR is achieved by recognition of biosynthetic errors by Msh proteins (MutS homologs). The recognition of single base mismatches and small insertion/deletion loops (IDL) by the Msh2-Msh6 heterodimer is an important initial event in the MMR pathway. Several studies have suggested that the prokaryotic MutS homodimer and its eukaryotic counterpart Msh2-Msh6 bind duplex DNA containing a mismatch or IDL with a higher affinity than homoduplex DNA. However, the exact mechanism by which Msh2-Msh6 distinguishes different types of mismatched base pairs from a large excess of canonical Watson-Crick base pairs is still unknown. In this study, we are utilizing DNA duplexes containing 6-methylisoxanthopterin (6-MI) a fluorescent nucleoside analog to measure the binding affinity of S. cerevisiae Msh2-Msh6 to different single base pair mismatches. For greater sensitivity this analog is incorporated into a pentamer sequence ATFAA (F = 6-MI), which exhibits enhanced fluorescence. Fluorescence anisotropy measurements performed with these intrinsically labeled duplexes reveal the following order for Msh2-Msh6 binding affinity to DNA mismatches: G:T > +T ≈ G:A > G:C. This order is consistent with previously reported affinities measured in the gel. We have further investigated DNA dynamics upon Msh2-Msh6 binding using time-resolved fluorescence spectroscopy. Specific placement of the probe at the mismatch site or adjacent to it reveals significant local motion prior to protein binding. We observe that those mismatches with the highest affinity exhibit the greatest amount of motion. Protein binding stabilizes mismatch local motion, which is potentially consistent with Phe intercalation at the site, as observed in Msh2-Msh6-DNA co-crystal structures.

Base‐Pairing Behavior of a Carbocyclic Janus‐AT Nucleoside Analogue Capable of Recognizing A and T within a DNA Duplex

Janus-type nucleosides are heterocycles with two faces, each of which is designed to complement the H-bonding interactions of natural nucleosides comprising a canonical Watson-Crick base pair. By intercepting all of the hydrogen bonds contained within the base pair, oligomeric Janus nucleosides are expected to achieve sequence-specific DNA recognition through the formation of J-loops that will be more stable than D-loops, which simply replaces one base-pair with another. Herein, we report the synthesis of a novel Janus-AT nucleoside analogue, JAT , affixed on a carbocyclic analogue of deoxyribose that was converted to the corresponding phosphoramidite. A single JAT was successfully incorporated into a DNA strand by solid phase for targeting both A and T bases, and characterized through biophysical and computational methods. Experimental UV-melting and circular dichroism data demonstrated that within the context of a standard duplex, JAT associates preferentially with T over A, and much more poorly with C and G. Density functional theory calculations confirm that the JAT structure is well suited to associate only with A and T thereby highlighting the importance of the electronic structure in terms of H-bonding. Finally, molecular dynamics simulations validated the observation that JAT can substitute more effectively as an A-analogue than as a T-analogue without substantial distortion of the B-helix. Overall, this new Janus nucleotide is a promising tool for the targeting of A-T base pairs in DNA, and will lead to the development of oligo-Janus-nucleotide strands for sequence-specific DNA recognition.

NMR characterisation of a highly conserved secondary structural RNA motif of Halobacterium halobium 23S rRNA

The highly conserved 29-mer RNA motif corresponding to the peptidyl transferase central circle region of the domain V of Halobacterium halobium 23S rRNA has been characterised by multidimensional NMR spectroscopy. The NMR structure has a good all atom average RMSD of 1.28 Å and a stable A-form helical conformation. The NMR structure differs from the X-ray crystal structure of an analogous motif, contained within the Escherichia coli ribosome, as none of the bases are flipped out and a number of non-canonical base pairs are formed in the solution structure. Thus in the observed NMR structure, the predicted A7 to U30 base pair is not seen and a non-canonical U6 to U30 base pair was formed in its place. Similarly the predicted A9 to U26 base pair was also not observed and another non-canonical A9 to A27 base pair was formed. It was also seen from the conformational analysis that the steps near the bulges had the greatest deviation from the canonical Watson-Crick base pair step parameters. Despite these differences, the 29-mer structure provides a working model of the RNA within the ribosome in a more natural solution state than that observed in the intact ribosome crystal structures, particularly around the A27 residue. The NMR structure determination of the 29-mer RNA motif provides a solid foundation for determining the NMR structure of the RNA-amicetin complex in the next step. To extend the above study, a fully (13)C and (15)N isotopically labelled 37-mer RNA version of the Halobacterium halobium RNA sample has been characterised using ultra high field 1 GHz spectroscopy. The results have been used to demonstrate the advantages conferred by the use of a 1 GHz spectrometer frequency over 800 MHz in terms of superior sensitivity and greater spectral dispersion achieved in the spectrum of the RNA.

Global and Local Conformational Studies of Mismatched Duplex DNA Upon Msh2-Msh6 Binding by Steady-State and Time-Resolved Fluorescence

The DNA mismatch repair (MMR) system guards the integrity of genetic material by scanning and correcting errors in a post-replicative manner and thus improves the fidelity of DNA replication by several orders of magnitude. In eukaryotic cells, the initiation of MMR is achieved by recognition of biosynthetic errors by Msh proteins (MutS homologs). Single base mismatches and small insertion/deletion loops (IDL) are recognized by the Msh2-Msh6 heterodimer followed by recruitment of MutL homologs (Mlh/Pms) and this ATP-dependent ternary complex further activates downstream MMR events. The exact mechanism by which Msh2-Msh6 distinguishes a mismatched base pair precisely from a large excess of canonical Watson-Crick base pairs in an efficient manner is still unknown. In this study we explore the specificity of Msh2-Msh6 binding that enables discrimination between different mismatches through measurement of binding affinity and protein-induced bending using fluorescence anisotropy, gel mobility shift assays and Forster Resonance Energy Transfer (FRET). These measurements yield the following order of binding affinity: G:T > +T > G:A > G:C and are in good agreement with previous results. The FRET efficiency of free and Msh2-Msh6-bound mismatched duplex DNA suggests that Msh2-Msh6 bends both +T and G:T duplex DNA relative to G:C homoduplex DNA, which appears to remain relatively straight in the bound form. We have also monitored local DNA base pair dynamics with time-resolved fluorescence intensity and anisotropy spectroscopy measurements. By using a fluorescent nucleoside analog, 6-methylisoxanthopterin incorporated into mismatched DNA, we can explicitly investigate single base pair dynamics. These experiments reveal that protein binding stabilizes the probe placed at the mismatch or located adjacent to the unpaired thymine. This stabilization is relatively local to the mismatch site and is not propagated down the helix.

Structural Variations of Single and Tandem Mismatches in RNA Duplexes: A Joint MD Simulation and Crystal Structure Database Analysis

Internal loops within RNA duplex regions are formed by single or tandem basepairing mismatches with flanking canonical Watson-Crick basepairs on both sides. They are the most common motif observed in RNA secondary structures and play integral functional and structural roles. In this report, we have studied the structural features of 1 × 1, 2 × 2, and 3 × 3 internal loops using all-atom molecular dynamics (MD) simulation technique with explicit solvent model. As MD simulation is intricately dependent on the choice of force-field and these are often rather approximate, we have used both the most popular force-fields for nucleic acids-CHARMM27 and AMBER94-for a comparative analysis. We find that tandem noncanonical basepairs forming 2 × 2 and 3 × 3 internal loops are considerably more stable than the single mismatches forming 1 × 1 internal loops, irrespective of the force field. We have also analyzed crystal structure database to study the conservation of these helical fragments in the corresponding sets of RNA structures. We observe that the nature of stability in MD simulations mimic their fluctuating natures in crystal data sets also, probably indicating reliable natures of both the force fields to reproduce experimental results. We also notice significant structural changes in the wobble G:U basepairs present in these double helical stretches, leading to a biphasic stability for these wobble pairs to release the deformational strains introduced by internal loops within duplex regions.

Steric Restrictions of RISC in RNA Interference Identified with Size-Expanded RNA Nucleobases

Understanding the interactions between small interfering RNAs (siRNAs) and the RNA-induced silencing complex (RISC), the key protein complex of RNA interference (RNAi), is of great importance to the development of siRNAs with improved biological and potentially therapeutic function. Although various chemically modified siRNAs have been reported, relatively few studies with modified nucleobases exist. Here we describe the synthesis and hybridization properties of siRNAs bearing size-expanded RNA (xRNA) nucleobases and their use as a novel and systematic set of steric probes in RNAi. xRNA nucleobases are expanded by 2.4 Å using benzo-homologation and retain canonical Watson-Crick base-pairing groups. Our data show that the modified siRNA duplexes display small changes in melting temperature (+1.4 to -5.0 °C); substitutions near the center are somewhat destabilizing to the RNA duplex, while substitutions near the ends are stabilizing. RNAi studies in a dual-reporter luciferase assay in HeLa cells revealed that xRNA nucleobases in the antisense strand reduce activity at some central positions near the seed region but are generally well tolerated near the ends. Most importantly, we observed that xRNA substitutions near the 3'-end increased activity over that of wild-type siRNAs. The data are analyzed in terms of site-dependent steric effects in RISC. Circular dichroism experiments show that single xRNA substitutions do not significantly distort the native A-form helical structure of the siRNA duplex, and serum stability studies demonstrated that xRNA substitutions protect siRNAs against nuclease degradation.

Interactions of Metal Ions with DNA and Some Applications

Nucleic acids play a critical role in life as we know it. It contains the necessary information required for the structure and function of a living organisms. Metal ions play a critical role in stabilizing conformations. In the well-known double helix structure of DNA, metal ions stabilize a particular conformation that ensures storage and propagation of genetic information. Metal ions, however, can interact with various sites on nucleic acids. Moreover, metal coordination can have a tremendous impact on the structure, conformation, stability and the electronic properties of the nucleic acids. The interactions are controlled by the relative affinity of metal ion coordination to the negatively charged phosphodiester backbone versus binding to other donor sites located in the nucleobases. The canonical Watson–Crick base pairs (A-T and G-C) as well as non-canonical base pairs (Hoogsteen and wobble) and mismatched pairs are often sites for metal ion interactions. In this review, an overview will be provided of the structure of different forms of nucleic acids (DNA and RNA) and the impact of different metal ions on their stability and structure. In addition, the recent applications of metal-DNA interactions in nanotechnology, biosensor and bioelectronics will also be discussed along with some therapeutic applications of metal complexes.

Expansion of the Genetic Alphabet: Unnatural Nucleobases and Their Applications

Nucleic acids are essential biomolecules that encode all of the information necessary for life. Specific pairing of A with T (or U) and C with G during replication, transcription, and translation is the key to effective transmission of genetic information between generations, as well as accurate conversion of genetic information into protein sequence. Given the magnitude of the tasks orchestrated by the Watson-Crick base pairing, it is striking to consider that biological systems accomplish these tasks using only four nucleobases. Realizing the powerful nature of base-pair recognition, researchers have been inspired to ask the question of whether the genetic code can be artificially expanded to generate biological systems having novel functions. It was this question that led Alex Rich in 1962 to propose the concept of orthogonal base pairing between iso-G and iso-C and inspired Professor Steven A. Benner in the late 1980s to expand the genetic alphabet from four to six letters. Benner's early research focused on the development of new base pairs having hydrogen bonding patterns orthogonal to those in the canonical Watson-Crick base pairs. In 1994, Professor Eric T. Kool opened a new functional dimension with the creation of nonhydrogen bonding unnatural nucleobase surrogates. Expansion of the genetic alphabet has dramatically increased the functional potential of DNA, for example, by enabling site-directed oligonucleotide labeling and in vitro selections with oligonucleotides having increased chemical diversity. Translation of an expanded DNA alphabet into RNA is a challenging task, but one which has potential to give rise to semisynthetic organisms with increased biodiversity. This special issue highlights recent accomplishments at the interface of organic chemistry and molecular biology which hold promise to further expand the potential of nucleic acids having unnatural nucleobases. Specifically, the reports in this special issue focus on the synthesis of unnatural nucleobases and nucleic acid backbones, the exploration of their structure and duplex stabilizing ability, and the polymerase mediated replication and transcription of DNA containing unnatural nucleobases. T. Lonnberg and a coworker report the synthesis and study of a bis(pyrazolyl)purine ribonucleoside having increased hydrophobic surface area and the ability to form complex with metal ions. The hydrogen bonding pattern of this nucleoside makes it complementary to thymine and uridine. The authors demonstrate that the bis(pyrazolyl) nucleobase is capable of forming a Pd2+-mediated base pair with uridine in the monomeric state. When incorporated into an oligonucleotide, the bis(pyrazolyl) nucleobase stabilizes DNA duplexes when paired with thymine, but this stabilization appears to result from increased π-stacking interactions rather than metal complexation. These studies open the door to applications using unnatural nucleobases to increase the binding affinity of probes and therapeutics targeted at native DNA and RNA. Much effort has focused on the incorporation of unnatural nucleobases into native DNA and RNA, and the availability of nonnative backbones such as LNA, PNA, and GNA opens the door for further expansion of nucleic acid structure and function. C. Forster and coworkers evaluate the structural properties of “all LNA” duplexes and demonstrate that these duplexes have a relaxed helical structure unique from that of DNA and RNA. J. M. Heemstra and coworkers report an expedient synthetic route to the Fmoc-protected PNA backbone, which serves as a key intermediate in the synthesis of PNA monomers having unnatural nucleobases. Together, these studies facilitate the exploration of nucleic acids having both nonnative backbones and unnatural nucleobases, which is in turn anticipated to provide molecules having novel structural and functional properties. The ability to use native or engineered replication and transcription machinery with nucleic acids containing unnatural nucleobases is critical to many in vitro and in vivo applications. I. Hirao and coworkers describe a two-unnatural-base-pair system capable of incorporating unnatural nucleobases into DNA via polymerase chain reaction, then into RNA via T7 transcription. Using this system, they demonstrate sequence-specific incorporation of a biotinylated nucleobase into a 260-mer RNA sequence. Generating modified RNA of this length is not feasible using purely synthetic methods; thus this work significantly improves access to site-specifically labeled RNA sequences. Additionally, P. J. Beuning and a coworker review the use of native A and Y family DNA polymerases for replication of DNA containing unnatural nucleobases. This review serves as an excellent resource for researchers seeking to utilize unnatural nucleobases and provides significant insight into how polymerases deal with both synthetic and damaged DNA nucleobases. Collectively, these reports demonstrate the potential of unnatural nucleobases for applications in chemistry and biology and offer valuable tools for further exploring nucleic acid structural diversity.

Theoretical investigation on DNA/RNA base pairs mediated by copper, silver, and gold cations

B3LYP density functional based computations were performed in order to characterize the interactions present in some Cu(+), Ag(+), and Au(+) metal ion-mediated DNA and RNA base pairs from both structural and electronic points of view. Examined systems involve as ligands canonical Watson-Crick, Hoogsteen and Wobble base pairs. Two artificial Hoogsteen base pairs were also taken into account. Binding energy values indicate that complexes involving silver cations are less stable than those in which copper or gold are present, and propose a similar behaviour for these two latter ions. The nature of the bond linking metal ions and bases was described by the NBO analysis that suggests metal coordinative interactions to be covalent. An evaluation of the dispersion contributions for the investigated systems was performed with the B3LYP-D3 functional.

Anharmonic vibrations of nucleobases: Structural basis of one- and two-dimensional infrared spectra for canonical and mismatched base pairs

Canonical Watson-Crick base pairs and four representative mismatched base pairs have been studied by quantum chemical computations. Detailed anharmonic vibrational analysis was carried out to reveal some vibrational signatures characteristic of structural aspects of the base monomers and dimers, which were well manifested in simulated 1D IR and 2D IR spectra. The degree of delocalization of the selected normal modes, represented by the potential energy distribution, was found to vary significantly from isolated bases to H-bonded dimers, and was accompanied by changes in anharmonicities of these modes. Examples are given for the generally accepted carbonyl stretching mode of base pairs appearing in the 6-μm wavelength region of IR spectra.

Mechanistic Studies of Hairpin to Duplex Conversion for Trinucleotide Repeat Sequences

The expansion of a trinucleotide repeat sequence, such as CAG/CTG, has been pinpointed as the molecular basis for a number of neurodegenerative disorders. It has been proposed that as part of the expansion process, these repetitive sequences adopt non-B conformations such as hairpins. However, the prevalence of these hairpins and their contributions to the DNA expansion have not been well defined. In this work, we utilized a molecular beacon strategy to examine the stability of the (CAG)(10) hairpin and also its behavior in the presence of the complementary (CTG)(10) hairpin. We find that the two hairpins represent kinetically trapped species that can coexist but irreversibly convert to duplex upon thermal induction. Furthermore, as monitored by fluorescence and optical analysis, modifications to the base composition of either the loop or stem region have a profound effect on the ability of the trinucleotide repeat hairpins to convert to duplex. Additionally, the rate of duplex formation is also reduced with these loop and stem-modified hairpins. These results demonstrate that the trinucleotide repeat hairpins can convert to duplex via two independent mechanisms as follows: the loop-loop interactions found in kissing hairpins or the stem-stem interactions of a cruciform.

The Acyclic 2,4-Diaminopyrimidine Nucleoside Phosphonate Acts as a Purine Mimetic in HIV-1 Reverse Transcriptase DNA Polymerization

The acyclic pyrimidine nucleoside phosphonate (ANP) phosphonylmethoxyethoxydiaminopyrimidine (PMEO-DAPym) differs from other ANPs in that the aliphatic alkyloxy linker is bound to the C-6 of the 2,4-diaminopyrimidine base through an ether bond, instead of the traditional alkyl linkage to the N-1 or N-9 of the pyrimidine or purine base. In this study, we have analyzed the molecular interactions between PMEO-DAPym-diphosphate (PMEO-DAPym-pp) and the active sites of wild-type (WT) and drug-resistant HIV-1 reverse transcriptase (RT). Pre-steady-state kinetic analyses revealed that PMEO-DAPym-pp is a good substrate for WT HIV-1 RT: its catalytic efficiency of incorporation (k(pol)/K(d)) is only 2- to 3-fold less than that of the corresponding prototype purine nucleotide analogs PMEA-pp or (R)PMPA-pp. HIV-1 RT recognizes PMEO-DAPym-pp as a purine base instead of a pyrimidine base and incorporates it opposite to thymine (in DNA) or uracil (in RNA). Molecular modeling demonstrates that PMEO-DAPym-pp fits into the active site of HIV-1 RT without significant perturbation of key amino acid residues and mimics an open incomplete purine ring that allows the canonical Watson-Crick base pairing to be maintained. PMEO-DAPym-pp is incorporated more efficiently than (R)PMPA-pp by mutant K65R HIV-1 RT and is not as efficiently excised as (R)PMPA by HIV-1 RT containing thymidine analog mutations. Overall, the data revealed that PMEO- DAPym represents the prototype compound of a novel class of pyrimidine acyclic nucleoside phosphonates that are recognized as a purine nucleotide and should form the rational basis for the design and development of novel purine nucleo(s)(t)ide mimetics as potential antiviral or antimetabolic agents.

Base-pairing mediated non-covalent polymers

The naturally occurring nucleic acid bases (nucleobases) adenine, thymine (uracil), guanine, and cytosine are widely appreciated for their ability to stabilize canonical Watson-Crick base-pairing motifs, as well as a number of other well-characterized arrangements, such as Hoogsteen and wobble heterodimers, and a variety of homodimers. In this tutorial review, the use of these kinds of interactions to form synthetic polymeric and oligomeric ensembles is summarized. Particular emphasis will be placed on synthetic analogues of guanine that stabilize the formation of well-defined higher order aggregates, as well as de novo polymeric systems whose properties are modulated by the presence of nucleobase derivatives incorporated within or attached to the chain-defining backbone. In both cases, nucleobase-nucleobase interactions serve to underlie the chemistry, establish the structural morphology, and enable the development of bioinspired, environmentally responsive materials.

Direct detection of N−H⋯N hydrogen bonds in biomolecules by NMR spectroscopy

A nuclear magnetic resonance (NMR) experiment is described for the direct detection of N-H[...]N hydrogen bonds (H-bonds) in 15N isotope-labeled biomolecules. This quantitative HNN-COSY (correlation spectroscopy) experiment detects and quantifies electron-mediated scalar couplings across the H-bond (H-bond scalar couplings), which connect magnetically active (15)N nuclei of the H-bond donor and acceptor. Detectable H-bonds comprise the imino H-bonds in canonical Watson-Crick base pairs, many H-bonds in unusual nucleic acid base pairs and H-bonds between protein backbone or side-chain N-H donor and N acceptor moieties. Unlike other NMR observables, which provide only indirect evidence of the presence of H-bonds, the H-bond scalar couplings identify all partners of the H-bond, the donor, the donor proton and the acceptor in a single experiment. The size of the scalar couplings can be related to H-bond geometries and as a time average to H-bond dynamics. The time required to detect the H-bonds is typically less than 1 d at millimolar concentrations for samples of molecular weight < or = approximately 25 kDa. A C15N/13C-labeled potato spindle tuber viroid T1 RNA domain is used as an example to illustrate this procedure.

Purine sensing by riboswitches

Structured mRNA elements called riboswitches control gene expression by binding to small metabolites. Over a dozen riboswitch classes have been characterized that target a broad range of molecules and vary widely in size and secondary structure. Four of the known riboswitch classes recognize purines or modified purines. Three of these classes are closely related in conserved sequence and secondary structure, but members of these classes selectively recognize guanine, adenine or 2'-deoxyguanosine. Members of the fourth riboswitch class adopt a distinct structure to form a selective binding pocket for the guanine analogue preQ(1) (7-aminomethyl-7-deazaguanine). All four classes of purine-sensing riboswitches are most likely to recognize their respective metabolites by utilizing a riboswitch residue to make a canonical Watson-Crick base-pair with the ligand. This review will provide a summary of the purine-sensing riboswitches, as well as discuss the complex functions and applications of these RNAs.

DNA–Metal Base Pairs

Recent developments show encouraging results for the use of DNA as a construction material for nanometer-sized objects. Today, however, DNA-based molecular nanoarchitectures are constructed with mainly unmodified or at best end-modified oligonucleotides, thus shifting the development of functionalized DNA structures into the limelight. One of most recent developments in this direction is the substitution of the canonical Watson-Crick base pairs by metal complexes. In this way "metal-base pairs" are created, which could potentially impart magnetic or conductive properties to DNA-based nanostructures. This review summarizes research which started almost 45 years ago with the investigation of how metal ions interact with unmodified DNA and which recently culminated in the development of artificial ligand-like nucleobases so far able to coordinate up to ten metal ions inside a single DNA duplex in a programmable fashion.