Base-pairing Properties Research Articles

Base pairing involving artificial bases in vitro and in vivo.

Herein we report the synthesis of N8-glycosylated 8-aza-deoxyguanosine (N8-8-aza-dG) and 8-aza-9-deaza-deoxyguanosine (N8-8-aza-9-deaza-dG) nucleotides and their base pairing properties with 5-methyl-isocytosine (d-isoCMe), 8-amino-deoxyinosine (8-NH2-dI), 1-N-methyl-8-amino-deoxyinosine (1-Me-8-NH2-dI), 7,8-dihydro-8-oxo-deoxyinosine (8-Oxo-dI), 7,8-dihydro-8-oxo-deoxyadenosine (8-Oxo-dA), and 7,8-dihydro-8-oxo-deoxyguanosine (8-Oxo-dG), in comparison with the d-isoCMe:d-isoG artificial genetic system. As demonstrated by Tm measurements, the N8-8-aza-dG:d-isoCMe base pair formed less stable duplexes as the C:G and d-isoCMe:d-isoG pairs. Incorporation of 8-NH2-dI versus the N8-8-aza-dG nucleoside resulted in a greater reduction in Tm stability, compared to d-isoCMe:d-isoG. Insertion of the methyl group at the N1 position of 8-NH2-dI did not affect duplex stability with N8-8-aza-dG, thus suggesting that the base paring takes place through Hoogsteen base pairing. The cellular interpretation of the nucleosides was studied, whereby a lack of recognition or mispairing of the incorporated nucleotides with the canonical DNA bases indicated the extent of orthogonality in vivo. The most biologically orthogonal nucleosides identified included the 8-amino-deoxyinosines (1-Me-8-NH2-dI and 8-NH2-dI) and N8-8-aza-9-deaza-dG. The 8-oxo modifications mimic oxidative damage ahead of cancer development, and the impact of the MutM mediated recognition of these 8-oxo-deoxynucleosides was studied, finding no significant impact in their in vivo assay.

Evaluating Hydrogen Bonds and Base Stacking of Single, Tandem and Terminal GU Mismatches in RNA with a Mesoscopic Model.

Guanine-Uracil (GU) mismatches are crucial to the stability of the RNA double helix and need to be considered in RNA folding algorithms for numerous biotechnological applications. Yet despite its importance, many aspects of GU base pairs are still poorly understood. There is also a lack of parametrization which prevents it to be considered in mesoscopic models. Here, we adapted the mesoscopic Peyrard-Bishop model to deal with context-dependent hydrogen bonds of GU mismatches and calculated the model parameters related to hydrogen bonding and base stacking from available experimental melting temperatures. The context-dependence causes a proliferation of parameters which made the problem computationally very demanding. We were able to overcome this problem by systematically regrouping the parameters during the minimization procedure. Our results not only provide the much needed parametrization but also answer several questions about the general properties of GU base pairs, as they can be associated straightforwardly to hydrogen bonding and base stacking. In particular, we found a very small Morse potential for tandem 5'-GU-3', which confirms a single hydrogen bond for this configuration, answering a long-standing question over conflicting experimental findings. Terminal GU base pairs are known to increase the duplex stability, but it is not clear why. Our results suggest that the increased terminal stability is mostly due to stronger hydrogen bonding.

Nucleosides with Transposed Base or 4'-Hydroxymethyl Moieties and Their Corresponding Oligonucleotides.

This review focuses on 4'-hydroxymethyl- or nucleobase-transposed nucleosides, nucleotides, and nucleoside phosphonates, their stereoisomers, and their close analogues. The biological activities of all known 4'-hydroxymethyl- or nucleobase-transposed nucleosides, nucleotides, and nucleoside phosphonates as potential antiviral or anticancer agents are compiled. The routes that have been taken for the chemical synthesis of such nucleoside derivatives are described, with special attention to the innovative strategies. The enzymatic synthesis, base-pairing properties, structure, and stability of oligonucleotides containing nucleobase- or 4'-hydroxymethyl-transposed nucleotides are discussed. The use of oligonucleotides containing nucleobase- or 4'-hydroxymethyl-transposed nucleotides as small oligonucleotide (e.g., human immunodeficiency virus integrase) inhibitors, in applications such as antisense therapy, silencing RNA (siRNA), or aptamer selections, is detailed.

To edit or not to edit: regulation of ADAR editing specificity and efficiency.

Hundreds to millions of adenosine (A)-to-inosine (I) modifications are present in eukaryotic transcriptomes and play an essential role in the creation of proteomic and phenotypic diversity. As adenosine and inosine have different base-pairing properties, the functional consequences of these modifications or 'edits' include altering coding potential, splicing, and miRNA-mediated gene silencing of transcripts. However, rather than serving as a static control of gene expression, A-to-I editing provides a means to dynamically rewire the genetic code during development and in a cell-type specific manner. Interestingly, during normal development, in specific cells, and in both neuropathological diseases and cancers, the extent of RNA editing does not directly correlate with levels of the substrate mRNA or the adenosine deaminase that act on RNA (ADAR) editing enzymes, implying that cellular factors are required for spatiotemporal regulation of A-to-I editing. The factors that affect the specificity and extent of ADAR activity have been thoroughly dissected in vitro. Yet, we still lack a complete understanding of how specific ADAR family members can selectively deaminate certain adenosines while others cannot. Additionally, in the cellular environment, ADAR specificity and editing efficiency is likely to be influenced by cellular factors, which is currently an area of intense investigation. Data from many groups have suggested two main mechanisms for controlling A-to-I editing in the cell: (1) regulating ADAR accessibility to target RNAs and (2) protein-protein interactions that directly alter ADAR enzymatic activity. Recent studies suggest cis- and trans-acting RNA elements, heterodimerization and RNA-binding proteins play important roles in regulating RNA editing levels in vivo. WIREs RNA 2016, 7:113-127. doi: 10.1002/wrna.1319.

New Insights into the Biological Role of Mammalian ADARs; the RNA Editing Proteins.

The ADAR proteins deaminate adenosine to inosine in double-stranded RNA which is one of the most abundant modifications present in mammalian RNA. Inosine can have a profound effect on the RNAs that are edited, not only changing the base-pairing properties, but can also result in recoding, as inosine behaves as if it were guanosine. In mammals there are three ADAR proteins and two ADAR-related proteins (ADAD) expressed. All have a very similar modular structure; however, both their expression and biological function differ significantly. Only two of the ADAR proteins have enzymatic activity. However, both ADAR and ADAD proteins possess the ability to bind double-strand RNA. Mutations in ADARs have been associated with many diseases ranging from cancer, innate immunity to neurological disorders. Here, we will discuss in detail the domain structure of mammalian ADARs, the effects of RNA editing, and the role of ADARs in human diseases.

Calorimetric and Spectroscopic Analysis of the Thermal Stability of Short Duplex DNA-Containing Sugar and Base-Modified Nucleotides.

Base- and sugar-modified analogs of DNA and RNA are finding ever expanding use in medicine and biotechnology as tools to better tailor structured oligonucleotides by altering their thermal stability, nuclease resistance, base-pairing specificity, antisense activity, or cellular uptake. Proper deployment of these chemical modifications generally requires knowledge of how each affects base-pairing properties and thermal stabilities. Here, we describe in detail how differential scanning calorimetry and UV spectroscopy may be used to quantify the melting thermodynamics of short dsDNA containing chemically modified nucleosides in one or both strands. Insights are provided into why and how the presence of highly stable base pairs containing modified nucleosides can alter the nature of calorimetry or melting spectroscopy data, and how each experiment must therefore be conducted to ensure high-quality melting thermodynamics data are obtained. Strengths and weaknesses of the two methods when applied to chemically modified duplexes are also addressed.

Bifacial Base-Pairing Behaviors of 5-Hydroxyuracil DNA Bases through Hydrogen Bonding and Metal Coordination.

A novel bifacial ligand-bearing nucleobase, 5-hydroxyuracil (U(OH) ), which forms both a hydrogen-bonded base pair (U(OH) -A) and a metal-mediated base pair (U(OH) -M-U(OH) ) has been developed. The U(OH) -M-U(OH) base pairs were quantitatively formed in the presence of lanthanide ions such as Gd(III) when U(OH) -U(OH) pairs were consecutively incorporated into DNA duplexes. This result established metal-assisted duplex stabilization as well as DNA-templated assembly of lanthanide ions. Notably, a duplex possessing U(OH) -A base pairs was destabilized by addition of Gd(III) ions. This observation suggests that the hybridization behaviors of the U(OH) -containing DNA strands are altered by metal complexation. Thus, the U(OH) nucleobase with a bifacial base-pairing property holds great promise as a component for metal-responsive DNA materials.

In vivo detection and replication studies of α-anomeric lesions of 2'-deoxyribonucleosides.

DNA damage, arising from endogenous metabolism or exposure to environmental agents, may perturb the transmission of genetic information by blocking DNA replication and/or inducing mutations, which contribute to the development of cancer and likely other human diseases. Hydroxyl radical attack on the C1′, C3′ and C4′ of 2-deoxyribose can give rise to epimeric 2-deoxyribose lesions, for which the in vivo occurrence and biological consequences remain largely unexplored. Through independent chemical syntheses of all three epimeric lesions of 2′-deoxyguanosine (dG) and liquid chromatography-tandem mass spectrometry analysis, we demonstrated unambiguously the presence of substantial levels of the α-anomer of dG (α-dG) in calf thymus DNA and in DNA isolated from mouse pancreatic tissues. We further assessed quantitatively the impact of all four α-dN lesions on DNA replication in Escherichia coli by employing a shuttle-vector method. We found that, without SOS induction, all α-dN lesions except α-dA strongly blocked DNA replication and, while replication across α-dA was error-free, replicative bypass of α-dC and α-dG yielded mainly C→A and G→A mutations. In addition, SOS induction could lead to markedly elevated bypass efficiencies for the four α-dN lesions, abolished the G→A mutation for α-dG, pronouncedly reduced the C→A mutation for α-dC and triggered T→A mutation for α-dT. The preferential misincorporation of dTMP opposite the α-dNs could be attributed to the unique base-pairing properties of the nucleobases elicited by the inversion of the configuration of the N-glycosidic linkage. Our results also revealed that Pol V played a major role in bypassing α-dC, α-dG and α-dT in vivo. The abundance of α-dG in mammalian tissue and the impact of the α-dNs on DNA replication demonstrate for the first time the biological significance of this family of DNA lesions.

Synthesis of 5-N-tert-butylaminoxylcytidine and EPR studies on its base pairing properties

5-N-tert-Butylaminoxylcytidine was synthesized as a new spin-labelled cytidine to detect guanosine by EPR spectroscopy. Metal-halogen exchange reaction of TBS-protected N-benzoyl-5-iodocytidine followed by a reaction with 2-methyl-2-nitrosopropane afforded 5-N-tert-butylhydroxylaminocytidine with considerable affinity for guanosine and its oxidation gave 5-N-tert-butylaminoxylcytidine. Changes in EPR parameters monitored its selective base pairing with guanosine derivatives and the formation of a 2:2 complex with 8-oxoguanosine.

How the Intercalation of Phenanthroline Affects the Structure, Energetics, and Bond Properties of DNA Base Pairs: Theoretical Study Applied to Adenine–Thymine and Guanine–Cytosine Tetramers

The effects of phenanthroline (phen) intercalation on the structure, energetics, and bonding of adenine-thymine and guanine-cytosine tetramers (A-T/T-A and G-C/C-G) were studied through density functional theory (DFT) using functionals that were recently improved to consider the effect of dispersion forces. Our results given by energy decomposition analysis show that the dispersion contribution, ΔEdisp, is the most important contribution to the interaction energy, ΔEint. However, it is not enough to compensate the Pauli repulsion term, ΔEPauli, and the roles of the orbital contribution, ΔEorb, and, in particular, the electrostatic contribution, ΔEelstat, become crucial for the stabilization of the structures in the intercalation process. On the other hand, for G-C/C-G systems, hydrogen-bonding (HB) interactions are more important than stacking (S) interactions, whereas for A-T/T-A systems, HB and S become competitive. Moreover, intercalation produces important changes not only in the hydrogen bonds of base pairs, because S and HB are deeply connected, but also in other characteristic geometric parameters of the base pairs.

DNA Duplex Stability of the Thio-Iso-Guanine•Methyl-Iso-Cytosine Base Pair

We report the synthesis, incorporation into oligonucleotides, and base-pairing properties of the 2-thio-variant of iso-guanine. Iso-guanine is the purine component of a nonstandard base pair with 5-methyl-iso-cytosine. The 2-thio-iso-guanine•5-methyl-iso-cytosine base pair is found to have similar stability to an adenine•thymine pair.

Intermolecular 'cross-torque': the N4-cytosine propargyl residue is rotated to the 'CH'-edge as a result of Watson-Crick interaction

Propargyl groups are attractive functional groups for labeling purposes, as they allow CuAAC-mediated bioconjugation. Their size minimally exceeds that of a methyl group, the latter being frequent in natural nucleotide modifications. To understand under which circumstances propargyl-containing oligodeoxynucleotides preserve base pairing, we focused on the exocyclic amine of cytidine. Residues attached to the exocyclic N4 may orient away from or toward the Watson–Crick face, ensuing dramatic alteration of base pairing properties. ROESY-NMR experiments suggest a uniform orientation toward the Watson–Crick face of N4-propargyl residues in derivatives of both deoxycytidine and 5-methyl-deoxycytidine. In oligodeoxynucleotides, however, UV-melting indicated that N4-propargyl-deoxycytidine undergoes standard base pairing. This implies a rotation of the propargyl moiety toward the ‘CH’-edge as a result of base pairing on the Watson–Crick face. In oligonucleotides containing the corresponding 5-methyl-deoxycytidine derivative, dramatically reduced melting temperatures indicate impaired Watson–Crick base pairing. This was attributed to a steric clash of the propargyl moiety with the 5-methyl group, which prevents back rotation to the ‘CH’-edge, consequently preventing Watson–Crick geometry. Our results emphasize the tendency of an opposing nucleic acid strand to mechanically rotate single N4-substituents to make way for Watson–Crick base pairing, providing no steric hindrance is present on the ‘CH’-edge.

1',5'-Anhydro-L-ribo-hexitol Adenine Nucleic Acids (α-L-HNA-A): Synthesis and Chiral Selection Properties in the Mirror Image World.

The synthesis and a preliminary investigation of the base pairing properties of (6' → 4')-linked 1',5'-anhydro-L-ribo-hexitol nucleic acids (α-L-HNA) have herein been reported through the study of a model oligoadenylate system in the mirror image world. Despite its considerable preorganization due to the rigidity of the "all equatorial" pyranyl sugar backbone, α-L-HNA represents a versatile informational biopolymer, in view of its capability to cross-communicate with natural and unnatural complements in both enantiomeric forms. This seems the result of an inherent flexibility of the oligonucleotide system, as witnessed by the singular formation of iso- and heterochiral associations composed of regular, enantiomorphic helical structures. The peculiar properties of α-L-HNA (and most generally of the α-HNA system) provide new elements in our understanding of the structural prerequisites ruling the stereoselectivity of the hybridization processes of nucleic acids.

Isoguanine and 5‐Methyl‐Isocytosine Bases, In Vitro and In Vivo

The synthesis, base-pairing properties and in vitro and in vivo characteristics of 5-methyl-isocytosine (isoCMe) and isoguanine (isoG) nucleosides, incorporated in an HNA(h) (hexitol nucleic acid)–DNA(d) mosaic backbone, are described. The required h-isoG phosphoramidite was prepared by a selective deamination as a key step. As demonstrated by Tm measurements the hexitol sugar showed slightly better mismatch discrimination against dT. The d-isoG base mispairing follows the order T>G>C while the h-isoG base mispairing follows the order G>C>T. The h- and d-isoCMe bases mainly mispair with G. Enzymatic incorporation experiments show that the hexitol backbone has a variable effect on selectivity. In the enzymatic assays, isoG misincorporates mainly with T, and isoCMe misincorporates mainly with A. Further analysis in vivo confirmed the patterns of base-pair interpretation for the deoxyribose and hexitol isoCMe/isoG bases in a cellular context, through incorporation of the bases into plasmidic DNA. Results in vivo demonstrated that mispairing and misincorporation was dependent on the backbone scaffold of the base, which indicates rational advances towards orthogonality.

Noncoding regions of C. elegans mRNA undergo selective adenosine to inosine deamination and contain a small number of editing sites per transcript

ADARs (Adenosine deaminases that act on RNA) “edit” RNA by converting adenosines to inosines within double-stranded regions. The primary targets of ADARs are long duplexes present within noncoding regions of mRNAs, such as introns and 3′ untranslated regions (UTRs). Because adenosine and inosine have different base-pairing properties, editing within these regions can alter splicing and recognition by small RNAs. However, despite numerous studies identifying multiple editing sites in these genomic regions, little is known about the extent to which editing sites co-occur on individual transcripts or the functional output of these combinatorial editing events. To begin to address these questions, we performed an ultra-deep sequencing analysis of 4 Caenorhabditis elegans 3′ UTRs that are known ADAR targets. Synchronous editing events were determined for the long duplexes in vivo. Furthermore, the validity of each editing event was confirmed by sequencing the same regions of mRNA from worms that lack A-to-I editing. This analysis identified a large number of editing sites that can occur within each 3′ UTR, but interestingly, each individual transcript contained only a small fraction of these A-to-I editing events. In addition, editing patterns were not random, indicating that an editing event can affect the efficiency of editing at subsequent adenosines. Furthermore, we identified specific sites that can be both positively and negatively correlated with additional sites leading to mutually exclusive editing patterns. These results suggest that editing in noncoding regions is selective and hyper-editing of cellular RNAs is rare.

Binding to gold nanoclusters alters the hydrogen bonding interactions and electronic properties of canonical and size-expanded DNA base pairs

Structural perturbations, in terms of size expansion and metal binding, lead to exciting electronic properties which can be exploited in designing novel nano-electronic devices.

DFT investigation of the vibrational properties of AT base pairs in the presence of Ca2+ and Mn2+ ions

DFT investigation of the vibrational properties of AT base pairs in the presence of Ca2+ and Mn2+ ions

Watson–Crick base pairing, electronic and photophysical properties of triazole modified adenine analogues: a computational study

Triazole adenine nucleobase analogues show fluorescence in the UV-Vis region and form Watson–Crick base pairing with thymine nucleobases.

Sequence Design for a Test Tube of Interacting Nucleic Acid Strands.

We describe an algorithm for designing the equilibrium base-pairing properties of a test tube of interacting nucleic acid strands. A target test tube is specified as a set of desired "on-target" complexes, each with a target secondary structure and target concentration, and a set of undesired "off-target" complexes, each with vanishing target concentration. Sequence design is performed by optimizing the test tube ensemble defect, corresponding to the concentration of incorrectly paired nucleotides at equilibrium evaluated over the ensemble of the test tube. To reduce the computational cost of accepting or rejecting mutations to a random initial sequence, the structural ensemble of each on-target complex is hierarchically decomposed into a tree of conditional subensembles, yielding a forest of decomposition trees. Candidate sequences are evaluated efficiently at the leaf level of the decomposition forest by estimating the test tube ensemble defect from conditional physical properties calculated over the leaf subensembles. As optimized subsequences are merged toward the root level of the forest, any emergent defects are eliminated via ensemble redecomposition and sequence reoptimization. After successfully merging subsequences to the root level, the exact test tube ensemble defect is calculated for the first time, explicitly checking for the effect of the previously neglected off-target complexes. Any off-target complexes that form at appreciable concentration are hierarchically decomposed, added to the decomposition forest, and actively destabilized during subsequent forest reoptimization. For target test tubes representative of design challenges in the molecular programming and synthetic biology communities, our test tube design algorithm typically succeeds in achieving a normalized test tube ensemble defect ≤1% at a design cost within an order of magnitude of the cost of test tube analysis.

Synthesis of phosphoramidites of isoGNA, an isomer of glycerol nucleic acid.

IsoGNA, an isomer of glycerol nucleic acid GNA, is a flexible (acyclic) nucleic acid with bases directly attached to its linear backbone. IsoGNA exhibits (limited) base-pairing properties which are unique compared to other known flexible nucleic acids. Herein, we report on the details of the preparation of isoGNA phosphoramidites and an alternative route for the synthesis of the adenine derivative. The synthetic improvements described here enable an easy access to isoGNA and allows for the further exploration of this structural unit in oligonucleotide chemistry thereby spurring investigations of its usefulness and applicability.