Nucleobase Mimic Research Articles

Exceptional Nuclease Resistance of DNA and RNA with the Addition of Small-Molecule Nucleobase Mimics.

Nucleases present a formidable barrier to the application of nucleic acids in biology, significantly reducing the lifetime of nucleic acid-based drugs. Here, we develop a novel methodology to protect DNA and RNA from nucleases by reconfiguring their supramolecular structure through the addition of a nucleobase mimic, cyanuric acid. In the presence of cyanuric acid, polyadenine strands assemble into triple helical fibers known as the polyA/CA motif. We report that this motif is exceptionally resistant to nucleases, with the constituent strands surviving for up to 1 month in the presence of serum. The conferred stability extends to adjacent non-polyA sequences, albeit with diminishing returns relative to their polyA sections due to hypothesized steric clashes. We introduce a strategy to regenerate stability through the introduction of free polyA strands or positively charged amino side chains, enhancing the stability of sequences of varied lengths. The proposed protection mechanism involves enzyme failure to recognize the unnatural polyA/CA motif, coupled with the motif's propensity to form long, bundling supramolecular fibers. The methodology provides a fundamentally new mechanism to protect nucleic acids from degradation using a supramolecular approach and increases lifetime in serum to days, weeks, or months.

An expeditious synthesis of novel DNA nucleobase mimics of (+)-anisomycin

A glycal based expeditious synthesis of novel nucleoside analogues of (+)-anisomycin is reported. Readily available tri-O-benzyl-D-glucal was converted to a partially protected trihydroxypyrrolidine that is used as a common scaffold for the introduction of various nucleobases at the primary hydroxyl centre. Nucleoside analogues possessing all four DNA bases have been synthesized. Selective acetylation at C3 position was carried out with two of these unnatural nucleosides in order to mimic the structure of (+)-anisomycin. Cytotoxicity studies of some of these nucleosides showed that they display weaker activity on HeLa cells than Ara-C.

Tuning Exciton Coupling of Merocyanine Nucleoside Dimers by RNA, DNA and GNA Double Helix Conformations.

Exciton coupling between two or more chromophores in a specific environment is a key mechanism associated with color tuning and modulation of absorption energies. This concept is well exemplified by natural photosynthetic proteins, and can also be achieved in synthetic nucleic acid nanostructures. Here we report the coupling of barbituric acid merocyanine (BAM) nucleoside analogues and show that exciton coupling can be tuned by the double helix conformation. BAM is a nucleobase mimic that was incorporated in the phosphodiester backbone of RNA, DNA and GNA oligonucleotides. Duplexes with different backbone constitutions and geometries afforded different mutual dye arrangements, leading to distinct optical signatures due to competing modes of chromophore organization via electrostatic, dipolar, π–π‐stacking and hydrogen‐bonding interactions. The realized supramolecular motifs include hydrogen‐bonded BAM–adenine base pairs and antiparallel as well as rotationally stacked BAM dimer aggregates with distinct absorption, CD and fluorescence properties.

Tuning Exciton Coupling of Merocyanine Nucleoside Dimers by RNA, DNA and GNA Double Helix Conformations

AbstractExciton coupling between two or more chromophores in a specific environment is a key mechanism associated with color tuning and modulation of absorption energies. This concept is well exemplified by natural photosynthetic proteins, and can also be achieved in synthetic nucleic acid nanostructures. Here we report the coupling of barbituric acid merocyanine (BAM) nucleoside analogues and show that exciton coupling can be tuned by the double helix conformation. BAM is a nucleobase mimic that was incorporated in the phosphodiester backbone of RNA, DNA and GNA oligonucleotides. Duplexes with different backbone constitutions and geometries afforded different mutual dye arrangements, leading to distinct optical signatures due to competing modes of chromophore organization via electrostatic, dipolar, π–π‐stacking and hydrogen‐bonding interactions. The realized supramolecular motifs include hydrogen‐bonded BAM–adenine base pairs and antiparallel as well as rotationally stacked BAM dimer aggregates with distinct absorption, CD and fluorescence properties.

Effects of Nucleobase Amino Acids on the Binding of Rob to Its Promoter DNA: Differential Alteration of DNA Affinity and Phenotype.

Nucleic acid binding proteins have been studied extensively, but the nature of the interactions that control their affinity, selectivity, and DNA and RNA functions is still not well understood. To understand the nature and functional consequences of such interactions, we introduced nucleobase amino acids at specific positions of the transcriptional regulator Rob protein in vivo and succeeded in demonstrating that an alteration of the protein-DNA affinity can affect specific phenotypes associated with Rob protein-DNA interactions. Previously, we inserted different nucleobase amino acids in lieu of Arg40; this residue is known (via X-ray crystallography) to interact with the micF DNA promoter A-box residue Gua6. The interactions predominantly involved Watson-Crick-like H bonding. The present study focused primarily on the micF DNA promoter B-box; the crystallographically determined interaction involves H bonding between the agmatine moiety of Arg90 within an HTH motif of Rob and a phosphate oxygen anion to the 5'-side of Thy14. We had two main goals, the first of which was to demonstrate enhanced Rob-binding to the micF promoter DNA and the functional consequences resulting from the interaction of micF DNA with Rob analogues containing Arg90 nucleobase mimics. The second was to explore the possible functional consequences of enhancing the protein-DNA affinity with nucleobase replacements, which mechanistically mediate interactions differently than those reported to be operative for specific protein-DNA interactions. Nucleobase replacement at position 90 with Arg isosteres enhanced the Rob protein-micF DNA affinity in parallel with increasing antibiotic and Hg2+ resistance, while aromatic amino acid replacements increased the affinity but not the antibiotic or Hg2+ resistance. The demonstration of an increased affinity through strong base stacking interactions was notable.

Spontaneous Symmetry Breaking in the Formation of Supramolecular Polymers: Implications for the Origin of Biological Homochirality.

Aqueous solutions of the achiral, monomeric, nucleobase mimics (2,4,6-triaminopyrimidine, TAP, and a cyanuric acid derivative, CyCo6) spontaneously assemble into macroscopic homochiral domains of supramolecular polymers. These assemblies exhibit a high degree of chiral amplification. Addition of a small quantity of one handedness of a chiral derivative of CyCo6 generates exclusively homochiral structures. This system exhibits the highest reported degree of chiral amplification for dynamic helical polymers or supramolecular helices. Significantly, homochiral polymers comprised of hexameric rosettes with structural features that resemble nucleic acids are formed from mixtures of cyanuric acid (Cy) and ribonucleotides (l-, d-pTARC) that arise spontaneously from the reaction of TAP with the sugars. These findings support the hypothesis that nucleic acid homochirality was a result of symmetry breaking at the supramolecular polymer level.

Spontaneous Symmetry Breaking in the Formation of Supramolecular Polymers: Implications for the Origin of Biological Homochirality

AbstractAqueous solutions of the achiral, monomeric, nucleobase mimics (2,4,6‐triaminopyrimidine, TAP, and a cyanuric acid derivative, CyCo6) spontaneously assemble into macroscopic homochiral domains of supramolecular polymers. These assemblies exhibit a high degree of chiral amplification. Addition of a small quantity of one handedness of a chiral derivative of CyCo6 generates exclusively homochiral structures. This system exhibits the highest reported degree of chiral amplification for dynamic helical polymers or supramolecular helices. Significantly, homochiral polymers comprised of hexameric rosettes with structural features that resemble nucleic acids are formed from mixtures of cyanuric acid (Cy) and ribonucleotides (l‐, d‐pTARC) that arise spontaneously from the reaction of TAP with the sugars. These findings support the hypothesis that nucleic acid homochirality was a result of symmetry breaking at the supramolecular polymer level.

Nucleobase Containing Synthetic Polymers: Advancing Biomimicry via Controlled Synthesis and Self-Assembly

The hydrogen-bonding recognition interactions of nucleobases are a fundamental property of nucleic acid chemistry and associated transcription, translation, and replication functions. Nucleobase interactions are central in protein biosynthesis, yielding sequence- and stereospecific macromolecules capable of assembly into precisely defined, complex shapes and morphologies that make up the machinery of life. As the understanding of nucleobases and their significance developed in the past century, chemists have inevitably sought to extend their function from a biological setting onto wholly synthetic platforms. Recent advances point to a burgeoning area of study which may soon bear fruit in some of the holy grails of polymer synthesis, namely sequence (and stereo) control, single chain manipulation, and controlled polymer folding. This Perspective seeks to summarize recent developments in the area of nucleobase containing polymers (including nucleobase mimics), with particular emphasis on controlled polymerization, self-assembly, and templating polymerization.

A Universal Nucleoside with Strong Two-Band Switchable Fluorescence and Sensitivity to the Environment for Investigating DNA Interactions

With the aim of developing a new tool to investigate DNA interactions, a nucleoside analogue incorporating a 3-hydroxychromone (3HC) fluorophore as a nucleobase mimic was synthesized and incorporated into oligonucleotide chains. In comparison with existing fluorescent nucleoside analogues, this dye features exceptional environmental sensitivity switching between two well-resolved fluorescence bands. In labeled DNA, this nucleoside analogue does not alter the duplex conformation and exhibits a high fluorescence quantum yield. This probe is up to 50-fold brighter than 2-aminopurine, the fluorescent nucleoside standard. Moreover, the dual emission is highly sensitive to the polarity of the environment; thus, a strong shielding effect of the flanking bases from water was observed. With this nucleoside, the effect of a viral chaperone protein on DNA base stacking was site-selectively monitored.

Click fleximers: a modular approach to purine base-expanded ribonucleoside analogues

The synthesis of nucleoside analogues incorporating 4-(5-pyrimidinyl)-1,2,3-triazole aglycons as expanded purine nucleobase mimics were accessed using the copper-catalyzed azide-alkyne Huisgen cycloaddition between a ribosyl azide and 5-alkynylpyrimidines. Depending on the nature of the alkyne employed, other nucleoside analogues that possess fluorescence or potential metal-binding properties were prepared. Computational studies were undertaken on the purine analogues and indicate that the heterocycles of the unfused nucleobase prefer a coplanar arrangement and the anti-glycosidic conformer is favoured in most instances.

Synthesis of non glycosidic nucleobase-sugar mimetics

Abstract Biologically active organic molecules acting as nucleoside mimics are frequently encountered in pharmaceutical research. They are either synthetic heterocycles, which miss the sugar-derived interactions with the active site of the nucleoside-binding protein, or natural products containing a glycosidic linkage, which may cause bioavailability and metabolic stability problems. We report here the concept of synthetic full nucleoside mimics, including both a N-containing nucleobase-like portion and a sugar-like moiety, where the latter consists of 5- and 6-membered carbacycles connected by a more stable and drug-like C N bond to the nucleobase mimic. Compounds 14, 16 (indolinones), 21 and 23 (benzimidazolones) have been prepared as model compounds.

N-Arylmethyl substituted iminoribitol derivatives as inhibitors of a purine specific nucleoside hydrolase

A key enzyme within the purine salvage pathway of parasites, nucleoside hydrolase, is proposed as a good target for new antiparasitic drugs. We have developed N-arylmethyl-iminoribitol derivatives as a novel class of inhibitors against a purine specific nucleoside hydrolase from Trypanosoma vivax. Several of our inhibitors exhibited low nanomolar activity, with 1,4-dideoxy-1,4-imino- N-(8-quinolinyl)methyl- d-ribitol (UAMC-00115, K i 10.8 nM), N-(9-deaza-adenin-9-yl)methyl-1,4-dideoxy-1,4-imino- d-ribitol ( K i 4.1 nM), and N-(9-deazahypoxanthin-9-yl)methyl-1,4-dideoxy-1,4-imino- d-ribitol ( K i 4.4 nM) being the three most active compounds. Docking studies of the most active inhibitors revealed several important interactions with the enzyme. Among these interactions are aromatic stacking of the nucleobase mimic with two Trp-residues, and hydrogen bonds between the hydroxyl groups of the inhibitors and amino acid residues in the active site. During the course of these docking studies we also identified a strong interaction between the Asp40 residue from the enzyme and the inhibitor. This is an interaction which has not previously been considered as being important.

Scope and limitations of the SCS-MP2 method for stacking and hydrogen bonding interactions

Fluorobenzenes are pi-acceptor synthons that form pi-stacked structures in molecular crystals as well as in artificial DNAs. We investigate the competition between hydrogen bonding and pi-stacking in dimers consisting of the nucleobase mimic 2-pyridone (2PY) and all fluorobenzenes from 1-fluorobenzene to hexafluorobenzene (n-FB, with n = 1-6). We contrast the results of high level ab initio calculations with those obtained using ultraviolet (UV) and infrared (IR) laser spectroscopy of isolated and supersonically cooled dimers. The 2PY.n-FB complexes with n = 1-5 prefer double hydrogen bonding over pi-stacking, as diagnosed from the UV absorption and IR laser depletion spectra, which both show features characteristic of doubly H-bonded complexes. The 2-pyridone.hexafluorobenzene dimer is the only pi-stacked dimer, exhibiting a homogeneously broadened UV spectrum and no IR bands characteristic for H-bonded species. MP2 (second-order Møller-Plesset perturbation theory) calculations overestimate the pi-stacked dimer binding energies by about 10 kJ/mol and disagree with the experimental observations. In contrast, the MP2 treatment of the H-bonded dimers appears to be quite accurate. Grimme's spin-component-scaled MP2 approach (SCS-MP2) is an improvement over MP2 for the pi-stacked dimers, reducing the binding energy by approximately 10 kJ/mol. When applied to explicitly correlated MP2 theory (SCS-MP2-R12 approach), agreement with the corresponding coupled-cluster binding energies [at the CCSD(T) level] is very good for the pi-stacked dimers, within +/- 1 kJ/mol for the 2PY complexes with 1-fluorobenzene, 1,2-difluorobenzene, 1,2,4,5-tetrafluorobenzene, pentafluorobenzene and hexafluorobenzene. Unfortunately, the SCS-MP2 approach also reduces the binding energy of the H-bonded species, leading to disagreement with both coupled-cluster theory and experiment. The SCS-MP2-R12 binding energies follow the SCS-MP2 binding energies closely, being about 0.5 and 0.7 kJ/mol larger for the H-bonded and pi-stacked forms, respectively, in an augmented correlation-consistent polarized valence quadruple-zeta basis. It seems that the SCS-MP2 and SCS-MP2-R12 methods cannot provide sufficient accuracy to replace the CCSD(T) method for intermolecular interactions where H-bonding and pi-stacking are competitive.

Synthesis and Properties of Oligonucleotides with Iodo-Substituted Aromatic Aglycons: Investigation of Possible Halogen Bonding Base Pairs

Ab initio calculations of halogen bond energies of artificial base pairs constructed between iodinated aromatic nucleobase mimics and nitrogen-containing acceptor molecules such as pyridine and imidazole suggest that modified base pairs are converted to optimized planar base pairs with weak Delta E values of -0.19 to -3.93 kcal/mol. To evaluate the contribution of halogen bonding toward duplex stabilization of such modified nucleobase mimics introduced into artificial base pairs, we synthesized three C-nucleoside analogues 1-3 with several iodinated aromatic rings and an imidazole nucleoside derivative 4 and incorporated them into oligodeoxynucleotides. Hybridization studies of modified oligodeoxynucleotides incorporating iodoaromatic bases showed their unique universal base-like ability; however, no indication of halogen bond formation was observed. A more sophisticated design is required for the development of new base pairs stabilized by halogen bonding.

Hydrogen Bonding of the Nucleobase Mimic 2-Pyridone to Fluorobenzenes: An ab Initio Investigation

The hydrogen-bonded complexes of the nucleobase mimic 2-pyridone (2PY) with seven different fluorinated benzenes (1-, 1,2-, 1,4-, 1,2,3-, 1,3,5-, 1,2,3,4-, and 1,2,4,5-fluorobenzene) are important model systems for investigating the relative importance of hydrogen bonding versus pi-stacking interactions in DNA. We have shown by supersonic-jet spectroscopy that these dimers are hydrogen bonded and not pi-stacked at low temperature (Leist, R.; Frey, J. A.; Leutwyler, S. J. Phys. Chem. A 2006, 110, 4180). Their geometries and binding energies D(e) were calculated using the resolution of identity (RI) Møller-Plesset second-order perturbation theory method (RIMP2). The most stable dimers are bound by antiparallel N-H...F-C and C-H...O=C hydrogen bonds. The binding energies are extrapolated to the complete basis set (CBS) limit, , using the aug-cc-pVXZ basis set series. The CBS binding energies range from -D(e,CBS) = 6.4-6.9 kcal/mol and the respective dissociation energies from -D(0,CBS) = 5.9-6.3 kcal/mol. In combination with experiment, the latter represent upper limits to the dissociation energies of the pi-stacked isomers (which are not observed experimentally). The individual C-H...O=C and N-H...F-C contributions to D(e) can be approximately separated. They are nearly equal for 2PY.fluorobenzene; each additional F atom strengthens the C-H...O=C hydrogen bond by approximately 0.5 kcal/mol and weakens the C-F...H-N hydrogen bond by approximately 0.3 kcal/mol. The single H-bond strengths and lengths correlate with the gas-phase acid-base properties of the C-H and C-F groups of the fluorobenzenes.

The degenerate properties of modified purine and pyrimidine DNA bases: a density functional study

Density-functional theory was used to study the properties (binding geometries and affinities for the natural DNA bases) of various degenerate nucleobases, which bind without discrimination to the purines or pyrimidines. The data for purine mimics ( Z and K) indicates that although stronger binding strengths are calculated for pairs with cytosine compared with thymine, cytosine binds to a less stable tautomer of the nucleobase mimic. Indeed, the energy differences between the binding strengths and the tautomers effectively cancel and thereby provide a possible explanation for the observed degenerate properties of these molecules. Similar trends are found for the pyrimidine mimics ( M and P); however, the energy differences do not cancel, even upon inclusion of environmental effects.