Nucleobase Moieties Research Articles

Recognition of Mixed-Sequence DNA Duplexes: Design Guidelines for Invaders Based on 2′-O-(Pyren-1-yl)methyl-RNA Monomers

The development of agents that recognize mixed-sequence double-stranded DNA (dsDNA) is desirable because of their potential as tools for detection, regulation, and modification of genes. Despite progress with triplex-forming oligonucleotides, peptide nucleic acids, polyamides, and other approaches, recognition of mixed-sequence dsDNA targets remains challenging. Our laboratory studies Invaders as an alternative approach toward this end. These double-stranded oligonucleotide probes are activated for recognition of mixed-sequence dsDNA through modification with +1 interstrand zippers of intercalator-functionalized nucleotides such as 2'-O-(pyren-1-yl)methyl-RNA monomers and have recently been shown to recognize linear dsDNA, DNA hairpins, and chromosomal DNA. In the present work, we systematically studied the influence that the nucleobase moieties of the 2'-O-(pyren-1-yl)methyl-RNA monomers have on the recognition efficiency of Invader duplexes. Results from thermal denaturation, binding energy, and recognition experiments using Invader duplexes with different +1 interstrand zippers of the four canonical 2'-O-(pyren-1-yl)methyl-RNA A/C/G/U monomers show that incorporation of these motifs is a general strategy for activation of probes for recognition of dsDNA. Probe duplexes with interstrand zippers comprising C and/or U monomers result in the most efficient recognition of dsDNA. The insight gained from this study will drive the design of efficient Invaders for applications in molecular biology, nucleic acid diagnostics, and biotechnology.

Synthesis of Phosphonoglycine Backbone Units for the Development of Phosphono Peptide Nucleic Acids

AbstractA series of phosphono‐modified backbone mimics based on achiral and chiral N‐(dihydroxypropyl)glycine units were obtained by sequential addition of phosphonate and nucleobase moieties to suitably protected dihydroxypropylamines. Simple synthetic strategies enabled the preparation of various target derivatives that will be useful as building blocks for the preparation of new synthetic polymers containing a phosphonate internucleotide linkage in place of the standard phosphodiester bond.

2′,4′-BNA bearing a chiral guanidinopyrrolidine-containing nucleobase with potent ability to recognize the CG base pair in a parallel-motif DNA triplex

In order to expand the target sequence used in triplex DNA formation, seven novel nucleotide analogues were synthesized and incorporated into triplex-forming oligonucleotides by post-elongation modification approaches. Among them, , equipped with a suitable restricted conformation of sugar and nucleobase moieties, was found to have the highest sequence-selectivity and affinity towards CG base pairs within double-stranded DNA.

Nucleobase-Functionalized 1,6-Dithiapyrene-Type Electron-Donors: Supramolecular Assemblies by Complementary Hydrogen-Bonds and π-Stacks

Synthesis, crystal structures and redox properties of 1,6-dithiapyrene (DTPY)-type electron-donors functionalized with nucleobases (uracil, cytosine and adenine) were investigated. The electrochemical measurements showed that the uracil-substituted derivatives were slightly stronger electron-donors than DTPY, and the cytosine- and adenine-substitution caused a slight weakening of the electron-donating ability. In the crystal structures, DTPY-nucleobases constructed multidimensional assemblies by complementary hydrogen-bonds on the nucleobase moieties and π-stacks and S···S interactions on the DTPY skeleton. The uracil derivative formed two kinds of hydrogen-bonded pairs with different H-bonding modes (Watson–Crick and reverse Watson–Crick types), both of which were further linked through π-stacks on the DTPY skeleton to construct one-dimensional alternating columns. In the CH2Cl2 solvated crystal, the uracil derivative built up a two-dimensional π-layer by the complementary hydrogen-bonds and π-stacks. In...

2-Methylwyosine, a Nucleoside With Restricted Anti Conformation in the East Region Enforced by Nucleobase Moiety Modification: Synthesis and Conformational Analysis by NMR and Molecular Dynamics

GRAPHICAL ABSTRACT We synthesized a new 2-methyl derivative of wyosine using a multistep procedure starting from guanosine. We examined different synthetic paths and optimized the conditions for each step. Based on MD calculations and analysis of the 3 J HH and J C1′H1′ of the ribose moiety, we discovered that the sugar part adopted conformation specific for the East region rarely occurring in solution. This unusual conformational preference is probably due to steric repulsions between the methyl group at position 2 and the 5′-CH2OH group. We observed that N-glycosidic bond stability weakened 14-fold upon the introduction of the methyl group in position 2 compared with wyosine.

New Approach for Designing Single-Chain Magnets: Organization of Chains via Hydrogen Bonding between Nucleobases

Two one-dimensional (1D) manganese complexes, [Mn(2)(naphtmen)(2)(L)](ClO(4))·2Et(2)O·2MeOH·H(2)O (1) and [Mn(2)(naphtmen)(2)(HL)](ClO(4))(2)·MeOH (2), were synthesized by using a bridging ligand with a nucleobase moiety, 6-amino-9-β-carboxyethylpurine, and a salen-type manganese(III) dinuclear complex, [Mn(2)(naphtmen)(2)(H(2)O)(2)](ClO(4))(2) (naphtmen(2-) = N,N'-(1,1,2,2-tetramethylethylene)bis(naphthylideneiminato) dianion). In 1 and 2, the carboxylate-bridged Mn(III) dinuclear units are alternately linked by two kinds of weak Mn···O interactions into 1D chains. As a result, canted antiferromagnetic and ferromagnetic interactions are alternately present along the chains, leading to a 1D chain with non-cancellation of anisotropic spins. Since the chains connected via H-bonds between nucleobase moieties are magnetically isolated, both 1 and 2 act as single-chain magnets (SCMs). More importantly, this result shows the smaller canting angles hinder long-range ordering in favor of SCM dynamics.

Synthesis of novel nucleobase-terminated organosilane and its self-assembly on a substrate

A novel nucleobase-terminated organosilane with nucleobase and ethoxysilane moieties was synthesized by a simple method in which bromouracil reacts with amino-terminated organosilane without solvents. The uracil-terminated organosilane was self-assembled on a hydroxylated surface, and then the uracil-concentrated surface was obtained. The resulting uracil-terminated self-assembled layers were characterized by X-ray photoelectron spectroscopy and ellipsometry. Adenine complementary to uracil was effectively adsorbed onto the resultant uracil-concentrated surface by multiple hydrogen bonds. Herein, a simple method is provided for synthesizing a nucleobase-terminated organosilane and modifying a hydroxylated surface to a nucleobase-concentrated surface. Our method, which uses the self-assembly of nucleobase-terminated organosilanes, facilitates the construction of bioconjugated devices for separating and sensing nucleobases and nucleic acids. A novel nucleobase-terminated organosilane with both nucleobase and ethoxysilane moieties was synthesized by a simple method that includes only a reaction of bromouracil with amino-terminated organosilane without using solvents. The uracil-terminated organosilane was self-assembled on a hydroxylated surface, followed by chemical modifications to uracil-concentrated surface. Adenine complementary to uracil was effectively adsorbed onto the resultant uracil-concentrated surface by multiple hydrogen bonds. This paper provides with a simple method for synthesizing a nucleobase-terminated organosilane and for modifying a hydroxylated surface to a nucleobase-concentrated surface.

ChemInform Abstract: Development of Highly Nuclease‐Resistant Chemically‐Modified Oligonucleotides

AbstractReview: 67 refs.

Hydrogelation and spontaneous fiber formation of 8-aza-7-deazaadenine nucleoside ‘click’ conjugates

Nucleoside hydrogels based on benzyl azide ‘click’ conjugates of 8-aza-7-deaza-2′-deoxyadenosine bearing 7-ethynyl, 7-octa-(1,7-diynyl), and 7-tri-prop-2-ynyl-amine side chains were synthesized ( 1, 3, 4). The cycloaddition adduct with the shortest linker ( 1) yields the most powerful hydrogelator forming stable gels at a concentration of 0.3 wt % of 1 in water. One molecule of 1 catches 7500 water molecules. Cycloaddition of the 8-aza-7-deaza-7-azido-2′-deoxyadenosine ( 9) and 3-phenyl-1-propyne ( 10) leads to the isomeric conjugate 2, with a C–N connectivity between the nucleobase and triazole moiety. This gel is less stable than that of the adduct 1. Both gels show a similar stability over a wide pH range (4.0–10.0). Xerogels of 1 and 2 studied by scanning electron microscopy (SEM) reveal that both click adducts ( 1 and 2) form long fibers spontaneously.

Direct Evidence for Tautomerization of the Uracil Moiety within the Pb2+/Uridine-5′-monophosphate Complex: A Combined Tandem Mass Spectrometry and IRMPD study

The structure of the [Pb(UMP)-H](+) (UMP = uridine-5'-monophosphate) complex was studied in the gas phase by combining electrospray ionization (ESI), tandem mass spectrometry, and mid-infrared multiple photon dissociation (IRMPD) spectroscopy. The results obtained show that Pb(2+) ions interact not only with the deprotonated phosphate group but also with a carbonyl group of the nucleobase moiety by folding of the mononucleotide, resulting in macrochelates that are not likely to be present in solution. Comparison between the IRMPD and DFT-computed spectra suggests that the ESI-generated complex likely corresponds to a mixture of several structures, and establishes the enolic tautomers as the most abundant species for the [Pb(UMP)-H](+) ion, while the very weak IRMPD signal observed at ∼1763 cm(-1) points to a minor population of oxo forms. Our data also suggest that losing the nucleobase residue under CID conditions does not necessarily mean a lack of interaction between the metal and the nucleobase moiety, as commonly reported in the literature for large oligonucleotides.

Learning from Directed Evolution: Thermus aquaticus DNA Polymerase Mutants with Translesion Synthesis Activity

DNA is being constantly damaged by endo- and exogenous agents such as reactive oxygen species, chemicals, radioactivity, and ultraviolet radiation. Additionally, DNA is inherently labile, and this can result in, for example, the spontaneous hydrolysis of the glycosidic bond that connects the sugar and the nucleobase moieties in DNA; this results in abasic sites. It has long been obscure how cells achieve DNA synthesis past these lesions, and only recently has it been discovered that several specialized DNA polymerases are involved in translesion synthesis. The underlying mechanisms that render one DNA polymerase competent in translesion synthesis while another DNA polymerase fails are still indistinct. Recently two variants of Taq DNA polymerase that exhibited higher lesion bypass ability than the wild-type enzyme were identified by directed-evolution approaches. Strikingly, in both approaches it was independently found that substitution of a single nonpolar amino acid side chain by a cationic side chain increases the capability of translesion synthesis. Here, we combined both mutations in a single enzyme. We found that the KlenTaq DNA polymerase that bore both mutations superseded the wild-type as well as the respective single mutants in translesion-bypass proficiency. Further insights in the molecular basis of the detected gain of translesion-synthesis function were obtained by structural studies of DNA polymerase variants caught in processing canonical and damaged substrates. We found that increased positive charge of the surface potential in the area proximal to the negatively charged substrates promotes translesion synthesis by KlenTaq DNA polymerase, an enzyme that has very limited naturally evolved capability to perform translesion synthesis. Since expanded positively charged surface potential areas are also found in naturally evolved translesion DNA polymerases, our results underscore the impact of charge on the proficiency of naturally evolved translesion DNA polymerases.

C5‐Functionalized DNA, LNA, and α‐L‐LNA: Positional Control of Polarity‐Sensitive Fluorophores Leads to Improved SNP‐Typing

Single nucleotide polymorphisms (SNPs) are important markers in disease genetics and pharmacogenomic studies. Oligodeoxyribonucleotides (ONs) modified with 5-[3-(1-pyrenecarboxamido)propynyl]-2'-deoxyuridine monomer X enable detection of SNPs at non-stringent conditions due to differential fluorescence emission of matched versus mismatched nucleic acid duplexes. Herein, the thermal denaturation and optical spectroscopic characteristics of monomer X are compared to the corresponding locked nucleic acid (LNA) and α-L-LNA monomers Y and Z. ONs modified with monomers Y or Z result in a) larger increases in fluorescence intensity upon hybridization to complementary DNA, b) formation of more brightly fluorescent duplexes due to markedly larger fluorescence emission quantum yields (Φ(F)=0.44-0.80) and pyrene extinction coefficients, and c) improved optical discrimination of SNPs in DNA targets. Optical spectroscopy studies suggest that the nucleobase moieties of monomers X-Z adopt anti and syn conformations upon hybridization with matched and mismatched targets, respectively. The polarity-sensitive 1-pyrenecarboxamido fluorophore is, thereby, either positioned in the polar major groove or in the hydrophobic duplex core close to quenching nucleobases. Calculations suggest that the bicyclic skeletons of LNA and α-L-LNA monomers Y and Z influence the glycosidic torsional angle profile leading to altered positional control and photophysical properties of the C5-fluorophore.

Desulfurization of 2-thiouracil nucleosides: Conformational studies of 4-pyrimidinone nucleosides

4-Pyrimidinone ribofuranoside ( H 2o 4U ) and 4-pyrimidinone 2′-deoxyribofuranoside ( dH 2o 4U ) were synthesized by the oxidative desulfurization of parent 2–thiouracil nucleosides with m-chloroperbenzoic acid. The crystal structures of H 2o 4U and dH 2o 4U and their conformations in solution were determined and compared with corresponding 2-thiouracil and uracil nucleosides. The absence of a large 2-thiocarbonyl/2-carbonyl group in the nucleobase moiety results in C2′- endo puckering of the ribofuranose ring (S conformer) in the crystal structure of H 2o 4U , which is not typical of RNA nucleosides. Interestingly, the hydrogen bonding network in the crystals of dH 2o 4U stabilizes the sugar moiety conformation in the C3′- endo form (N conformer), rarely found in DNA nucleosides. In aqueous solution, dH 2o 4U reveals a similar population of the C2′- endo conformation (65%) to that of 2′-deoxy-2-thiouridine (62%), while the 62% population of the S conformer for H 2o 4U is significantly different from that of the parent 2-thiouridine, for which the N conformer is dominant (71%). Such a difference may be of biological importance, as the desulfurization process of natural tRNA 2-thiouridines may occur under conditions of oxidative stress in the cell and may influence the decoding process.

Development of Highly Nuclease-resistant Chemically-modified Oligonucleotides

Chemical modification of therapeutic oligodeoxyribonucleotides (ODNs) is necessary to avoid not only degradation by endo- and exo-nucleases but also recognition by sensors such as an innate immune system. We have been developing modified nucleosides having an aminoalky linker at the pyrimidine nucleobase or sugar moiety. ODNs containing 5-N-(6-aminohexyl)carbamoyl-2'-deoxyuridine (7) were thermally stabilized about 3°C per modification and were about 160 times more stable to hydrolysis by snake venom phosphodiesterase (a 3'-exonuclease) than unmodified ODNs, but not by endonucleases. On the other hand, ODNs containing 4'-C-(aminoethyl)thymidine (14b), which was synthesized by a newly developed radical cyclization-ring-enlargement reaction by us, were 87 times more stable to hydrolysis by DNase I (an endonuclease) and 133 times more stable in 50% human serum than unmodified ODNs. The highly stereoselective synthesis of 4'-thioribonuclesides ((S)Ns) was also developed using a Pummerer reaction. Human thrombin RNA aptamer (CII-1-37) containing 4'-thiouridine and 4'-thiocytidine was obtained by SELEX with a K(d) value of 4.7 nM, while a previously known RNA aptamer (RNA-24) has a K(d) value of 85 nM. Studies of the modification pattern-RNAi activity relationships by using (S)Ns have been carried out against luciferase genes. We found that siRNAs, which have 4 residues of (S)Ns on both ends of the sense strand and 4 residues on the 3'-end of the antisense strand, were the most effective. 4'-ThioRNA is about 1100 times more stable in 50% human plasma than unmodified RNA. However, oligoribonucleotides ((SM)ONs) containing 2'-O-methyl-4'-thioribonucleosides were 9800 times more stable in 50% human plasma than unmodified RNA. Since (SM)ON duplexes were thermally more stable than unmodified ON duplexes, therefore they would be quite suitable to use for oligonucleotide therapeutics.

Toward the Supramolecular Cyclodextrin Dimers Using Nucleobase Pairs

The synthesis of eleven new cyclodextrin derivatives having nucleobase moiety - thymin-1-yl, adenin-9-yl, and guanin- 9-yl - is described. These two moieties are linked by different spac- ers, such as aminoethyl and 1,2,3-triazolyl group. Direct nucleo- philic substitution and 1,3-dipolar cycloaddition were performed in good yields (13-73%) for some of the synthesized compounds.

Structure of Pb2+/dCMP and Pb2+/CMP complexes as characterized by tandem mass spectrometry and IRMPD spectroscopy

The structure of Pb2+/cytidine-5′-monophosphate (CMP) and Pb2+/deoxycytidine-5′-monophosphate (dCMP) complexes were probed in the gas phase by combining electrospray ionization, tandem mass spectrometry and mid-infrared multiple-photon dissociation (IRMPD) spectroscopy. The fragment ions detected upon collision suggest that the metal interacts with both the phosphate group and the cytosine. This finding is further confirmed by IRMPD spectroscopy. The IRMPD spectrum of the ESI-generated [Pb(dCMP)−H]+ and [Pb(CMP)−H]+ complexes is indeed in very good agreement with DFT-computed infrared absorption spectra of macrochelate forms, in which the Pb2+ ion not only interacts with the phosphate group but also with the carbonyl group of the nucleobase moiety, by folding of the mononucleotides. The structures thus characterized therefore differ from those proposed in solution and deduced from potentiometric studies. Our data also suggest that losing the nucleobase residue under CID conditions does not necessarily mean a lack of interaction between the metal and the nucleobase moiety, as commonly mentioned in the literature for large oligonucleotides.

Electron Attachment to Hydrated Oligonucleotide Dimers: Guanylyl-3′,5′-Cytidine and Cytidylyl-3′,5′-Guanosine

The dinucleoside phosphate deoxycytidylyl-3',5'-deoxyguanosine (dCpdG) and deoxyguanylyl-3',5'-deoxycytidine (dGpdC) systems are among the largest to be studied by reliable theoretical methods. Exploring electron attachment to these subunits of DNA single strands provides significant progress toward definitive predictions of the electron affinities of DNA single strands. The adiabatic electron affinities of the oligonucleotides are found to be sequence dependent. Deoxycytidine (dC) on the 5' end, dCpdG, has larger adiabatic electron affinity (AEA, 0.90 eV) than dC on the 3' end of the oligomer (dGpdC, 0.66 eV). The geometric features, molecular orbital analyses, and charge distribution studies for the radical anions of the cytidine-containing oligonucleotides demonstrate that the excess electron in these anionic systems is dominantly located on the cytosine nucleobase moiety. The pi-stacking interaction between nucleobases G and C seems unlikely to improve the electron-capturing ability of the oligonucleotide dimers. The influence of the neighboring base on the electron-capturing ability of cytosine should be attributed to the intensified proton accepting-donating interaction between the bases. The present investigation demonstrates that the vertical detachment energies (VDEs) of the radical anions of the oligonucleotides dGpdC and dCpdG are significantly larger than those of the corresponding nucleotides. Consequently, reactions with low activation barriers, such as those for O-C sigma bond and N-glycosidic bond breakage, might be expected for the radical anions of the guanosine-cytosine mixed oligonucleotides.

Potential Supramolecular Cyclodextrin Dimers Using Nucleobase Pairs

The synthesis of six new cyclodextrin derivatives having nucleobase moiety is described. These two moieties are linked by different spacers, such as aminoethyl and 1,2,3-triazolyl groups. Example of association constants for complexation of adenine and thymine derivatives: K AT = 385 M-¹ using NMR methodology is reported. Study of interaction between four cyclodextrin derivatives and one adamantyl guest is described by ITC.

RNA phosphodiester backbone dynamics of a perdeuterated cUUCGg tetraloop RNA from phosphorus-31 NMR relaxation analysis

We have analyzed the relaxation properties of all (31)P nuclei in an RNA cUUCGg tetraloop model hairpin at proton magnetic field strengths of 300, 600 and 900 MHz in solution. Significant H, P dipolar contributions to R (1) and R (2) relaxation are observed in a protonated RNA sample at 600 MHz. These contributions can be suppressed using a perdeuterated RNA sample. In order to interpret the (31)P relaxation data (R (1), R (2)), we measured the (31)P chemical shift anisotropy (CSA) by solid-state NMR spectroscopy under various salt and hydration conditions. A value of 178.5 ppm for the (31)P CSA in the static state (S (2) = 1) could be determined. In order to obtain information about fast time scale dynamics we performed a modelfree analysis on the basis of our relaxation data. The results show that subnanosecond dynamics detected around the phosphodiester backbone are more pronounced than the dynamics detected for the ribofuranosyl and nucleobase moieties of the individual nucleotides (Duchardt and Schwalbe, J Biomol NMR 32:295-308, 2005; Ferner et al., Nucleic Acids Res 36:1928-1940, 2008). Furthermore, the dynamics of the individual phosphate groups seem to be correlated to the 5' neighbouring nucleobases.

Synthesis and biological evaluation of novel neamine–nucleoside conjugates potentially targeting to RNAs

Eighteen novel neamine–nucleoside conjugates with ethylenediamine–lysine or ethylenediamine–arginine as the linker were synthesized and their potential binding to A site of 16S RNA and TAR RNA was evaluated using SPR (surface plasmon resonance). Compared with neamine, compounds 10i and 10q show 6.3 and 4.8 times potential in binding to A site of 16S RNA and eight and six times potential in binding to TAR RNA, respectively. According to the data of SPR, it indicates that amino acid residue and nucleobase moieties of the designed neamine–nucleosides conjugates exhibit the important contributions for the binding to A site of 16S RNA and TAR RNA. The molecular docking study on the interaction between the ligands and A site of 16S RNA is in agreement with the experimental data. The novel type of modification may provide a promising way for the development of neamine derivatives effectively targeting to RNAs.