Nucleobase Analogues Research Articles

Synthesis, characterization and cytotoxic properties of platinum(II) complexes containing the nucleosides adenosine and cytidine

Cytidine (cyt) and adenosine (ado) react with cis-[L 2Pt(μ-OH)] 2(NO 3) 2 (L = PMe 3, PPh 3) in various solvents to give the nucleoside complexes cis-[L 2Pt{cyt(− H), N 3 N 4 }] 3(NO 3) 3 (L = PMe 3, 1) , cis-[L 2Pt{cyt(− H), N 4 }(cyt, N 3 )]NO 3 (L = PPh 3, 2), cis-[L 2Pt{ado(− H), N 1 N 6 }] 2(NO 3) 2 (L = PMe 3, 3) and cis-[L 2Pt{ado(− H), N 6 N 7 }]NO 3 (L = PPh 3, 4). When the condensation reaction is carried out in solution of nitriles (RCN, R = Me, Ph) the amidine derivatives cis-[(PPh 3) 2PtNH=C(R){cyt(− 2H)}]NO 3 (R = Me, 5a; R = Ph, 5b) and cis-[(PPh 3) 2PtNH=C(R){ado(− 2H)}]NO 3 (R = Me, 6a: R = Ph, 6b) are quantitatively formed. The coordination mode of these nucleosides, characterized in solution by multinuclear NMR spectroscopy and mass spectrometry, is similar to that previously observed for the nucleobases 1-methylcytosine (1-MeCy) and 9-methyladenine (9-MeAd). The cytotoxic properties of the new complexes, and those of the nucleobase analogs, cis-[(PPh 3) 2PtNH=C(R){1-MeCy(− 2H)}]NO 3 (R = Me, 7a: R = Ph, 7b), cis-[(PPh 3) 2PtNH=C(R){9-MeAd(− 2H)}]NO 3 (R = Me, 8a: R = Ph, 8b) have been investigated in a wide panel of human cancer cells. Interestingly, whereas the Pt(II) nucleoside complexes ( 1– 4) did not show appreciable cytotoxicity, the corresponding amidine derivatives ( 7a, 7b, 8a, 8b, 5b, and 6b) exhibited a significant in vitro antitumor activity.

Determinants of Precatalytic Conformational Transitions in the DNA Cytosine Methyltransferase M.HhaI

The DNA methyltransferase M.HhaI is an excellent model for understanding how recognition of a nucleic acid substrate is translated into site-specific modification. In this study, we utilize direct, real-time monitoring of the catalytic loop position via engineered tryptophan fluorescence reporters to dissect the conformational transitions that occur in both enzyme and DNA substrate prior to methylation of the target cytosine. Using nucleobase analogues in place of the target and orphan bases, the kinetics of the base flipping and catalytic loop closure rates were determined, revealing that base flipping precedes loop closure as the rate-determining step prior to methyl transfer. To determine the mechanism by which individual specific hydrogen bond contacts at the enzyme-DNA interface mediate these conformational transitions, nucleobase analogues lacking hydrogen bonding groups were incorporated into the recognition sequence to disrupt the major groove recognition elements. The consequences of binding, loop closure, and catalysis were determined for four contacts, revealing large differences in the contribution of individual hydrogen bonds to DNA recognition and conformational transitions on the path to catalysis. Our results describe how M.HhaI utilizes direct readout contacts to accelerate extrication of the target base that offer new insights into the evolutionary history of this important class of enzymes.

Influence of C-5 substituted cytosine and related nucleoside analogs on the formation of benzo[a]pyrene diol epoxide-dG adducts at CG base pairs of DNA.

Endogenous 5-methylcytosine (MeC) residues are found at all CG dinucleotides of the p53 tumor suppressor gene, including the mutational ‘hotspots’ for smoking induced lung cancer. MeC enhances the reactivity of its base paired guanine towards carcinogenic diolepoxide metabolites of polycyclic aromatic hydrocarbons (PAH) present in cigarette smoke. In the present study, the structural basis for these effects was investigated using a series of unnatural nucleoside analogs and a representative PAH diolepoxide, benzo[a]pyrene diolepoxide (BPDE). Synthetic DNA duplexes derived from a frequently mutated region of the p53 gene (5′-CCCGGCACCC GC[15N3,13C1-G]TCCGCG-3′, + strand) were prepared containing [15N3, 13C1]-guanine opposite unsubstituted cytosine, MeC, abasic site, or unnatural nucleobase analogs. Following BPDE treatment and hydrolysis of the modified DNA to 2′-deoxynucleosides, N2-BPDE-dG adducts formed at the [15N3, 13C1]-labeled guanine and elsewhere in the sequence were quantified by mass spectrometry. We found that C-5 alkylcytosines and related structural analogs specifically enhance the reactivity of the base paired guanine towards BPDE and modify the diastereomeric composition of N2-BPDE-dG adducts. Fluorescence and molecular docking studies revealed that 5-alkylcytosines and unnatural nucleobase analogs with extended aromatic systems facilitate the formation of intercalative BPDE–DNA complexes, placing BPDE in a favorable orientation for nucleophilic attack by the N2 position of guanine.

Supramolecular Porous Network Formed by Molecular Recognition between Chemically Modified Nucleobases Guanine and Cytosine

The involvement of surfaces in the origin of the first genetic molecules on Earth has long been suggested. Prior to the emergence of nucleic acid polymerases in the prebiotic soup, the self-assembly of primitive nucleobase building blocks may have relied on surface-mediated recognition events which catalyzed the formation of a covalent backbone in prototype oligonucleotides that subsequently may have functioned as templates in a primitive copying mechanism. This initial replication process may have been catalyzed by surfaces or chemical substances in solution—including RNA itself, as postulated in the RNA world hypothesis. Today, the role and the relative importance of the basic, fundamental driving forces for nucleic acid replication such as base pairing, base stacking, and steric effects are still under intense debate. Watson–Crick hydrogen bonding has traditionally been thought to be a prerequisite for high-fidelity DNA replication. However, recent studies on nucleobase analogues with the same size and shape as the natural ones but without relevant hydrogen-bonding groups have revealed that these analogues can recognize each other with high fidelity when incorporated into DNA sequences in vivo. Watson–Crick hydrogen bonding thus seems not to be a requisite for the selectivity of base pairing in DNA replication. However, in the absence of polymerases in the prebiotic soup, Watson– Crick hydrogen bonding may have played a more crucial role in the molecular recognition between the nucleobase building blocks at surfaces and for further polymerization. In support of this postulation, molecular recognition between complementary bases, most likely driven by hydrogen bonding alone, has already been observed both at the liquid/solid (HOPG) interface and on the noble Au(111) surface under extreme ultrahigh vacuum (UHV) conditions. These previous experiments were, however, conducted with nucleobases alone, and hence did not take the presence of deoxyribose into account. It is therefore of utmost importance to explore the role that Watson–Crick hydrogen bonding plays at surfaces in chemical structures that mimic nucleotides so as to address the fundamental question of how the polymerization of nucleotides may have started in the prebiotic soup in the absence of enzymes. The development of the scanning tunneling microscopy (STM) technique has advanced our understanding of supramolecular self-assembly systems on surfaces and has allowed intermolecular interactions to be explored at the submolecular scale. Herein we show by using a combination of high-resolution STM imaging and density functional theory (DFT) that sequential co-deposition of N-aryl-modified nucleobases cytosine (C) and guanine (G) onto the Au(111) surface under UHV conditions results in the formation of highly ordered supramolecular porous networks, where Watson–Crick hydrogen bonding between chemically modified C and G molecules plays the primary role in their stabilization. As the N-arylation of the nucleobases has been performed on the nitrogen atom normally attached to the sugar moiety in DNA or RNA (Scheme 1), these N-aryl-modified nucleobases thus represent two-dimensional (2D) structural mimics of naturally occurring nucleotides. The current results outline a new route for directing the self-assembly of nucleobase-derived nanostructures at the surface. Furthermore, the observed [*] Prof. W. Xu, Dr. M. F. Jacobsen, Dr. M. Yu, Prof. E. Laegsgaard, Prof. I. Stensgaard, Prof. T. R. Linderoth, Prof. J. Kjems, Prof. K. V. Gothelf, Prof. F. Besenbacher Interdisciplinary Nanoscience Center (iNANO) and Center for DNA Nanotechnology (CDNA), Department of Physics and Astronomy, Department of Chemistry, and Department of Molecular Biology, Aarhus University 8000 Aarhus C (Denmark) E-mail: fbe@inano.au.dk

Improved specificity of gene silencing by siRNAs containing unlocked nucleobase analogs

siRNAs confer sequence specific and robust silencing of mRNA. By virtue of these properties, siRNAs have become therapeutic candidates for disease intervention. However, their use as therapeutic agents can be hampered by unintended off-target effects by either or both strands of the siRNA duplex. We report here that unlocked nucleobase analogs (UNAs) confer desirable properties to siRNAs. Addition of a single UNA at the 5′-terminus of the passenger strand blocks participation of the passenger strand in RISC-mediated target down-regulation with a concomitant increase in guide strand activity. Placement of a UNA in the seed region of the guide strand prevents miRNA-like off-target silencing without compromising siRNA activity. Most significantly, combined substitution of UNA at the 3′-termini of both strands, the addition of a UNA at the 5′-terminus of the passenger strand, and a single UNA in the seed region of the guide strand, reduced the global off-target events by more than 10-fold compared to unmodified siRNA. The reduction in off-target events was specific to UNA placement in the siRNA, with no apparent new off-target events. Taken together, these results indicate that when strategically placed, UNA substitutions have important implications for the design of safe and effective siRNA-based therapeutics.

A New Unnatural Base Pair System between Fluorophore and Quencher Base Analogues for Nucleic Acid-Based Imaging Technology

In the development of orthogonal extra base pairs for expanding the genetic alphabet, we created novel, unnatural base pairs between fluorophore and quencher nucleobase analogues. We found that the nucleobase analogue, 2-nitropyrrole (denoted by Pn), and its 4-substitutions, such as 2-nitro-4-propynylpyrrole (Px) and 4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole (NH(2)-hx-Px), act as fluorescence quenchers. The Pn and Px bases specifically pair with their pairing partner, 7-(2,2'-bithien-5-yl)imidazo[4,5-b]pyridine (Dss), which is strongly fluorescent. Thus, these unnatural Dss-Pn and Dss-Px base pairs function as reporter-quencher base pairs, and are complementarily incorporated into DNA by polymerase reactions as a third base pair in combination with the natural A-T and G-C pairs. Due to the static contact quenching, the Pn and Px quencher bases significantly decreased the fluorescence intensity of Dss by the unnatural base pairings in DNA duplexes. In addition, the Dss-Px pair exhibited high efficiency and selectivity in PCR amplification. Thus, this new unnatural base pair system would be suitable for detection methods of target nucleic acid sequences, and here we demonstrated the applications of the Dss-Pn and Dss-Px pairs as molecular beacons and in real-time PCR. The genetic alphabet expansion system with the replicable, unnatural fluorophore-quencher base pair will be a useful tool for sensing and diagnostic applications, as well as an imaging tool for basic research.

Detailed analytical study of radiolysis products of simple organic compounds as a methodological approach to investigate prebiotic chemistry—Part 1

Aqueous solutions of simple organic C1 compounds (methanol and acetonitrile) and ammonia, presumably present on primordial Earth, were subjected to 60Co gamma irradiation (total dose 800 kGy). The irradiation gave a complex mixture of organic compounds leading interestingly to a positive balance of synthesis vs. degradation reactions. In particular, if acetonitrile was used, nucleobase analogues could be detected among products. Highly sensitive and powerful analytical techniques (e.g. GC–MS, HPLC-MS) made this investigation feasible at a reasonable cost in terms of time and results. Plausible reaction pathways leading to major compounds were proposed, supported by literature data.

The crystal structure of non-modified and bipyridine-modified PNA duplexes.

Peptide nucleic acid (PNA) is a synthetic analogue of DNA that commonly has an N-aminoethyl glycine backbone. The crystal structures of two PNA duplexes, one containing eight standard nucleobase pairs (GGCATGCC)(2), and the other containing the same nucleobase pairs and a central pair of bipyridine ligands, have been solved with a resolution of 1.22 and 1.10 Å, respectively. The non-modified PNA duplex adopts a P-type helical structure similar to that of previously characterized PNAs. The atomic-level resolution of the structures allowed us to observe for the first time specific modes of interaction between the terminal lysines of the PNA and the backbone and the nucleobases situated in the vicinity of the lysines, which are considered an important factor in the induction of a preferred handedness in PNA duplexes. Our results support the notion that whereas PNA typically adopts a P-type helical structure, its flexibility is relatively high. For example, the base-pair rise in the bipyridine-containing PNA is the largest measured to date in a PNA homoduplex. The two bipyridines bulge out of the duplex and are aligned parallel to the major groove of the PNA. In addition, two bipyridines from adjacent PNA duplexes form a π-stacked pair that relates the duplexes within the crystal. The bulging out of the bipyridines causes bending of the PNA duplex, which is in contrast to the structure previously reported for biphenyl-modified DNA duplexes in solution, where the biphenyls are π stacked with adjacent nucleobase pairs and adopt an intrahelical geometry. This difference shows that relatively small perturbations can significantly impact the relative position of nucleobase analogues in nucleic acid duplexes.

DNA Polymerase Recognition of Unnatural Base Pair: 1-Methoxy-3-methylbenzene and 6-Methyl-1(2H)-isoquinolinone

The faithful replication of DNA by DNA polymerases is necessary for the expansion of both the genetic alphabet and the genetic code. 1 The stability and sequence specificity of a natural DNA duplex are based on the complementary and orthogonal Watson-Crick hydrogen-bonding patterns of dA:dT and dC:dG base pairs. Although inter-nucleobase hydrogen bonding and shape complementarity are important for the stability and faithful copying of DNA, there is no reason to assume that the pairing of two unnatural bases could not be driven by other intermolecular interactions, for example, those based on hydrophobic interactions and van der Waals forces. Some groups have used this concept to explore the development of unnatural nucleotides bearing nucleobase analogs that pair via hydrophobic interactions and packing forces. 2-5 Recently, Romesberg’s group found that the heteropair formed between 1-methoxy-3-methylbenzene 1 and 6-methyl-1(2H)-isoquinolinethione 2 is a very promising, predominantly hydrophobic base pair. This base pair is synthesized by the insertion of one unnatural triphosphate opposite the other unnatural nucleotide in the template, and it is then extended by the insertion of the next correct dNTP (natural triphosphates), with relatively high efficiency and fidelity, by diverse DNA polymerases (Figure 1). 5 However, the slowest step in the replication of DNA containing this heteropair is the incorporation of 1 triphosphate opposite 2 in the template. Also, dG inserts competitively opposite 2 in the template, which may limit the use of this heteropair in biological and biotechnological applications. We are therefore interested in the modifications of 1 or 2 to optimize the formation of unnatural heteropairs. Herein, we report the DNA replication results for two nucleotides, 6-methyl-1(2H)-isoquinolinone 3, in which an oxygen atom is substituted for the sulfur atom in 2 to compare the effect of the oxygen atom on replication properties, and 7-methyl-1(2H)isoquinolinone 4, in which, as well as replacing the sulfur atom in 2 by an oxygen atom, the methyl substituent is moved from position 6 to position 7. The substitution at position 7 is expected to bury the methyl group within the duplex, as shown

Synthesis, Characterization, and Binding Property of Isoelectronic Analogues of Nucleobases, B(6)-Substituted 5-Aza-6-borauracils and -thymines

As isoelectronic BN-containing analogues of 6-substituted uracil and thymine, a series of B(6)-substituted 5-aza-6-borauracils (U(BN)s) and -thymines (T(BN)s) were synthesized and fully characterized. The crystallographic and spectroscopic analyses of the analogues revealed that the framework and hydrogen-bonding pattern of T(BN)s were similar to those of the original nucleobase, thymine.

Abstract 1697: A study on the reactivity of activated polycyclic aromatic hydrocarbons (PAH) with guanines base pared to C-5 substituted cytosines

Abstract All CG dinucleotides within exons 5-8 of the human p53 tumor suppressor gene contain endogenous 5-methylcytosine (MeC). Guanine residues within these sites (e.g. codons 157, 158, 245, 248, and 273) are the major mutational hotspots for smoking induced lung cancer, suggesting that MeC mediates the reactivity of neighboring guanine bases towards tobacco carcinogens. Previous studies have shown that the reactivity of a carcinogenic diol epoxide metabolite of the human carcinogen, benzo[a]pyrene (B[a]P), towards the exocyclic amino group of guanine, is increased when guanine is base paired to MeC as compared to unmethylated cytosine. In the present study, the structural basis for the enhanced reactivity of BPDE towards MeC: G base pairs was investigated using a stable isotope labeling approach and a series of MeC structural analogs, including 5-ethyl-dC, 5-propyl-dC, N4-ethyl-dC, 5-chloro-dC, 5-bromo-dC, 5-iodo-dC, 5-propynyl-dC, difluorotoluene, pyrrolo-dC, phenylpyrrolo-dC, and diaminonaphthyl-derived nucleoside. Synthetic DNA duplexes derived from the frequently mutated region of the p53 tumor suppressor gene (5′-CCCGGCACCCGC[15N3, 13C1-G]TCCGCG-3′, from exon 5) were prepared containing [15N3, 13C1]-labeled guanine opposite C, MeC, or nucleobase analogs. Circular dichroism (CD) and UV melting studies have shown that C-5 substituents on cytosine do not disrupt the structure and stability of the DNA duplex. Following treatment with (±)-anti-benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide [(±)-anti-BPDE], (-)-anti-benzo[a]pyrene-s-7,t-8-dihydrodiol-t-9,10-epoxide [(-)-anti-BPDE], or related PAH diol epoxides, 5-methyl chrysene diol epoxide, benzo[c]phenanthrene diol epoxide, benzo[g]chrysene diol epoxide, and benzo[a,l]pyrene diol epoxide, and enzymatic hydrolysis of the adducted DNA to 2′-deoxynucleosides, the amounts of stereoisomeric N2-guanine adducts formed at the labeled site were determined by capillary HPLC-ESI+-MS/MS. We found that the presence of 5-methylcytosine and nucleobases with extended aromatic systems increases the reactivity of the partner guanine towards BPDE and other PAH diolepoxides, while 5-fluoro-dC and 5-iodo-dC lead to a decreased reactivity. Furthermore, the presence of C-5-cytosine analog modifies the stereoisomeric composition of the resulting adducts. Low temperature fluorescence and molecular docking studies reveal that the presence of MeC and unnatural base analogs with extended aromatic systems facilitate the formation of the pre-covalent BPDE-DNA complexes which place BPDE in a favorable orientation for trans attack by the N2 position of guanine. These results provide a mechanistic insight into the origins of increased reactivity of PAHs towards MeCG dinucleotides. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1697.

Abstract 581: Development of RNA interference-based therapeutics for bladder cancer and hepatocellular carcinoma

Abstract Bladder cancer and hepatocellular carcinoma are two of many cancers refractory to current treatments. Small interfering RNAs (siRNAs) are a new therapeutic modality able to specifically silence expression of targets not accessible via current small molecule and antibody options. MDRNA is developing UsiRNAs, a novel siRNA construct containing unlocked nucleobase analogs, with improved specificity for RNA interference (RNAi). Delivery of UsiRNAs to target tissues is achieved using proprietary Di-alkylated Amino Acid (DiLA2)-based liposomes. Survivin, overexpressed in many cancers, is involved in cell division and inhibition of apoptosis. In orthotopic and xenograft models of liver cancer, systemic administration of survivin UsiRNA-DiLA2 liposomes resulted in approximately 60% and 70% reductions in survivin mRNA, respectively, and > 50% decreases in tumor weight. Local intravesical administration in an orthotopic bladder cancer model resulted in 90% inhibition of mRNA expression and substantial tumor growth inhibition. Polo-like kinase 1 (PLK-1), also elevated in many tumors, regulates cell cycle progression and mitosis. Treatment of bladder cancer and liver cancer cell lines with PLK-1 UsiRNA/DiLA2 liposomes leads to significant caspase activation and corresponding apoptotic cell death. In vivo studies with PLK-1 UsiRNA are in progress. These UsiRNAs and UsiRNAs directed against additional targets are being evaluated in vitro and in orthotopic and xenograft models of cancer as single agents, in combinations of target-specific UsiRNAs, and with existing small molecule and antibody therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 581.

A Unique Fluorescent Base Analogue for the Expansion of the Genetic Alphabet

Fluorescent nucleobase analogues are useful in a wide variety of biology and biotechnology tools as molecular probes and reporters for nucleic acids. Here we present a novel fluorescent purine analogue, 7-(2,2'-bithien-5-yl)-imidazo[4,5-b]pyridine (denoted as Dss). The nucleoside triphosphates of Dss can be site-specifically incorporated into DNA and RNA by polymerases, opposite its pairing partner, pyrrole-2-carbaldehyde (Pa), in DNA templates. Despite its high specificity in replication and transcription, Dss in oligonucleotides functions as a universal base that pairs with all four natural bases with nearly equal thermal stabilities. Thus, Dss would be a powerful tool for fluorescent base replacements at specific positions in functional DNA and RNA molecules.

Identification and Functional Characterization of the First Nucleobase Transporter in Mammals: IMPLICATION IN THE SPECIES DIFFERENCE IN THE INTESTINAL ABSORPTION MECHANISM OF NUCLEOBASES AND THEIR ANALOGS BETWEEN HIGHER PRIMATES AND OTHER MAMMALS

Nucleobases are important compounds that constitute nucleosides and nucleic acids. Although it has long been suggested that specific transporters are involved in their intestinal absorption and uptake in other tissues, none of their molecular entities have been identified in mammals to date. Here we describe identification of rat Slc23a4 as the first sodium-dependent nucleobase transporter (rSNBT1). The mRNA of rSNBT1 was expressed highly and only in the small intestine. When transiently expressed in HEK293 cells, rSNBT1 could transport uracil most efficiently. The transport of uracil mediated by rSNBT1 was sodium-dependent and saturable with a Michaelis constant of 21.2 microM. Thymine, guanine, hypoxanthine, and xanthine were also transported, but adenine was not. It was also suggested by studies of the inhibitory effect on rSNBT1-mediated uracil transport that several nucleobase analogs such as 5-fluorouracil are recognized by rSNBT1, but cytosine and nucleosides are not or only poorly recognized. Furthermore, rSNBT1 fused with green fluorescent protein was mainly localized at the apical membrane, when stably expressed in polarized Madin-Darby canine kidney II cells. These characteristics of rSNBT1 were almost fully in agreement with those of the carrier-mediated transport system involved in intestinal uracil uptake. Therefore, it is likely that rSNBT1 is its molecular entity or at least in part responsible for that. It was also found that the gene orthologous to the rSNBT1 gene is genetically defective in humans. This may have a biological and evolutional meaning in the transport and metabolism of nucleobases. The present study provides novel insights into the specific transport and metabolism of nucleobases and their analogs for therapeutic use.

Photophysical and structural properties of the fluorescent nucleobase analogues of the tricyclic cytosine (tC) family

Fundamental insight into the unique fluorescence and nucleobase-mimicking properties of the fluorescent nucleobase analogues of the tC family is not only vital in explaining the behaviour of these probes in nucleic acid environments, but will also be profitable in the development of new and improved fluorescent base analogues. Here, temperature-dependent fluorescence quantum yield measurements are used to successfully separate and quantify the temperature-dependent and temperature-independent non-radiative excited-state decay processes of the three nucleobase analogues tC, tC(O) and tC(nitro); all of which are derivatives of a phenothiazine or phenoxazine tricyclic framework. These results strongly suggest that the non-radiative decay process dominating the fast deactivation of tC(nitro) is an internal conversion of a different origin than the decay pathways of tC and tC(O). tC(nitro) is reported to be fluorescent only in less dipolar solvents at room temperature, which is explained by an increase in excited-state dipole moment along the main non-radiative decay pathway, a suggestion that applies in the photophysical discussion of large polycyclic nitroaromatics in general. New insight into the ground and excited-state potential energy surfaces of the isolated tC bases is obtained by means of high level DFT and TDDFT calculations. The S(0) potential energy surfaces of tC and tC(nitro) possess two global minima corresponding to geometries folded along the middle sulfur-nitrogen axis separated by an energy barrier of 0.05 eV as calculated at the B3LYP/6-311+G(2d,p) level. The ground-state potential energy surface of tC(O) is also predicted to be shallow along the bending coordinate but with an equilibrium geometry corresponding to the planar conformation of the tricyclic framework, which may explain some of the dissimilar properties of tC and tC(O) in various confined (biological) environments. The S(1) equilibrium geometries of all three base analogues are predicted to be planar. These results are discussed in the context of the tC bases positioned in double-stranded DNA scenarios.

Fluorescent nucleoside analogue displays enhanced emission upon pairing with guanine.

A fluorescent nucleobase analogue, 7-aminoquinazoline-2,4-(1H,3H)-dione, is incorporated into a DNA oligonucleotide and senses mismatched pairing by displaying G-specific fluorescence enhancement.

The First All-Nucleobase Analog FRET Pair

The fluorescent nucleobase analogs of the tricyclic cytosine (tC) family are promising nucleic acid probes capable of being inserted into double-stranded DNA as a replacement for one of the natural bases without perturbing the overall double helical structure. The high fluorescence quantum yields in both single- and double stranded DNA, combined with a rigid and well-defined position inside the DNA double helix, make these molecules particularly well suited as fluorescence resonance energy transfer (FRET) probes in nucleic acid studies. Recently we reported the first all-nucleobase analog FRET-pair, consisting of tCO as the donor and the newly developed tCnitro as acceptor which will be the focus of this presentation.1-3 The FRET-pair successfully monitors distances covering up to more than one turn of the DNA duplex and, more importantly, the rigid stacking of the two base analogs, and consequently excellent control of the their exact positions, results in a very high control of the orientation factor in the FRET efficiency. A set of DNA strands containing the FRET-pair at wisely chosen locations will, thus, make it possible to accurately distinguish distance- from orientation-changes using FRET. We believe the development of this new tool opens up a wide range of possibilities in the structural investigation of nucleic acids, e.g. in characterizing DNA-protein complexes and in monitoring the inherent dynamics and the structural changes of nucleic acids in response to all kinds of stimuli.

Characterization of Nucleobase Analogue FRET Acceptor tCnitro

The fluorescent nucleobase analogues of the tricyclic cytosine (tC) family, tC and tC(O), possess high fluorescence quantum yields and single fluorescence lifetimes, even after incorporation into double-stranded DNA, which make these base analogues particularly useful as fluorescence resonance energy transfer (FRET) probes. Recently, we reported the first all-nucleobase FRET pair consisting of tC(O) as the donor and the novel tC(nitro) as the acceptor. The rigid and well-defined position of this FRET pair inside the DNA double helix, and consequently excellent control of the orientation factor in the FRET efficiency, are very promising features for future studies of nucleic acid structures. Here, we provide the necessary spectroscopic and photophysical characterization of tC(nitro) needed in order to utilize this probe as a FRET acceptor in nucleic acids. The lowest energy absorption band from 375 to 525 nm is shown to be the result of a single in-plane polarized electronic transition oriented approximately 27 degrees from the molecular long axis. This band overlaps the emission bands of both tC and tC(O), and the Forster characteristics of these donor-acceptor pairs are calculated for double-stranded DNA scenarios. In addition, the UV-vis absorption of tC(nitro) is monitored in a broad pH range and the neutral form is found to be totally predominant under physiological conditions with a pK(a) of 11.1. The structure and electronic spectrum of tC(nitro) is further characterized by density functional theory calculations.

Evolving a polymerase for hydrophobic base analogues.

Hydrophobic base analogues (HBAs) have shown great promise for the expansion of the chemical and coding potential of nucleic acids but are generally poor polymerase substrates. While extensive synthetic efforts have yielded examples of HBAs with favorable substrate properties, their discovery has remained challenging. Here we describe a complementary strategy for improving HBA substrate properties by directed evolution of a dedicated polymerase using compartmentalized self-replication (CSR) with the archetypal HBA 5-nitroindole (d5NI) and its derivative 5-nitroindole-3-carboxamide (d5NIC) as selection substrates. Starting from a repertoire of chimeric polymerases generated by molecular breeding of DNA polymerase genes from the genus Thermus, we isolated a polymerase (5D4) with a generically enhanced ability to utilize HBAs. The selected polymerase. 5D4 was able to form and extend d5NI and d5NIC (d5NI(C)) self-pairs as well as d5NI(C) heteropairs with all four bases with efficiencies approaching, or exceeding, those of the cognate Watson-Crick pairs, despite significant distortions caused by the intercalation of the d5NI(C) heterocycles into the opposing strand base stack, as shown by nuclear magnetic resonance spectroscopy (NMR). Unlike Taq polymerase, 5D4 was also able to extend HBA pairs such as Pyrene: varphi (abasic site), d5NI: varphi, and isocarbostyril (ICS): 7-azaindole (7AI), allowed bypass of a chemically diverse spectrum of HBAs, and enabled PCR amplification with primers comprising multiple d5NI(C)-substitutions, while maintaining high levels of catalytic activity and fidelity. The selected polymerase 5D4 promises to expand the range of nucleobase analogues amenable to replication and should find numerous applications, including the synthesis and replication of nucleic acid polymers with expanded chemical and functional diversity.

Self-Assembly of Artificial Nucleobase 1H-Benzimidazole-4,7-dione at the Liquid/Solid Interface

Self-assembly at the liquid/solid interface of an electrochemically active DNA nucleobase analogue, 1H-benzoimidazole-4,7-dione (Q), has been studied by means of scanning tunneling microscopy (STM). High-resolution STM images revealed the formation of well-ordered two-dimensional (2D) supramolecular nanostructures when the Q molecules are adsorbed onto the graphite surface from a 1-octanol solution. Detailed analysis shows that the observed 2D nanostructures are mainly dominated by hydrogen-bonded Q molecules. Since Q can be considered as a molecule mimicking the nucleobase guanine (G), which is known to form Watson-Crick base pairs with its complementary nucleobase cytosine (C), we have examined the binding ability of Q with C realized by available hydrogen-bonding sites on both Q and C molecules. Upon deposition of a mixture of Q and C molecules onto a graphite surface, one might expect that hydrogen-bonded QC dimers were observed in a new 2D self-assembled structure governed by inter- and intramolecular hydrogen-bonding interactions between Q and C molecules. However, our STM experiments showed that no well-ordered structures are formed and instead phase separation occurs where large-scale homodomains are formed consisting of the individual QQ and CC dimers. To gain further insight into the possible molecular arrangements of the Q and C nucleobases in the mixture phase, the high-resolution STM images are compared with the results from ab initio density functional theory (DFT) calculations.