Six unnatural nucleotides featuring fluorine-substituted phenyl nucleobase analogues have been synthesized, incorporated into DNA, and characterized in terms of the structure and replication properties of the self-pairs they form. Each unnatural self-pair is accommodated in B-form DNA without detectable structural perturbation, and all are thermally stable and selective to roughly the same degree. Furthermore, the efficiency of polymerase-mediated mispair synthesis is similar for each unnatural nucleotide in the template. In contrast, the efficiency of polymerase-mediated self-pair extension is highly dependent on the specific fluorine substitution pattern. The most promising unnatural base pair candidate of this series is the 3-fluorobenzene self-pair, which is replicated with reasonable efficiency and selectivity.
Expansion of the genetic alphabet has been a long time goal of chemical biology. A third DNA base pair that is stable and replicable would have a great number of practical applications and would also lay the foundation for a semi-synthetic organism. We have reported that DNA base pairs formed between deoxyribonucleotides with large aromatic, predominantly hydrophobic nucleobase analogs, such as propinyl isocarbostyril (dPICS), are stable and efficiently synthesized by DNA polymerases. However, once incorporated into the primer, these analogs inhibit continued primer elongation. More recently, we have found that DNA base pairs formed between nucleobase analogs that have minimal aromatic surface area in addition to little or no hydrogen-bonding potential, such as 3-fluoro benzene (d3FB), are synthesized and extended by DNA polymerases with greatly increased efficiency. Here we show that the rate of synthesis and extension of the self pair formed between two d3FB analogs is sufficient for in vitro DNA replication. To better understand the origins of efficient replication, we examined the structure of DNA duplexes containing either the d3FB or dPICS self pairs. We find that the large aromatic rings of dPICS pair in an intercalative manner within duplex DNA, while the d3FB nucleobases interact in an edge-on manner, much closer in structure to natural base pairs. We also synthesized duplexes containing the 5-methyl substituted derivatives of d3FB (d5Me3FB) paired opposite d3FB or the unsubstituted analog (dBEN). In all, the data suggest that structure, electrostatics and dynamics can all contribute to the extension of unnatural primer termini. The results also help explain the replication properties of many previously examined unnatural base pairs and should help design unnatural base pairs that are better replicated.
The stability and replication of DNA containing self-pairs formed between unnatural nucleotides bearing benzofuran, benzothiophene, indole, and benzotriazole nucleobases are reported. These nucleobase analogues are based on a similar scaffold but have different hydrogen-bond donor/acceptor groups that are expected to be oriented in the duplex minor groove. The unnatural base pairs do not appear to induce major structural distortions and are accommodated within the constraints of a B-form duplex. The differences between these unnatural base pairs are manifest only in the polymerase-mediated extension step, not in base-pair stability or synthesis. The benzotriazole self-pair is extended with an efficiency that is only 200-fold less than a correct natural base pair. The data are discussed in terms of available polymerase crystal structures and imply that further modifications may result in unnatural base pairs that can be both efficiently synthesized and extended, resulting in an expanded genetic alphabet.
The storage and replication of genetic information requires bases that pair stably and selectively in duplex DNA and are also good substrates for DNA polymerases. Though the four natural bases pair by hydrogen bonding, 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 hydrophobicity. [1, 2] To investigate this issue, a wide variety of hydrophobic base pairs has been characterized. [3,4] For example, ICS (see Figure 1) and its derivative PICS, with a propynyl group at C7, form self-pairs in duplex DNA that are as stable as natural base pairs. The unnatural triphosphates are also inserted opposite themselves in the COMMUNICATIONS 3841 conversion by removal of ethylene gas, [3] ROIMP gives high conversion under gentle reflux conditions for two reasons. First, ROMP of monomer B is efficient in making the initial homopolycycloalkene chains. Second, the formation of 1,2-disubstituted a,b-unsaturated carbonyl compounds is thermodynamically favored by more than 3 kcal mol À1 per bond. [11] These enthalpic factors, combined with the loss of ethylene, drive the reaction to high conversion. Furthermore, the unfavorable oligomerization of diacrylates, in which the intermediate is an unstable enoic carbene, leads to high A,B-alternation. [12] Therefore, ROIMP has benefits of both chain-growth and step-growth polymerization, leading to high molecular weight and high selectivity.To optimize conversion, other polymerization conditions were investigated. It was found that 0.1±0.5 m solutions in CH 2 Cl 2 at 40 8C give the best results. In contrast to ROMP, increasing the concentration beyond 0.5 m resulted in lower conversion. Switching to toluene or 1,2-dichloroethane as solvent also gave lower conversion at either 40 8C or 608C. Although there is precedence for CH 2 Cl 2 being the best solvent for cross metathesis of functionalized olefins, [12] the concentration dependence for ROIMP is somewhat surprising, since concentrations of 0.1±0.5 m are considered dilute conditions for conventional step-growth-polymerization reactions.Controlling the molecular weight of polymers is a very important issue since polymers with different molecular weights often exhibit different properties. For alternating copolymers produced by ROIMP, molecular weight can be roughly controlled by changing the relative stoichiometry of the two monomers. For example, using 0.96 equivalents of cyclooctene to 1.0 equivalent of hydroquinone diacrylate gave a copolymer of 17 800 g mol À1 with 98 % A,B-alternation (PDI ¼ 1.64), whereas a copolymer of 45 200 g mol À1 and 95.5 % A,B-alternation (PDI ¼ 1.69) was obtained by increasing the cyclooctene to 1.06 equivalents. These results show that when compared with the 1:1 case (Table 1, entry 7), an excess of hydroquinone diacrylate shortens the polymer chain, but an excess of cyclooctene gives higher molecular weight as a result of the oligomeric blocks of polycyclooctene.In conclusion, we have demonstrated a n...
Six new unnatural nucleobases have been synthesized and characterized in terms of stability and selectivity of self-pairing in duplex DNA and efficiency and fidelity of self-pairing during polymerase-mediated replication. Each nucleobase has a conserved ring structure but differs from the others in its specific pattern of substitution with oxygen and sulfur atoms. Heteroatom derivatization within the conserved scaffold is shown to have only moderate effects on unnatural self-pair synthesis by the polymerase; larger effects were observed on the thermal stability and polymerase-mediated extension of the self-pairs. The largest effects of heteroatom substitution were on the stability and synthesis of mispairs between the unnatural and natural bases. Certain heteroatom substitutions were found to have a general effect while others were found to have effects that were specific for a particular unnatural or natural base. The data are useful for designing stable and replicable third base pairs and for understanding the contributions of nucleobase shape, polarity, and polarizability to the stability and replication of DNA.
E. coli DNA photolyase is a monomeric light-harvesting enzyme that utilizes a methenyltetrahydrofolate (MTHF) antenna cofactor to harvest light energy for the repair of thymine dimers in DNA. For this purpose, the enzyme evolved to bind the cofactor and red-shift its absorption maximum by 25 nm. Using the crystal structure as a guide, we mutated each protein residue that contacts the cofactor in an effort to identify the interactions responsible for this selective stabilization of the cofactor's excited state. Hydrogen bonding, packing, and electrostatic interactions were examined. Remarkably, a single residue, Glu109, appears to play an important, if not exclusive, role in inducing the observed red-shift. Thus, this protein, the simplest light-harvesting system known, appears to have evolved a remarkably simple mechanism to tune the photophysical properties of the antenna cofactor appropriately for biological function.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.