Polymerase Recognition of Unnatural Base Pairs

Abstract

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...

Expanding the Scope of Replicable Unnatural DNA: Stepwise Optimization of a Predominantly Hydrophobic Base Pair

“…Previous structure-activity relationship (SAR) data indicate that the ortho methoxy group of the d MMO2 scaffold is necessary for efficient replication, 8 , 12 , 13 and that substituents at the adjacent meta position are not well tolerated. 14–16 Thus modification at the para - and remaining meta -position of the d MMO2 scaffold appears to be most promising for optimization.…”

Major Groove Substituents and Polymerase Recognition of a Class of Predominantly Hydrophobic Unnatural Base Pairs

“…For over two decades, efforts have been focused on the development of unnatural base pairs where pairing is mediated by orthogonal H-bonding patterns, and progress along this route continues. [1-4] However, we [5-16] and others, [17-21] have demonstrated that hydrophobic and packing forces are also sufficient to underlie the efficient and selective replication of an unnatural base pair. If sufficiently well replicated, such unnatural base pairs could be used as part of an expanded genetic system and would allow for an increase in the information potential of a genome, but will likely be useful immediately for in vitro applications, such as the site-specific labeling of enzymatically synthesized DNA or RNA with novel functionality ( i.e .…”

Optimization of an Unnatural Base Pair toward Natural-Like Replication

“…the methoxy group of d MMO2 and the sulfur atom of d 5SICS , are essential for polymerase recognition 10,11,13. These substituents likely act as H-bond acceptors in the developing minor groove where they are required to engage polymerase-based H-bond donors 28–31.…”