Polymerase Evolution:  Efforts toward Expansion of the Genetic Code

Abstract

Genetic information is encoded by, but potentially not limited to a four-letter alphabet. A variety of predominantly hydrophobic nucleobase analogs that form self-pairs in DNA have been examined as third base pair candidates. For example, the PICS self-pair is both stable in duplex DNA and synthesized by some wild-type polymerases with reasonable efficiency. These efforts to expand the genetic code are expected to be facilitated by optimizing both the unnatural nucleobase analogs and the polymerases that replicate them. Here, we report the use of an activity-based selection system to evolve a DNA polymerase that more efficiently replicates DNA containing the PICS self-pair. The selection system is based on the co-display on phage of DNA polymerase libraries and a DNA substrate containg the self-pair. Only polymerases that accept the unnatural substrate incorporate a biotin-dUTP to the attached primer and may then be isolated on a streptavidin solid support. A mutant of Sf polymerase, P2, was evolved which both inserts dPICSTP opposite dPICS in the template and extends the unnatural primer terminus by incorporation of the next correct natural dNTP, where the parental enzyme catalyzes neither step at detectable rates. P2 was found to be a triple mutant of Sf, with the mutations F598I, I614F, and Q489H. The evolved properties of P2, as well as the observed mutations are consistent with an increased affinity for the DNA primer-template containing the selfpair.The biological system of information storage, based on the selective Watson-Crick hydrogen bonding (H-bonding) of adenine with thymine (dA:dT base pair) and guanine with cytosine (dG:dC base pair), has been conserved throughout nature. Expansion of the alphabet to contain a third base pair would allow additional information to be encoded in DNA and would also enable a variety of in vitro experiments using unnatural nucleic acids. 1 A priori, there is no reason to assume that the requirements for duplex stability and replication must limit the genetic To whom correspondence should be addressed. E-mail floyd@scripps.edu. One strategy for improving the replication of these unnatural base pairs is based on their further derivatization with heteroatoms or alkyl substituents. 6, 7 This strategy mimics the optimization of the natural bases in nature. However, in nature, base pair optimization proceeded simultaneously with DNA polymerase evolution. Thus, any expansion of the genetic code is expected to be facilitated by optimizing both the unnatural nucleobase analogs and the polymerases that replicate them. Here, we report our initial efforts towards the directed evolution of polymerases that more efficiently synthesize DNA containing the PICS self-pair. NIH Public AccessPreviously, we reported a phage display selection system designed to evolve DNA or RNA polymerases with novel activities. 8,9 We used the selection system to evolve variants of Sf that efficiently synthesize RNA 8 or DNA containing C2'-O-methyl modified nucleotides. 9The selection system is ba...

Discovery, Characterization, and Optimization of an Unnatural Base Pair for Expansion of the Genetic Alphabet

“…extension, tends to be relatively inefficient and generally limits the utility of these base pairs. Recent efforts to modify either the nucleobases [20][21][22][23][24] or the DNA polymerase 25 have significantly improved the rate of extension of unnatural base pairs and have demonstrated that efficient extension by the exonuclease deficient Klenow fragment of E. coli DNA Pol I (Kf) likely requires a minimally distorted primer terminus with a suitably positioned minor groove H-bond acceptor in the primer nucleobase. Unfortunately, these strategies have yet to yield a viable base pair candidate as the modifications that facilitate extension have also been found to limit synthesis and increase mispairing.…”

2‐Phenanthrenyl–DNA: Synthesis, Pairing, and Fluorescence Properties

“…Most of these investigations were driven by the search for additional base pairs to be used for the extension of the genetic alphabet, [1][2][3][4][5][6][7][8] as tools in biotechnology, [9][10][11] for probing recognition, fidelity, and nucleotide processing by DNA polymerases, [12][13][14][15] or for designing novel genetic systems. [16,17] Of special interest amongst these artificial constructs are aromatic base replacements that interact with each other, specifically without the formation of hydrogen bonds, merely on the basis of edge-on or face-on hydrophobic or stacking interactions.…”

A general strategy for the evolution of bond-forming enzymes using yeast display

“…In principle, chemical complementation using an adapted yeast three-hybrid assay is reaction-independent (14) but requires membrane-permeable substrates and offers limited control over reaction conditions because the bond-forming event must take place intracellularly. Phage-display and mRNA-display systems that are general for any bond-forming reaction have been used to evolve enzymes including DNA polymerases (15) and RNA ligases (16). These approaches also offer advantages of larger library sizes and significant control over reaction conditions because the enzymes are displayed extracellularly or expressed in the absence of a host cell.…”