DNA polymerases engineered by directed evolution to incorporate non-standard nucleotides

Abstract

DNA polymerases have evolved for billions of years to accept natural nucleoside triphosphate substrates with high fidelity and to exclude closely related structures, such as the analogous ribonucleoside triphosphates. However, polymerases that can accept unnatural nucleoside triphosphates are desired for many applications in biotechnology. The focus of this review is on non-standard nucleotides that expand the genetic "alphabet." This review focuses on experiments that, by directed evolution, have created variants of DNA polymerases that are better able to accept unnatural nucleotides. In many cases, an analysis of past evolution of these polymerases (as inferred by examining multiple sequence alignments) can help explain some of the mutations delivered by directed evolution.

Keywords: AEGIS; CSR; DNA polymerases; directed evolution; non-standard nucleotides; protein engineering.

FIGURE 1

Structure of Taq DNA polymerase showing the conserved motifs: (A) (LLVALDYSQIELR) – yellow; (B) (RRAAKTINFGVLY) – orange; (C) (LLQVHDELVLE) – pink. PDB ID 4DLG, which includes Taq beginning from the RNaseH portion till the end, in complex with a DNA primer and a DNA template, halted by/at a ddCTP.

FIGURE 2

Evolution can be modeled as a walk across a fitness landscape, here presented as a two-dimensional representation of a multiple dimensional hypersurface; analogous to a topographic map, peaks (+) indicate the locations where function exist while dips (–) represent regions with lack of function. Illustrated through an analogy to a word game, a meaningful (functional) string of letters (here “word”) must be reached starting from another string (“gene”) via stepwise replacement of single letters, where every intermediate along the path must itself also be a functional word. Solid arrows indicate a path of accepted mutations while dashed arrows illustrate deleterious mutations that produce non-functional proteins.

FIGURE 3

Compartmentalized self replication (CSR) system experiments start with the creation of a library of genes encoding variants of a polymerase. Members of this library are introduced into E. coli cells by electroporation. Here, just two variant genes (red and blue) are represented. These genes drive the expression of mutant polymerases in each E. coli cell, each of which is isolated in its own water-in-oil-emulsion droplet. (B) The first cycle of PCR breaks the cell wall of the E. coli, exposing the expressed polymerase molecules and their gene to the contents of a water droplet containing all of the necessary components necessary for a PCR amplification: (i) primers, (ii) dNTPs, (iii) a mutated gene of the polymerase, and (iv) the enzyme expressed by this gene (C). During PCR, any polymerases active under the selective pressure (blue) amplify their respective genes, enriching the pool of mutants having the desired properties; inactive polymerases (red) fail to do so (D). The emulsion is then broken and the amplified genes enriched in those encoding polymerases having the desired behaviors are extracted and inserted in a plasmid vector [circular DNA; E]. These then enter the cycle of selection again (A). After repeating these cycles an enriched pool of variants of the original gene are produced.

FIGURE 4

The Z:P pair from an artificially expanded genetic information system (AEGIS, left) showing in green the orbitals containing the unshared electron pairs presented to the minor groove of DNA. The natural C:G pair (right) showing these unshared pairs of electrons in the standard C:G pair. The pattern of hydrogen bonding is indicated A, hydrogen bond acceptor and D, hydrogen bond donor.