Directed polymerase evolution

Abstract

Polymerases evolved in nature to synthesize DNA and RNA, and they underlie the storage and flow of genetic information in all cells. The availability of these enzymes for use at the bench has driven a revolution in biotechnology and medicinal research; however, polymerases did not evolve to function efficiently under the conditions required for some applications and their high substrate fidelity precludes their use for most applications that involve modified substrates. To circumvent these limitations, researchers have turned to directed evolution to tailor the properties and/or substrate repertoire of polymerases for different applications, and several systems have been developed for this purpose. These systems draw on different methods of creating a pool of randomly mutated polymerases and are differentiated by the process used to isolate the most fit members. A variety of polymerases have been evolved, providing new or improved functionality, as well as interesting new insight into the factors governing activity.

Reverse Transcription of Threose Nucleic Acid by a Naturally Occurring DNA Polymerase

“…[1][2][3] Although such systems have been used to evolve XNA molecules with ligand binding and catalytic activities, [4][5][6] the current generation of engineered polymerases function with reduced activity relative to natural polymerases. [1][2][3] Although such systems have been used to evolve XNA molecules with ligand binding and catalytic activities, [4][5][6] the current generation of engineered polymerases function with reduced activity relative to natural polymerases.…”

“…Recent advances in polymerase engineering have enabled the replication of aw ide range of xenonucleic acid (XNA) polymers with backbones tructures that are unique relative to those found in natural DNA and RNA. [1][2][3] Although such systems have been used to evolve XNA molecules with ligand binding and catalytic activities, [4][5][6] the current generation of engineered polymerases function with reduced activity relative to natural polymerases. [7,8] The development of XNA polymerasesw ith strong reverse transcriptase activity,i np articular, has posed a significant bottleneck in the in vitro replicationo fX NA polymers.…”

Design and Discovery of New Combinations of Mutant DNA Polymerases and Modified DNA Substrates

“…First, we examined the ability of SFM4‐3, SFM4‐6, SFM4‐9, and these three enzymes to synthesize 2′‐F substituted DNA, by incubating the enzymes with 2′F‐NTPs (Figure ). We chose 2′F DNA because it has been shown to have nuclease resistance, and we previously showed it to be the most efficiently in the synthesis of M‐DNA by SFM19. SFM4‐3, SFM4‐6, and SFM4‐9 had not been previously examined for their ability to synthesize fully 2′F‐substituted DNA.…”

Expanding functional protein sequence space using generative adversarial networks

“…Due to the exponential decline in protein fitness with the number of random mutations 11,35 or the number of parent molecules that are used to generate the recombination libraries 36,37 , current methods are fundamentally limited in the sequence space that can be explored by their use. The expanded functional sequence diversity provided by ProteinGAN (Figure 2b,c) may also provide suitable, non-natural, starting points for protein engineering 38 , with great potential for applications in biocatalysis 39 . We speculate that further development of the ProteinGAN framework will enable even greater leaps in sequence space and may also make the method applicable to smaller enzyme families.…”