Engineering polymerases for applications in synthetic biology

Abstract

DNA polymerases play a central role in biology by transferring genetic information from one generation to the next during cell division. Harnessing the power of these enzymes in the laboratory has fueled an increase in biomedical applications that involve the synthesis, amplification, and sequencing of DNA. However, the high substrate specificity exhibited by most naturally occurring DNA polymerases often precludes their use in practical applications that require modified substrates. Moving beyond natural genetic polymers requires sophisticated enzyme-engineering technologies that can be used to direct the evolution of engineered polymerases that function with tailor-made activities. Such efforts are expected to uniquely drive emerging applications in synthetic biology by enabling the synthesis, replication, and evolution of synthetic genetic polymers with new physicochemical properties.

Computational investigations of polymerase enzymes: Structure, function, inhibition, and biotechnology

“…These different DNA Pol families vary significantly in fidelity and processivity depending on their functional roles. For instance, A‐ and B‐family DNA Pols, which are involved in DNA lesion bypass and mitochondrial DNA replication and repair, 49 have the highest fidelity (10 −5 to 10 −6 without exonuclease activity) 50,51 and highest processivity (>100 nt/s) 25 . Thermostable DNA Pols from these families, such as DNA Pol I (A‐family) from Thermus aquaticus (Taq), are also employed in PCR applications 25 .…”

“…Importantly, Pols are also utilized in biotechnological applications, 25–28 including the Nobel‐Prize‐winning (1993, Chemistry) polymerase chain reaction (PCR) method invented by Kary B. Mullis 29,30 . The PCR method is used for applications such as DNA amplification, 31 site‐directed mutagenesis, 32 diagnosis of infectious diseases, 33 and analyses of clinical, 34 forensic, 35 and environmental 36 samples.…”

“…The PCR method is used for applications such as DNA amplification, 31 site‐directed mutagenesis, 32 diagnosis of infectious diseases, 33 and analyses of clinical, 34 forensic, 35 and environmental 36 samples. Pols are also applied in synthetic biology to build synthetic genetic polymers for the storage and propagation of genetic information 25–28 . Examples of synthetic nucleotides (i.e., xeno‐nucleic acids) include threose nucleic acids, 37 1,5‐anhydrohexitol nucleic acids, 38 and locked nucleic acids 39 .…”

Multi-modal engineering ofBstDNA polymerase for thermostability in ultra-fast LAMP reactions

“…There have been various approaches advanced for engineering DNA polymerases for improved function in a variety of settings (36,37), but these typically focus on a single method for improvement or a single property of the polymerase. It has been hypothesized that impacting different kinetic properties of a protein can lead to additive improvements in protein function (1,2), a hypothesis that also implicitly underlies the many highly successful examples in which DNA shuffling is used to improve protein function (38,39).…”

Building better polymerases: Engineering the replication of expanded genetic alphabets

“…To date, progress in engineering DNA polymerases has been encouraging (105). In our work aimed at improving the ability of these enzyme to replicate UBPs (65,92,98), ZP Klentaq takes advantage of increased domain flexibility to improve the replication efficiency of Z:P pairs, even though both WT Klentaq and ZP Klentaq grip the template-primer duplex in a similar manner prior to and following incorporation of the AEGIS UBP.…”