Thermophilic Nucleic Acid Polymerases and Their Application in Xenobiology

Abstract

Thermophilic nucleic acid polymerases, isolated from organisms that thrive in extremely hot environments, possess great DNA/RNA synthesis activities under high temperatures. These enzymes play indispensable roles in central life activities involved in DNA replication and repair, as well as RNA transcription, and have already been widely used in bioengineering, biotechnology, and biomedicine. Xeno nucleic acids (XNAs), which are analogs of DNA/RNA with unnatural moieties, have been developed as new carriers of genetic information in the past decades, which contributed to the fast development of a field called xenobiology. The broad application of these XNA molecules in the production of novel drugs, materials, and catalysts greatly relies on the capability of enzymatic synthesis, reverse transcription, and amplification of them, which have been partially achieved with natural or artificially tailored thermophilic nucleic acid polymerases. In this review, we first systematically summarize representative thermophilic and hyperthermophilic polymerases that have been extensively studied and utilized, followed by the introduction of methods and approaches in the engineering of these polymerases for the efficient synthesis, reverse transcription, and amplification of XNAs. The application of XNAs facilitated by these polymerases and their mutants is then discussed. In the end, a perspective for the future direction of further development and application of unnatural nucleic acid polymerases is provided.

Polyamines promote xenobiotic nucleic acid synthesis by modified thermophilic polymerase mutants

“…Polymerase mutants, derived from thermophilic DNA polymerases such as Taq, 91N, Tgo, and KOD DNA polymerase have been developed for XNA synthesis. 4,5,[22][23][24][25][26] Based on KOD DNA polymerase, we previously developed the polymerase mutant, KOD DGLNK (KOD: N210D/Y409G/A485L/D614N/E664K), for the synthesis of locked nucleic acid (LNA), also called 2 0 ,4 0 -bridged nucleic acid (2 0 ,4 0 -BNA). 5 Some XNAs, such as arabino nucleic acid (ANA) 27 (Fig.…”

“…Polymerase mutants, derived from thermophilic DNA polymerases such as Taq, 91N, Tgo, and KOD DNA polymerase have been developed for XNA synthesis. 4,5,[22][23][24][25][26] Based on KOD DNA polymerase, we previously developed the polymerase mutant, KOD DGLNK (KOD: N210D/Y409G/A485L/D614N/E664K), for the synthesis of locked a National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), 7-6-8 Saito-Asagi, Ibaraki 567-0085, Osaka, Japan. E-mail: h-hoshino@nibiohn.go.jp b Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita 565-0871, Osaka, Japan.…”

Synthesis, Reverse Transcription, Replication, and Inter-Transcription of 2′-Modified Nucleic Acids with Evolved Thermophilic Polymerases: Efforts toward Multidimensional Expansion of the Central Dogma

“…To obtain efficient XNA polymerases, extensive efforts have been made on engineering DNA and RNA polymerases with various protein engineering methods, including directed evolution, rational design, and semi-rational design. 1,17 DNA polymerases from different families have different features in their structures, activities, and functions and play different roles in the replication and repair of the genomes of living organisms. 18,19 In principle, to construct a functional XNA replisome in vivo, DNA polymerases that are from several families and involved in genome replication have to be engineered to be efficient counterparts for XNAs.…”

“…However, due to their stringent substrate specificity, natural DNA and RNA polymerases are usually inefficient in the synthesis, reverse transcription, and replication of XNAs. To obtain efficient XNA polymerases, extensive efforts have been made on engineering DNA and RNA polymerases with various protein engineering methods, including directed evolution, rational design, and semi-rational design. , DNA polymerases from different families have different features in their structures, activities, and functions and play different roles in the replication and repair of the genomes of living organisms. , In principle, to construct a functional XNA replisome in vivo , DNA polymerases that are from several families and involved in genome replication have to be engineered to be efficient counterparts for XNAs . For in vitro applications, DNA polymerases that are from different families have found use in different scenarios, so XNA polymerases derived from these DNA polymerases are likely to be useful in different applications as well.…”

Direct Enzyme Engineering of B Family DNA Polymerases for Biotechnological Approaches

“…Table 2 summarizes the amino acid substitutions of engineered DNA polymerases with altered substrate specificity. Other recent reviews are available that provide a broad overview of different aspects and structural analysis of engineered DNA polymerases for XNA synthesis [5,6,9,69]. An initial attempt to compare the substrate specificity, thermal stability, reverse transcriptase activity, and fidelity of laboratory-evolved polymerases that were established to synthesize RNA and five different examples of XNA polymers was performed in [59].…”