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.

Keywords: nucleic acid polymerases; thermophilic enzymes; thermophilic organisms; xeno nucleic acids (XNAs); xenobiology.

Figure 1

Phylogenetic tree of representative thermophilic DNAPs. Green: family A DNAPs; pale yellow: family B DNAPs.

Figure 2

Expansion of the central dogma with XNAs and XNAPs. Green arrows: replication; blue arrows: transcription or reverse transcription.

Figure 3

Representative methods employed for the generation of XNAPs. (a) Methods for the construction of polymerase libraries. I: error-prone PCR; II: site-directed saturation mutagenesis; III: gene shuffling by StEP; IV: gene shuffling by DNase I digestion and PCR reassembly; (b) methods for the selection of polymerase mutants. I: phage display; II: CSR; III: CST.

Figure 4

Summary of the relationships and mutations of some representative engineered XNAPs. The mutated amino acids in the engineered XNAPs are indicated in red.

Figure 5

Distribution of the mutation sites in the engineered XNAPs. The mutation sites in engineered (a) Taq DNAP (green, PDB: 1TAU); (b) Tgo DNAP (cyan, PDB: 7B07); (c) KOD DNAP (yellow, PDB: 4K8Z); and (d) 9°N DNAP (red, PDB: 6ISF). The DNA templates and DNA primers are shown in blue and orange, respectively.

Figure 6

Application of XNAs and XNAPs. Modifications endow XNAs with expanded structural and functional diversities, and XNAPs further broaden the application scope of XNAs. (a) XNAzymes; (b) modified antisense oligonucleotides; (c) modified guide RNAs for CRISPR/Cas9 system; (d) XNA aptamers; (e) genetic information storage in living organisms; (f) XNA materials.