Xenobiotic Nucleic Acid (XNA) Synthesis by Phi29 DNA Polymerase

Abstract

Phi29 DNA polymerase (DNAP) is the replicative enzyme of the Bacillus subtilis bacteriophage Phi29. Its extraordinary processivity and its ability to perform isothermal amplification of DNA are central to many molecular biology applications, including high-sensitivity detection and large-scale production of DNA. We present here Phi29 DNAP as an efficient catalyst for the production of various artificial nucleic acids (XNAs) carrying backbone modifications such as 1,5-anhydrohexitol nucleic acid (HNA), 2'-deoxy-2'-fluoro-arabinonucleic acid (FANA), and 2'-fluoro-2'-deoxyribonucleic acid (2'-fluoro-DNA). A full protocol for the synthesis of HNA polymers by an exonuclease-deficient variant (D12A) of Phi29 DNAP plus a detailed guide for the design and test of novel XNA synthetase reactions performed by Phi29 DNAP are provided. © 2018 by John Wiley & Sons, Inc.

Modified nucleic acids: replication, evolution, and next-generation therapeutics

“…This functional plasticity may be aided by high thermostability, which promotes greater tolerance for mutations that would otherwise be excessively destabilizing. However, A-family [82][83][84][85][86][87][88][89][90] and mesophilic polymerases such as Phi29 [91,92] as well as RNA polymerases [93][94][95][96][97] have also been engineered to accept XNA substrates with success (Table 1). However, the efficiency, fidelity, and kinetics of engineered polymerases on XNA substrates are usually compromised relative to the native enzymes and natural substrates.…”

Biotechnology Tools Derived from the Bacteriophage/Bacteria Arms Race

“…That is, although some bacteriophages make use of modified nucleobases and have evolved systems that lead to 100% incorporation of the modified bases in their genomes, their DNA polymerases have not specialised towards being able to only incorporate the modified nucleobases-they remain able to recognise unmodified triphosphates. [14][15][16][17][18][19][20][21][22][23][24][25] *Address all correspondence to: vitor.pinheiro@kuleuven.be Still, the increased substrate flexibility of phage DNA polymerases may at least in part justify why a Bacillus subtilis Phi29 DNA polymerase required a single mutation for the synthesis of anhydrohexitol nucleic acids (HNA) [80] while an archaeal enzyme required in excess of seven mutations [81].…”

XNA Synthesis and Reverse Transcription by Engineered Thermophilic Polymerases

“…If maximum yield is desired, the best solution we have found to date is to purify the XNA (e.g., by PCR purification column or phenol:chloroform extraction and alcohol precipitation) and use the purified XNA as pre-annealed primer:template complex for a polishing reaction using Phi29 DNAP, which will readily extend to the end of templates (at least for HNA) as described elsewhere (Torres and Pinheiro, 2018). If maximum yield is desired, the best solution we have found to date is to purify the XNA (e.g., by PCR purification column or phenol:chloroform extraction and alcohol precipitation) and use the purified XNA as pre-annealed primer:template complex for a polishing reaction using Phi29 DNAP, which will readily extend to the end of templates (at least for HNA) as described elsewhere (Torres and Pinheiro, 2018).…”

“…XNA synthesis from mesophilic polymerases (Torres and Pinheiro, 2018), A-family RNA polymerases (Chen et al, 2016;Ghadessy, Ong, & Holliger, 2001) and single-subunit RNA polymerases (Ibach et al, 2013;Sousa & Padilla, 1995) have also been reported.…”

“…XNA synthesis from mesophilic polymerases (Torres and Pinheiro, ), A‐family RNA polymerases (Chen et al., ; Ghadessy, Ong, & Holliger, ) and single‐subunit RNA polymerases (Ibach et al., ; Sousa & Padilla, ) have also been reported. Among thermostable B‐family polymerases, engineering efforts have focused on DNAPs from Thermococcus gorgonarius (Cozens et al., , ; Liu et al, ; Pinheiro et al., ), Thermococcus kodakarensis (Larsen et al., ), and 9°N (Gardner & Jack, ), with mutations being portable between homologous enzymes, at least for some chemistries (Dunn, Otto, Fenton, & Chaput, ).…”