Abstract
Polymerase chain reaction (PCR) has been a defining tool in modern biology. Towards realizing mirror-image PCR, we have designed and chemically synthesized a mutant version of the 352-residue thermostable Sulfolobus solfataricus P2 DNA polymerase IV with l-amino acids and tested its PCR activity biochemically. To the best of our knowledge, this enzyme is the largest chemically synthesized protein reported to date. We show that with optimization of PCR conditions, the fully synthetic polymerase is capable of amplifying template sequences of up to 1.5 kb. The establishment of this synthetic route for chemically synthesizing DNA polymerase IV is a stepping stone towards building a d-enzyme system for mirror-image PCR, which may open up an avenue for the creation of many mirror-image molecular tools such as mirror-image systematic evolution of ligands by exponential enrichment.
Highlights
We have recently reported the realization of mirror-image genetic replication and transcription by a chemically synthesized 174-residue D-amino-acid African swine fever virus polymerase X (ASFV pol X) system [1]
We set out to explore synthetic routes to chemically synthesize the 352-residue thermostable Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), which was shown capable of PCR-amplifying sequences longer than 1 kb in the previous study [5]
Our previously reported mirror-image genetic replication system based on ASFV pol X lacked the processivity and thermal stability to PCR amplify L-DNA sequences [1]
Summary
We have recently reported the realization of mirror-image genetic replication and transcription by a chemically synthesized 174-residue D-amino-acid African swine fever virus polymerase X (ASFV pol X) system [1]. Because ASFV pol X is not thermostable, only a proofof-concept replication experiment was performed by supplying fresh enzymes in each cycle [1], making it impractical for many applications that require efficient amplification of L-nucleic acid sequences, such as. We reasoned that a mirror-image PCR system may be realized through development of synthetic routes for other more efficient and thermostable polymerases [1]. We set out to explore synthetic routes to chemically synthesize the 352-residue thermostable Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), which was shown capable of PCR-amplifying sequences longer than 1 kb in the previous study [5]. We showed that the fully synthetic polymerase can PCR amplify sequences from 110 bp to up to 1.5 kb, and can assemble short DNA oligonucleotides into longer gene sequences
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