Abstract
The construction of mirror-image biological systems may open the next frontier for biomedical technology development and discovery. Here we have designed and chemically synthesized a mutant version of the thermostable Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) consisting of d-amino acids. With a total peptide length of 358 amino acid residues, it is the largest chemically synthesized d-amino acid protein reported to date. We show that the d-polymerase is able to amplify a 120-bp l-DNA sequence coding for the Escherichia coli 5S ribosomal RNA gene rrfB by mirror-image polymerase chain reaction, and that both the natural and mirror-image systems operate with strict chiral specificity. The development of efficient miPCR systems may lead to many practical applications, such as mirror-image systematic evolution of ligands by exponential enrichment for the selection of therapeutically promising nuclease-resistant l-nucleic acid aptamers.
Highlights
We have previously reported the chemical synthesis of a 174-residue D-amino acid African Swine Fever Virus polymerase X (ASFV pol X) system [1], in which we demonstrated that two processes in the central dogma of molecular biology, the template-directed replication of DNA and transcription into RNA, could be catalyzed by a D-amino acid polymerase on an L-DNA template
We show that the mutant D-polymerase (D-Dpo4-5m) is able to amplify a 120-bp L-DNA sequence coding for the Escherichia coli 5S ribosomal RNA gene rrfB by mirror-image polymerase chain reaction (miPCR)
We show that the PCR efficiency of the mutant Dpo4 (Dpo4-5m) is comparable to that of the recombinant wild-type (WT) Dpo4 purified from E. coli, suggesting that these five point mutations (C31S, S86C, N123A, S207A and S313A) do not significantly affect the PCR efficiency of the enzyme (Supplementary Figure S1)
Summary
We have previously reported the chemical synthesis of a 174-residue D-amino acid African Swine Fever Virus polymerase X (ASFV pol X) system [1], in which we demonstrated that two processes in the central dogma of molecular biology, the template-directed replication of DNA and transcription into RNA, could be catalyzed by a D-amino acid polymerase on an L-DNA template The establishment of this mirror-image genetic replication and transcription system was a small step towards chemically synthesizing an alternative, mirror-image form of life in the laboratory [2, 3], whereas achieving this ultimate goal requires many more efficient molecular tools such as a thermostable enzyme capable of mirror-image polymerase chain reaction (miPCR). We show that the mutant D-polymerase (D-Dpo4-5m) is able to amplify a 120-bp L-DNA sequence coding for the Escherichia coli 5S ribosomal RNA (rRNA) gene rrfB by miPCR
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