Mirror-image polymerase chain reaction

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.

Sequencing Mirror-Image DNA Chemically

“…Using this approach, the sequences of therapeutically useful L-DNA aptamer drugs can now be validated, which is key to the safety of their clinical use. In addition, this approach can be used for confirming the L-DNA gene sequences enzymatically copied and assembled by synthetic mirror-image polymerases (Wang et al, 2016;Xu et al, 2017;Jiang et al, 2017;Pech et al, 2017), toward the realization of a mirror-image central dogma of molecular biology capable of genetic replication, transcription, and translation, and ultimately leading to the synthesis of mirror-image life. It may also facilitate the audacious endeavor to search for life forms with an opposite chirality on exoplanets.…”

“…In comparison, the sequencing of longer L-DNA molecules (typically of up to a couple of hundred nucleotides) is more feasible by the Maxam-Gilbert approach, albeit ultimately limited by the resolution of sequencing gel and occurrence of incomplete reactions (Franca et al, 2002). One of the disadvantages of the current approach is its low throughput compared with other modern sequencing techniques, which could be important for its application in a proposed MI-SELEX scheme (Xu et al, 2017;Jiang et al, 2017). Other drawbacks found in the classic Maxam-Gilbert sequencing approach, such as the technical complexity of using hazardous chemicals and requirement of end-labeling, may also hinder the convenient use of this approach.…”

“…Other drawbacks found in the classic Maxam-Gilbert sequencing approach, such as the technical complexity of using hazardous chemicals and requirement of end-labeling, may also hinder the convenient use of this approach. In practice, the 5 0 FAM labels can be introduced by the recently established mirror-image polymerase chain reaction (MI-PCR) system (Xu et al, 2017;Jiang et al, 2017;Pech et al, 2017) with an FAM-labeled primer. Alternatively, if a mirrorimage polynucleotide kinase could be chemically synthesized, it could also be used to radiolabel the 5 0 end of L-DNA molecules for sequencing.…”

“…Our inability to sequence the synthetic L-DNA aptamer drugs to ensure their quality has hindered their safe clinical use. In addition, a direct selection scheme for L-DNA aptamers against biological targets through a proposed mirror-image systematic evolution of ligands by exponential enrichment (MI-SELEX) approach is on the horizon (Xu et al, 2017;Jiang et al, 2017), but also requires the sequencing of enriched L-aptamer sequences from a randomized L-DNA pool. The rapid development of this field has created a need for a practical method to sequence mirrorimage DNA.…”

“…Most of the commonly used sequencing-by-synthesis methods are currently unsuitable because they require a particular polymerase capable of incorporating labeled L-di-deoxynucleotide triphosphates (L-ddNTPs) or L-deoxyribonucleotide triphosphates (dNTPs). Although a couple of mirror-image polymerase systems based on enzymes small enough for total chemical synthesis, such as the African swine fever virus polymerase X (ASFV pol X) (Wang et al, 2016) and the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) (Xu et al, 2017;Jiang et al, 2017;Pech et al, 2017), have been developed, they still suffer from issues such as poor fidelity or inability to incorporate labeled ddNTPs or dNTPs. While the next-generation nanopore DNA sequencing approach could be applied to sequencing mirror-image DNA in principle, it also requires a particular D-amino acid polymerase or helicase (which is not currently available) to help slow down the passing DNA (Derrington et al, 2015).…”