Towards the end goal of a mirror-image living entity, Denantiomers of key enzymes involved in the central dogma of molecular biology have been prepared (Figure ). Templatedirected polymerizations of the enantiomeric L-DNA and -RNA were first carried out using the D-protein enantiomer of African swine fever virus polymerase X (D-ASFV pol X). Composed of only 174 residues, ASFV pol X is the smallest DNA polymerase known, hence an ideal candidate for total chemical synthesis. The longest polynucleotide successfully replicated by D-ASFV pol x was 44 nucleotides in length but required fresh enzymes at each cycle due to its weak thermal stability. A significant advancement was achieved by preparing the 358-residue enantiomeric P2 DNA polymerase IV (Dpo4) from S. solfataricus, which is sufficiently stable to temperature flux and could perform mirror-image polymerase chain reaction (miPCR). The D-Dpo4 could successfully create a 120-bp L-DNA sequence encoding the E. coli 5S ribosomal RNA gene rrfB. Its thermostable variant D-Dpo4-3C could assemble a full L-DNA gene encoding protein Ssoo7d. Interestingly, a further-engineered variant D-Dpo4-5m-Y12S was reported to be capable of both transcription and reverse transcription, laying a strong foundation for the creation of mirror-image life. In order to create a lengthy enantiomeric gene with high fidelity, access to polymerase enzymes with a low error rate is essential, thus an enantiomeric derivative of Pyrococcus furiosus (Pfu) DNA polymerase has been deduced. Pfu is composed of 775 amino acids reaching 90 kDa in molecular weight, rendering its chemical synthesis challenging. To circumvent this issue, a split version of Pfu was prepared, consisting of N-(467-residue) and C-(308-residue) fragments. Due to the significant cost of D-isoleucine and its association with the aggregation of peptide fragments, most of them were replaced by other bulky residues including valine and leucine. The split polymerase D-Pfu could synthesize a 1.5-kb mirror-image gene from short, synthetic oligonucleotides. Interestingly, because of the inherent stability towards enzymatic cleavage, a trace amount of artificial L-DNA preserved in water from a local pond remained amplifiable by D-Pfu after one year, whereas D-DNA could not be amplified by L-Pfu after one day. This work is a clear leap forward in the pursuit of mirror-image biology. In addition, the miPCR platform is potentially useful in molecular discovery programs generating nuclease-resistant Lnucleotide aptamers for critical drug targets.