Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase

Abstract

All known polymerases copy genetic material by catalyzing phosphodiester bond formation. This highly conserved activity proceeds by a common mechanism, such that incorporated nucleoside analogs terminate chain elongation if the resulting primer strand lacks a terminal hydroxyl group. Even conservatively substituted 3′-amino nucleotides generally act as chain terminators, and no enzymatic pathway for their polymerization has yet been found. Although 3′-amino nucleotides can be chemically coupled to yield stable oligonucleotides containing N3′→P5′ phosphoramidate (NP) bonds, no such internucleotide linkages are known to occur in nature. Here, we report that 3′-amino terminated primers are, in fact, slowly extended by the DNA polymerase from B. stearothermophilus in a template-directed manner. When its cofactor is Ca2+ rather than Mg2+, the reaction is fivefold faster, permitting multiple turnover NP bond formation to yield NP-DNA strands from the corresponding 3′-amino-2′,3′-dideoxynucleoside 5′-triphosphates. A single active site mutation further enhances the rate of NP-DNA synthesis by an additional 21-fold. We show that DNA-dependent NP-DNA polymerase activity depends on conserved active site residues and propose a likely mechanism for this activity based on a series of crystal structures of bound complexes. Our results significantly broaden the catalytic scope of polymerase activity and suggest the feasibility of a genetic transition between native nucleic acids and NP-DNA.

CRISPR interference-guided modulation of glucose pathways to boost aconitic acid production in Escherichia coli

“…Apart from the versatile CRISPR tools [17,21], one promising strategy capable of coordinating cell growth and metabolites formation may be orthogonal expression system which allows decoupling of aconitic acid biosynthesis from TCA cycle. This hierarchical expression system comprises mainly orthogonal ribosomes [42] and XNA polymerase [43]. Recently, an orthogonal ribosome system has been exploited to decouple plasmid-based gene expression from host metabolisms [44].…”

Synthetic Life with Alternative Nucleic Acids as Genetic Materials

“…The classic phosphate diester linkages are substituted by different functional groups in sulfone-DNA [50], PS-DNA [51], PN-DNA [52], NP-DNA [53], triazole-DNA [54], and dPhoNA [55] ( Figure 5). like XNA in which the 4′-oxygen atom is substituted by a sulfur atom, the direct transcription can be performed in human cells.…”

“…NP-DNA can be synthesized from RNA template and extended up to 25 nucleotides inside a model protocell [65]. Enzymatic replication or reverse transcription of NP-DNA were performed by Szostak and coworkers, showing that NP-DNA might be a good candidate for the construction of synthetic life [52,66] Recently, Herdewijn's group reported the in vivo study of a synthetic genetic polymer bearing the P3 →N5 phosphoramidate linkages, which is denoted as PN-DNA [67]. Compared to DNA, PN-DNA is more stable under basic conditions and more acid-labile, such that the phosphoramidate linkage can be cleaved under acid conditions [68].…”

“…The classic phosphate diester linkages are substituted by different functional groups in sulfone-DNA [ 50 ], PS-DNA [ 51 ], PN-DNA [ 52 ], NP-DNA [ 53 ], triazole-DNA [ 54 ], and dPhoNA [ 55 ] ( Figure 5 ).…”

“…NP-DNA can be synthesized from RNA template and extended up to 25 nucleotides inside a model protocell [ 65 ]. Enzymatic replication or reverse transcription of NP-DNA were performed by Szostak and coworkers, showing that NP-DNA might be a good candidate for the construction of synthetic life [ 52 , 66 ]…”