Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase

Proc Natl Acad Sci U S A. 2020 Mar 31;117(13):7276-7283. doi: 10.1073/pnas.1922400117. Epub 2020 Mar 18.


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.

Keywords: NP-DNA; XNA; alternative genetic polymers; polymerases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amides / chemical synthesis
  • Amides / chemistry*
  • Amides / metabolism
  • DNA / chemical synthesis
  • DNA / chemistry*
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / metabolism
  • Magnetic Resonance Spectroscopy
  • Molecular Structure
  • Nucleic Acid Conformation
  • Oligonucleotides / chemistry
  • Phosphoric Acids / chemical synthesis
  • Phosphoric Acids / chemistry*
  • Phosphoric Acids / metabolism
  • Polymerization
  • RNA / chemistry


  • Amides
  • Oligonucleotides
  • Phosphoric Acids
  • RNA
  • DNA
  • phosphoramidic acid
  • DNA-Directed DNA Polymerase

Associated data

  • PDB/6UR2
  • PDB/6UR4
  • PDB/6UR9
  • PDB/6US5