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Polymerization of nucleic acids in biology utilizes 5′‐nucleoside triphosphates (NTPs) as substrates. The prebiotic availability of NTPs has been unresolved and other derivatives of nucleoside‐monophosphates (NMPs) have been studied. However, this latter approach necessitates a change in chemistries when transitioning to biology. Herein we show that diamidophosphate (DAP), in a one‐pot amidophosphorylation‐hydrolysis setting converts NMPs into the corresponding NTPs via 5′‐nucleoside amidophosphates (NaPs). The resulting crude mixture of NTPs are accepted by proteinaceous‐ and ribozyme‐polymerases as substrates for nucleic acid polymerization. This phosphorylation also operates at the level of oligonucleotides enabling ribozyme‐mediated ligation. This one‐pot protocol for simultaneous generation of NaPs and NTPs suggests that the transition from prebiotic‐phosphorylation and oligomerization to an enzymatic processive‐polymerization can be more continuous than previously anticipated.

Polymerization of nucleic acids in biology utilizes 5′‐nucleoside triphosphates (NTPs) as substrates. The prebiotic availability of NTPs has been unresolved and other derivatives of nucleoside‐monophosphates (NMPs) have been studied. However, this latter approach necessitates a change in chemistries when transitioning to biology. Herein we show that diamidophosphate (DAP), in a one‐pot amidophosphorylation‐hydrolysis setting converts NMPs into the corresponding NTPs via 5′‐nucleoside amidophosphates (NaPs). The resulting crude mixture of NTPs are accepted by proteinaceous‐ and ribozyme‐polymerases as substrates for nucleic acid polymerization. This phosphorylation also operates at the level of oligonucleotides enabling ribozyme‐mediated ligation. This one‐pot protocol for simultaneous generation of NaPs and NTPs suggests that the transition from prebiotic‐phosphorylation and oligomerization to an enzymatic processive‐polymerization can be more continuous than previously anticipated.