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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.

RNA‐catalyzed RNA ligation is widely believed to be a key reaction for primordial biology. However, since typical chemical routes towards activating RNA substrates are incompatible with ribozyme catalysis, it remains unclear how prebiotic systems generated and sustained pools of activated building blocks needed to form increasingly larger and complex RNA. Herein, we demonstrate in situ activation of RNA substrates under reaction conditions amenable to catalysis by the hairpin ribozyme. We found that diamidophosphate (DAP) and imidazole drive the formation of 2′,3′‐cyclic phosphate RNA mono‐ and oligonucleotides from monophosphorylated precursors in frozen water‐ice. This long‐lived activation enables iterative enzymatic assembly of long RNAs. Our results provide a plausible scenario for the generation of higher‐energy substrates required to fuel ribozyme‐catalyzed RNA synthesis in the absence of a highly evolved metabolism.

RNA‐katalysierte RNA‐Ligation wird häufig als Schlüsselreaktion für die Ursprünge der Biologie angesehen. Da jedoch typische Reaktionswege zur Aktivierung von RNA‐Substraten mit der Ribozymkatalyse inkompatibel sind, bleibt unklar, wie präbiotische Systeme aktivierte Bausteine in ausreichender Menge generieren und aufrechterhalten konnten, um zunehmend größere und komplexere RNAs zu bilden. Hier demonstrieren wir die In‐situ‐Aktivierung von RNA‐Substraten unter Bedingungen, die mit der katalytischen Aktivität von Haarnadelschleifen‐Ribozymen vereinbar sind. Wir fanden heraus, dass Diamidophosphat (DAP) und Imidazol die Entstehung von Mono‐ und Oligoribonukleotiden mit 2′,3′‐zyklischen Phosphaten aus monophosphorylierten Vorläufermolekülen in gefrorenem Wassereis ermöglichen können. Diese beständige Aktivierung erlaubt die schrittweise Entstehung langer RNA‐Stränge. Unsere Ergebnisse liefern ein plausibles Szenario für die Erzeugung energiereicher Substrate, die eine ribozymkatalysierte RNA‐Synthese in Abwesenheit eines hoch entwickelten Metabolismus gestatten.