Kopieren von RNA‐Sequenzen ohne Voraktivierung

Abstract

Der templatgesteuerte Einbau von Nukleotiden am Terminus eines Komplementärstrangs ist die Grundlage der Replikation. Im Fall von RNA kann dieser Vorgang in Abwesenheit von Enzymen stattfinden, wenn die Ribonukleotide vorher in eine Aktivspezies mit einer Abgangsgruppe überführt werden. Bisher war für diese Aktivierung ein separater chemischer Schritt nötig, was komplizierte präbiotisch plausible Szenarios erfordert. Wir zeigen nun, dass die Kombination eines Carbodiimids und eines Organokatalysators den annähernd quantitativen Einbau jedes der vier Ribonukleotide induziert. Bei In‐situ‐Aktivierung von Adenosinmonophosphat bilden sich gleichzeitig Oligomere in wässriger Lösung. Es können also Strangbildung und sequenzspezifische Kopiervorgänge ohne künstlichen Syntheseschritt stattfinden.

Spontaneous Formation of RNA Strands, Peptidyl RNA, and Cofactors

“…As mentioned above,signals for oligoadenylates had been observed in copying assays with in situ activation, [14] indicating that de novo strand formation was occurring in the absence of mineral surfaces as templates for oligomerization. [23][24][25] All ribonucleotides had shown some reactivity,b ut only AMP showed full conversion to oligomers after 7d at 0 8 8C.…”

“…c) Newly formed RNA strands, as determined by ion-exchange HPLC after 5d,t ogether with amass spectrum of one chromatographic fraction.d )Sequence-specific extension of primer by CMP, as detected by MALDI mass spectrometry after release from beads;bottom, right-hand side:r eaction kinetics of the copying on the immobilized template. [14] Conditions:0.15 m riboncleotides (each) and 0.125 m amino acid building block (each), condensation buffer,08 8C. acid, peaks in the ppm range typical for phosphoramidates appeared, indicating that the peptidyl RNAs formed are Nlinked.…”

“…Oligomerization of peptides and oligonucleotides requires activation to drive endergonic chain-growth reactions.A sr eported in Ref. [14],w eh ave found that ac ombination of the water-soluble carbodiimide EDC and alkylated heterocycles,s uch as 1-ethylimidazole,i nduces copying processes in RNAv ia in situ activation of ribonucleotides, without the need for chemical pre-activation. Carbodiimide is at automer of cyanamide,acompound formed under presumed prebiotic conditions.…”

“…Here we show that peptidyl RNAs form spontaneously when amino acids and ribonucleotides are exposed to am ixture of ac ondensing agent and ah eterocyclic catalyst, that is,c onditions inducing genetic copying. [14] Much like the intermediates of translation, the peptidyl RNAs are species in which the biomolecules that underlie encoded protein synthesis are covalently linked to one another.Further, under the same condensation conditions,c ofactors emerge through pyrophosphate-forming reactions.A denosine monophosphate is particularly reactive in the processes named above, helping to explain why it is found in so many pivotal biomolecules today.…”

“…Further, despite the competition for the condensing reagent, asequence-selective extension of the primer occurred in the same mixture ( Figure 3), with ar ate only modestly slower than those measured in the absence of the other components. [14] Next, we studied amixture of AMP and afree amino acid in condensation buffer at 0 8 8C. We chose glycine,t he most abundant amino acid in the product mixture of Miller.…”