Activation Chemistry
I. In situ phosphate activation
Active building block phosphorimidazolides can undergo hydrolysis quickly. Thus, we hypothesize there is a mechanism to continuously generate phosphorimidazolides from nucleotide monophosphates to sustain the chemical replication of RNA. We demonstrated that phosphorimidazolides and bridged dimers are available by prebiotically plausible in-situ mononucleotide activation either by a freeze-thaw cycles or small-molecule organocatalysis strategies. As the in-situ activation is compatible with nonenzymatic RNA copying, the continuously replication can be envisaged in a one-pot aqueous solution.
The hydrolysis of phosphorimidazolides not only reduces the effective concentration of activated mononucleotides but increases inactivated mononucleotides which would inhibit the primer extension due to the competitive base pairing on the template. We found that isocyanide-based bridge-forming activation accelerates the formation of imidzolium-bridged dimer and compensates the hydrolysis of activated species, leading to the higher efficiency of nonenzymatic RNA copying compared to that without bridge-forming activation chemistry.
Oligonucleotide helpers has been employed in nonenzymatic RNA copying to increase the efficiency. However, the effect of monomer-bridged-oligonucleotide species has never been evaluated quantitatively. Our kinetic study showed that monomer-bridged-trimers and monomer-bridged-tetramers are much superior substrates for nonenzymatic primer extension. Their higher reactivity and template affinity results faster template copying at lower substrate concentrations. These species can easily produce by incubating the mixed mononucleotides and oligonucleotides (< 4nt) under phospho-Passerini chemistry.
Read more
Zhang, S.J., Duzdevich, D., Szostak, J.W.*, 2020. Potentially Prebiotic Activation Chemistry Compatible with Nonenzymatic RNA Copying. J. Am. Chem. Soc. 142, 14810–14813. PDF
Zhang, S.J., Duzdevich, D., Ding, D., Szostak, J.W.*, 2022. Freeze-thaw cycles enable a prebiotically plausible and continuous pathway from nucleotide activation to nonenzymatic RNA copying. Proc. Natl. Acad. Sci. U.S.A. 119, e2116429119. PDF
Aitken, H.R.M., Wright, T.H., Radakovic, A., Szostak, J.W.*, 2023. Small-Molecule Organocatalysis Facilitates In Situ Nucleotide Activation and RNA Copying. J. Am. Chem. Soc. 145, 16142–16149. PDF
Ding, D., Zhang, S.J., Szostak, J.W.*, 2023. Enhanced nonenzymatic RNA copying with in-situ activation of short oligonucleotides. Nucleic Acids Research 51, 6528–6539. PDF
II. Assembly of Functional RNAs
The emergence of functional RNAs likely occurred via joining of constituent short fragments. We hypothesize that loop-closing ligation, a template free-RNA assembly pathway, can assemble functional RNAs. However, the model reaction used pre-activated oligonucleotides, which possess a short half-life and easily lose activity due to hydrolysis in the aqueous phase. Therefore, research on continuously short oligonucleotides activation and consecutive assembly into longer functional RNA is undergoing in our lab. In situ activation enables the inactivated oligonucleotides to be activated meanwhile it reactivate hydrolyzed oligonucleotides. Functional RNAs can thus emerge via a consecutive pre-organization, in situ phosphate activation, and loop-closing ligation process.
Read more
Wu, L.-F., Zhang, J., Cornwell-Arquitt, R., Hendrix, D.A., Radakovic, A., Szostak, J.W.*, 2024. Selective Nonenzymatic Formation of Biologically Common RNA Hairpins. Angew Chem Int Ed e202417370. PDF
Wu, L.-F., Liu, Z., Roberts, S.J., Su, M., Szostak, J.W.*, Sutherland, J.D.*, 2022. Template-Free Assembly of Functional RNAs by Loop-Closing Ligation. J. Am. Chem. Soc. 144, 13920–13927. PDF