Sliding over the Blocks in Enzyme-Free RNA Copying - One-Pot Primer Extension in Ice

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Abstract

<p>Template-directed polymerization of RNA in the absence of enzymes is the basis for an information transfer in the ‘RNA-world’ hypothesis and in novel nucleic acid based technology. Previous investigations established that only cytidine rich strands are efficient templates in bulk aqueous solutions while a few specific sequences completely block the extension of hybridized primers. We show that a eutectic water/ice system can support Pb<sup>2+</sup>/Mg<sup>2+</sup>-ion catalyzed extension of a primer across such sequences, i.e. AA, AU and AG, in a one-pot synthesis. Using mixtures of imidazole activated nucleotide 5′-monophosphates, the two first “blocking” residues could be passed during template-directed polymerization, i.e., formation of triply extended products containing a high fraction of faithful copies was demonstrated. Across the AG sequence, a mismatch sequence was formed in similar amounts to the correct product due to U·G wobble pairing. Thus, the template-directed extension occurs both across pyrimidine and purine rich sequences and insertions of pyrimidines did not inhibit the subsequent insertions. Products were mainly formed with 2′-5′-phosphodiester linkages, however, the abundance of <named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="gene" xlink:type="simple">3′–5′</named-content>-linkages was higher than previously reported for pyrimidine insertions. When enzyme-free, template-directed RNA polymerization is performed in a eutectic water ice environment, various intrinsic reaction limitations observed in bulk solution can then be overcome.</p>

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Löffler, P. M. G., Groen, J., Dörr, M., & Monnard, P. A. (2013). Sliding over the Blocks in Enzyme-Free RNA Copying - One-Pot Primer Extension in Ice. PLoS ONE, 8(9). https://doi.org/10.1371/journal.pone.0075617

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