Sliding over the blocks in enzyme-free RNA copying--one-pot primer extension in ice.
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(2+)/Mg(2+)-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 3'-5'-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.
Department of Physics, Chemistry and Pharmaceutical Sciences, University of Southern Denmark, Odense, Denmark.
Philipp M G
PM
Löffler
Joost
J
Groen
Mark
M
Dörr
Pierre-Alain
PA
Monnard
D017931:DNA Primers / Q000737:chemistry*; D007053:Ice; D007093:Imidazoles / Q000737:chemistry*; D012313:RNA / Q000138:chemical synthesis* / Q000737:chemistry; D012265:Ribonucleotides / Q000737:chemistry*
D017931:DNA Primers; D007053:Ice; D007093:Imidazoles; D012265:Ribonucleotides; D012313:RNA; C029899:imidazole