Non-Enzymatic Assembly of a Minimized RNA Polymerase Ribozyme

Central to the “RNA world” hypothesis of the origin of life is the emergence of an RNA catalyst capable of RNA replication. However, possible replicase ribozymes are quite complex and were likely predated by simpler non-enzymatic replication reactions. The templated polymerisation of phosphorimidazolide (Imp) activated ribonucleotides currently appears as the most tractable route to both generate and replicate short RNA oligomer pools from which a replicase could emerge. Herein we demonstrate the rapid assembly of complex ribozymes from such Imp-activated RNA fragment pools. Specifically, we show assembly of a newly selected minimal RNA polymerase ribozyme variant (150 nt) by RNA templated ligation of 5’-2-methylimidazole-activated RNA oligomers <30 nucleotides long. Our results provide support for the possibility that complex RNA structures could have emerged from pools of activated RNA oligomers and outlines a path for the transition from non-enzymatic/chemical to enzymatic RNA replication.


Table of Contents
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Oligonucleotides
Table S1 specifies the DNA and RNA oligonucleotides used in this study. All DNA and RNA oligonucleotides were purchased from Integrated DNA Technologies (IDT). DNA templates for RNA in-vitro transcription were generated by fill-in of two DNA oligonucleotides (in verse template sequence and primer P T7) using GoTaq (Promega) followed by DNA purification using QiaQuick (Qiagen). RNAs were transcribed using the MegaShortScript high-yield transcription kit (Ambion) and purified by RNeasy (Qiagen). Oligonucleotides were purified, if necessary, by denaturing PAGE on 10%, 15%, or 20% denaturing polyacrylamide gels containing 8 M urea. After visualisation by UV shadowing, oligonucleotides were excised from the gel, extracted overnight by crush and soak into 0.3 M NaOAc, followed by precipitation with absolute ethanol on dry ice, two washing steps with 70% ethanol anddried by speed vac. The oligonucleotide concentration was determined by Nanodrop (Thermo Fisher) at a wavelength of 260 nm.

Synthesis of 5'phosphorimidazole RNA oligonucleotides
5'-phosphorylated RNA oligonucleotides (IDT) were suspended in Millipore water to a concentration of 100 µM. 20 µl were added to 20 µl of 1 M 2-Methylimidazole (pH 6) and 5.7 mg of EDC and the mixture was incubated for 2h at 25°C. The activated RNA oligonucleotides were purified and concentrated using Vivaspin® 500 concentrators, 3000MWCO (Sartorius) and, after concentration measurement by Nanodrop, directly used in the templated ligation reaction.

Templated RNA ligation
Templated RNA ligation reactions were performed in 10 µl volumes with 5' fluorescently labelled primer (0.5 µM),5' 2-Methylimidazole activated RNA oligonucleotides (1.5 µM each), RNA or DNA template strands (50 µM each), 10 mM MgCl 2 and 50 mM CHES pH 9.The reactions were incubated for 24h at 37°C, stopped by the addition of equal volumes of 10 mM EDTA in 8 M urea, 0.05% bromophenol blue and full-length ligation products were isolated on 20% polyacrylamide/ 8 M urea gels, analysed using a Typhoon Trio scanner (GE Healthcare) and quantified using Image Quant software (Molecular Dynamics).

Functional activity and sequencing of full-length ligation products
Full-length ligation products were excised from the gel, extracted as described above and reverse transcribed and amplified by PCR using primers (P F and P F rev) and the SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase (Invitrogen). After in-vitro transcription RNA sequences were analysed for their primer extension capability (S6, S7). For sequencing, DNA products were inserted into pGEM®-T (Promega) vectors and sequenced (Sanger sequencing) by Genewiz.   Figure S3. Selection library. The partly randomised starting library for selection was generated from three previously selected RPR variants (Z, W, Y) with a distribution of 50% for Z and 25% for W and Y and each comprising a randomised linker region of 12, 18 and 24 nt. The diversity of the library was increased by 2 rounds of error prone PCR (25 cycles, 95˚C for 30 s, 50˚C for 30 s, 72˚C for 60 s) using the GeneMorph II Random Mutagenesis Kit (Agilent). Red marked parts in the starting library indicate fully or partly randomised nucleotides/regions.     Tables   Table S1. Table of DNA and RNA sequences. DNA sequences are coloured in grey and RNA sequences in black. IVT denotes RNA sequences generated by in vitro transcription. Primer binding sites in template sequences are underlined and 5' modifications (FITC: Fluorescein isothiocyanate, Bio: biotinylation, C18: 18-atom hexaethylene-glycol spacer, p: phosphorylation) are marked in red. Standard oligonucleotides for the CBT selection procedure are as described previously. [