Manganese(ii) promotes prebiotically plausible non-enzymatic RNA ligation reactions

Using different prebiotically plausible activating reagents, the RNA ligation yield was significantly increased in the presence of Mn(ii). The mechanism of the activation reaction has been investigated using 5′-AMP as an analogue.


Prebiotic synthesis pathway of organocatalysts
A prebiotically plausible synthesis of DCI involves heating cyanide in formamide 1 , these conditions also generate adenine.5-aminoimidazole-4-carbonitrile (AICN) is an intermediate in the synthesis of adenine under these conditions and has thus also been investigated in the context of ligation chemistry.In the presence of DCI, methyl isonitrile reacted with AICN, generating 1-methyladenine (Fig. S2), which co-crystallized with DCI (Fig. S3, Table S1).Hypoxanthine has been reported as a product resulting from adenine diazotization followed by hydrolysis 2,3 .

Passerini reaction in RNA reaction suppressed by DCI
We tested the formation of different azolide-5 ′ -AMPs using methyl isonitrile and acetaldehyde as activating reagents.Because Mn (II) is a paramagnetic metal ion which can interfere with NMR analysis, Mg (II) was used instead for these experiments and the results were analyzed by 31 P-NMR spectroscopy.5 ′ -AMP (10 mM), MgCl 2 (10 mM), DCI and/or 1-MeIm were dissolved in water and adjusted to pH 6, and acetaldehyde (100 mM) and methyl isonitrile (100 mM) were added sequentially afterwards.The mixture was transferred to a sealed NMR tube and analyzed by 31 P-NMR spectroscopy at a pre-determined time.As shown in Fig. S5, in the presence of DCI, the yield of DCI-5 ′ -AMP (Scheme 1c) was about 50 %, and it hydrolysed quite rapidly; after 1 hour, the yield of DCI-5 ′ -AMP dropped to 8 %.The stability of 1-MeIm-5 ′ -AMP (Scheme 1c) was different from DCI-5 ′ -AMP.As shown in Fig. S6, the formation of 1-MeIm-5 ′ -AMP was not complete after 5 minutes, and the maximum yield was 95 % after 1 hour of incubation.After 3 hours, there was 76 % of 1-MeIm-5 ′ -AMP left, which indicated 1-MeIm-5 ′ -AMP was much more stable than DCI-5 ′ -AMP.Furthermore, we tested the formation and stability of azolide-5 ′ -AMP in a mixed solution of DCI and 1-MeIm.As shown in Fig. S7, the yield of 1-MeIm-5 ′ -AMP was 82 % and the yield of DCI-5 ′ -AMP was 11 %; the reaction was finished in 5 minutes.Both 1-MeIm-5 ′ -AMP and DCI-5 ′ -AMP were hydrolysed afterwards, and after 3 hours almost all DCI-5 ′ -AMP was hydrolysed, but 74 % of 1-MeIm-5 ′ -AMP remained.The stability of 1-MeIm-5 ′ -AMP was similar to the experiments performed without DCI.These results indicated the formation and degradation of 1-MeIm-5 ′ -AMP were not affected by the addition of DCI.Interestingly, the Passerini reaction was completely suppressed by adding 1-MeIm and/or DCI.Encouraged by these results, we tested RNA ligation using methyl isonitrile and acetaldehyde as activating reagents and with different concentrations of DCI and/or 1-MeIm.As shown in Fig. S8, with a lower concentration of methyl isonitrile and acetaldehyde (Fig. S8, line a), both the ligation yield and the Passerini reaction yield were lower than with higher concentrations of activating reagents (Fig. S8, line  b), but the ratio of ligation and Passerini reaction products was similar.With addi-tional DCI (10 mM, Fig. S8, line c), both ligation and the Passerini reaction were not changed, but with an even higher concentration of DCI (100 mM, Fig. S8, line d), the ligation yield was increased and the Passerini reaction was suppressed.Similar reactions were repeated with MnCl 2 instead of MgCl 2 , and the results were similar (Fig. S8, line e and f).Because the junction of the nicked-duplex of RNA is more crowded than a mononucleotide, the Passerini reaction in RNA reaction cannot be completely suppressed.Nevertheless, our results show that significant suppression can be attained in the presence of DCI.Furthermore, we have tested the effect of the concentration of Mn(II) (Fig. S8, line g and h).Even with 0.1 mM of Mn(II), the yield of ligation doesn't drop significantly.

General methods
Reagents and solvents were obtained from Acros Organics, Alfa Aesar, Santa Cruz Biotechnology, Sigma-Aldrich, SYNTHON Chemicals GmbH & Co., KG and VWR International.Reagents and solvents were used without further purification unless otherwise stated.Phosphoramidites for RNA synthesis were purchased from Sigma-Aldrich or Link Technologies.The Cyanine3 (Cy3) labelled oligonucleotides were purchased in HPLC-purified Na + form from Integrated DNA Technologies.The non-labelled oligonucleotide was synthesized using an ÄKTA oligopilot plus 10 instrument (GE Healthcare).A Mettler Toledo SevenEasy pH Meter S20 combined with a Thermo Fisher Scientific Orion 8103BN Ross semi-micro pH electrode was used to measure and adjust the pH to the desired value.NMR spectra ( 1 H, and 31 P) were acquired using a Bruker Ultrashield 400 Plus instrument or a Bruker Ascend 400 instrument operating at 400.13, and 161.97, respectively.Samples consisting of H 2 O/D 2 O mixtures were analysed using HOD suppression to collect 1 H-NMR spectroscopy data.Chemical shifts (δ) are shown in ppm.Mass spectra were acquired on an Agilent 1200 LC-MS system equipped with an electrospray ionization (ESI) source and a 6130 quadrupole spectrometer (LC solvents: A, 0.2 % formic acid in H 2 O and B, 0.2 % formic acid in acetonitrile).Gel electrophoresis experiments using 20 % polyacrylamide and 8 M urea gels (8 cm × 8 cm × 1 mm) were typically run at 6 W in Tris/Borate/EDTA buffer.Fluorescence imaging was performed using an Amersham Typhoon imager (GE Healthcare) and quantified using Image Quant TL software (version 7.0).Oligonucleotide concentrations were determined by ultraviolet absorbance at 260 nm using a NanoDrop ND-1000 spectrophotometer.

Chemical synthesis of RNA oligomers
After automated synthesis, RNAs were first cleaved from the solid support by treating with 3 mL of a 1:1 mixture of NH 3 aqueous solution (28 % wt.) and CH 3 NH 2 ethanol so-lution (33 % wt.) at 55°C for 90 min in a tube with a sealed cap.The solid was removed by filtration and washed with 50 % EtOH/H 2 O.The solutions were combined and evaporated to dryness under reduced pressure.Silyl protecting groups were removed by treating the residues with 2 mL of a 1:1 mixture of triethylamine trihydrofluoride and DMSO at 65°C for 3 hours in a tube with a sealed cap.After brief cooling at -32°C, 40 mL of cold 50 mM NaClO 4 in acetone was added to the solution to precipitate the oligoribonucleotides.The resulting mixture was centrifuged and oligoribonucleotides was dried by lyophilization.The RNA was redissolved in 5 mL of water and passed through a Waters Sep-Pak C18 Cartridge with 10 g sorbent.The cartridge was prewashed with 50 mL of acetonitrile then 50 mL of water before sample loading, washed with 150 mL of H 2 O and 50 mL of 10 % aqueous acetonitrile.Eluates were checked for RNA content using a NanoDrop spectrophotometer.After lyophilizing, the resulting white powder was stored at -32°C for future use.

General procedure for desalting of RNA by ethanol precipitation
Oligonucleotides were desalted by addition of 2 M imidazole nitrate solution (pH 6.2, 1/10 the volume of the aliquot taken from the reaction), followed by a 3 M sodium acetate solution (pH 5.2, 1/10 the volume of the aliquot taken from the reaction) and absolute ethanol (to a final concentration of 75 % (v/v)).The resulting mixture was kept at -20°C for 3 h and then centrifuged for 30 min at 16,000g.The supernatant was removed, and the pellets were washed with 75 % (v/v) aqueous ethanol before additional centrifugation (10 min at 16,000g).The resulting pellets were air dried before being re-dissolved in water.

General procedure for oligonucleotide ligation reactions using methyl isonitrile
To an aqueous solution of RNAs, organocatalysts, and divalent metal ion was added nuclease-free water to 8 µL, then methyl isonitrile (400 mM, 2 µL of a 2 M aqueous stock solution) was added and the reaction was kept at room temperature.Aliquots of 3.0 µL were taken at the indicated time points and desalted by ethanol precipitation.For each aliquot, the resulting pellet was re-dissolved in 3.0 µL of nuclease-free water and 1.0 µL of the resulting solution was mixed with 4.0 µL of loading dye (95 % (v/v) 10 M urea in H 2 O, 5 % (v/v) EDTA solution (0.5 M in H 2 O, pH = 8.0), Blue bromophenol).The resulting mixture was analysed using polyacrylamide gel electrophoresis.

General procedure for oligonucleotide ligation reactions using methyl isonitrile and acetaldehyde
To an aqueous solution of RNAs, organocatalysts, and divalent metal ion was added nuclease-free water to 6 µL, then acetaldehyde (400 mM, 2 µL of a 2 M aqueous stock solution) was added, then methyl isonitrile (400 mM, 2 µL of a 2 M aqueous stock solution) was added and the reaction was kept at room temperature.Aliquots of 3.0 µL were taken at the indicated time points and desalted by ethanol precipitation.For each aliquot, the resulting pellet was re-dissolved in 3.0 µL of nuclease-free water and 1.0 µL of the resulting solution was mixed with 4.0 µL of loading dye (95 % (v/v) 10 M urea in H 2 O, 5 % (v/v) EDTA solution (0.5 M in H 2 O, pH = 8.0), Blue bromophenol).The resulting mixture was analysed using polyacrylamide gel electrophoresis.

General procedure for oligonucleotide ligation reactions using sodium hypochlorite and potassium cyanide
To an aqueous solution of RNAs, organocatalysis, potassium cyanide and divalent metal ion was added nuclease-free water to 6 µL, then sodium hypochlorite (20 mM, 4 µL of a 0.05 M aqueous stock solution) was added and the reaction was kept at room temperature.Aliquots of 1.0 µL were taken at the indicated time points was mixed with 4.0 µL of loading dye (95 % (v/v) 10 M urea in H 2 O, 5 % (v/v) EDTA solution (0.5 M in H 2 O, pH = 8.0), Blue bromophenol).The resulting mixture was analysed using polyacrylamide gel electrophoresis.

Comparison ligation reactions between cyanogen chloride and NCI
Three RNA components were mixed with imidazole (100 mM), potassium cyanide (50 mM), and MnCl 2 (10 mM) at pH 6.The resulting solution was separated into two parts, and to one of them sodium hypochlorite (20 mM) was added.Both mixtures were incubated at room temperature for 24 hours, 1 µL aliquot was diluted into a loading buffer, and to both of the remaining reaction solutions was added N-cyanoimidazole (100 mM) before incubation at room temperature for another 24 hours.A 1 µL aliquot of the resultant solution was diluted into a loading buffer and then analysed by PAGE.

X-ray crystallography for 1-methyladenine : 4,5-dicyanoimidazole: H 2 O
Single-crystal X-ray diffraction data were collected on a Bruker D8-QUEST diffractometer, equipped with an Incoatec IµS Cu microsource (λ= 1.5418 Å) and a PHOTON-III detector operating in shutterless mode.The crystal temperature was held at 180(2) K using an Oxford Cryosystems open-flow N 2 Cryostream.The control and processing software was Bruker APEX3.The structure was solved using SHELXT 5 and refined using SHELXL 5 .H atoms attached to C atoms were placed in idealised positions and refined as riding.H atoms attached to N or O atoms were located in the difference Fourier map and refined freely with isotropic displacement parameters.f) same as a, incubated for 24 hours; g) same as b, incubated for 24 hours; h) same as c, incubated for 24 hours; i) same as d, incubated for 24 hours; j) same as e, incubated for 24 hours.