NATuRe CHemISTRy
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A3′P 3 (10mM), A5′P 9 (10mM), Ac-ꢁ-Ala (100mM), glycine (50mM) and DCI
ꢄethods
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(20mM) was adjusted to pH 4 or 5.2 with HCl (1M) and NaOH (1M) solutions.
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 and were used without further puriꢃcation unless
otherwise stated. ꢆe 7nt RNA oligonucleotide (7nt primer, 5′-GAGAACC-3′),
Methyl isonitrile 1 (6 μl, final concentration to 200mM) was then added and the
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reaction was analysed using H and P NMR spectroscopy at 23°C. The pH had
changed only slightly (<0.2) by the time the isonitrile was totally consumed.
8
nt RNA oligonucleotide (8nt ligator, 5′-/phos/UACUGGCA/3Cy3Sp/-3′) and
3nt RNA oligonucleotide (13nt template, 5′-CCAGUAGGUUCUC-3′) were
Preparation of adenosine-2′-N-acetyl-ꢀ (or ꢁ)-alanine-3′-monophosphate
(ꢀ-standard or ꢁ-standard). 1,1′-Carbonyldiimidazole (8.9mg, 55μmol) was
added to a suspension of Ac-ꢁ-Ala or Ac-ꢂ-Ala (6.5mg, 50μmol) in acetonitrile
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+
purchased in HPLC-puriꢃed Na form from Integrated DNA Technologies. ꢆe
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nt RNA oligonucleotide (5′-/phos/UACUGGCA-3′) was synthesized using
(
50μl) and the mixture was vortexed for 5min at room temperature. The resulting
an ÄKTA oligopilot plus 10 instrument (GE Healthcare). All photochemical
reactions were carried out in Norell Suprasil quartz NMR tubes purchased from
Sigma-Aldrich using Hg lamps with principal emission at 360nm in a Rayonet
photochemical chamber reactor RPR-200, acquired from ꢆe Southern New
England Ultraviolet Company. A Mettler Toledo SevenEasy pH Meter S20
combined with a ꢆermo Fisher Scientiꢃc Orion 8103BN Ross semi-micro pH
electrode was used to measure and adjust the pH to the desired value. NMR spectra
solution, containing the acyl imidazolide derivative of N-acetyl-ꢁ (or ꢂ)-alanine,
was then added to a solution of A3′P 3 (9.8mg, 25μmol) in H O/D O (9:1, 450μl).
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2
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The mixture was quickly analysed using H and P NMR spectroscopy to confirm
that the desired 2′-acylated product had formed. The mixture was then used
immediately to spike other experiments.
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13
Chemical synthesis of 10. EDC (76.4mg, 400μmol) was added to 2ml of aqueous
solution containing A5′P 9 (8mg, 200μmol), Ac-Gly (47mg, 400μmol) and DCI 7
(
H, P and C) were acquired using a Bruker Ultrashield 400 Plus instrument
or a Bruker Ascend 400 instrument operating at 400.13, 161.97 and 100.62MHz,
respectively. Samples consisting of H O/D O mixtures were analysed using HOD
(
2.4mg, 20μmol) at pH 6.0 and at room temperature. The pH of the mixture was
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2
1
kept around 6 by adding 5M HCl solution. After 2h, the reaction was titrated to
suppression to collect H NMR spectroscopy data. ꢆe notations s, d, p and m
represent the multiplicities singlet, doublet, quintet and multiple, respectively.
Chemical shiꢈs (δ) are shown in ppm. Mass spectra were acquired on an Agilent
4 4
pH 4 and quenched by adding 40ml of cold NaClO solution (50mM NaClO in
acetone). The precipitate was collected by centrifugation, washed with cold acetone
and dried under a stream of nitrogen. The yield of the product 10e (40 to 70%) was
1
200 LC-MS system equipped with an electrospray ionization (ESI) source and
checked using 31P NMR spectroscopy at pH 5 in H
O/D O.
2 2
2
a 6130 quadrupole spectrometer (LC solvents: A, 0.2% formic acid in H O and
B, 0.2% formic acid in acetonitrile). Gel electrophoresis experiments using 20%
polyacrylamide and 8M urea gels (0.75mm thick, 20cm long) were typically run at
DCI catalysed hydrolysis of 10e. Synthesized 10e was dissolved in water (1ml) at
pH 5. Then, 150μl of this solution, 200μl of MES buffer (500mM, pH 5.2), 50μl
1
5W in Tris/Borate/EDTA buꢄer. Fluorescence imaging was performed using an
D O and a corresponding volume of DCI 7 solution (500mM, pH 5.2) were mixed
Amersham Typhoon imager (GE Healthcare) and quantiꢃed using Image Quant
TL soꢈware (version 7.0). Oligonucleotide concentrations were determined by
ultraviolet absorbance at 260nm using a NanoDrop ND-1000 spectrophotometer.
2
together to give final concentrations of DCI of 0mM, 10mM and 50mM. Water
was added to a final volume of 500μl if needed. The pH was checked to be 5.2,
otherwise it was adjusted with HCl (1M) and NaOH (1M) solutions. The reactions
were then monitored using 31P NMR spectroscopy at 23°C.
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Prebiotic synthesis of DCI 7. KCN (0.46mmol, 30mg, C-labelled or natural
abundance) was mixed with ammonium formate (0.06mmol, 4mg) in formamide
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Formation of oligopeptides from a dipeptide under activation chemistry. An
(
1ml) and the mixture was heated at 165°C for 2h (ref. ). The solvent was
2 2
aqueous solution (0.5ml, H O/D O, 9:1) of A5′P 9 (10mM), ꢁ-Ala-ꢁ-Ala (100mM)
evaporated under reduced pressure and the residue was extracted with hot water
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and DCI 7 (100mM) was adjusted to pH 5.2 with HCl (1M) and NaOH (1M)
solutions. Methyl isonitrile 1 (3μl, final concentration to 100mM) was then
added and the mixture was incubated at room temperature (white precipitate
was formed during the incubation). After 3 days, the precipitate was removed by
following evaporation. Then, the product mixture was analysed using H and
C
NMR spectroscopy (10% D O in H O). The formation of DCI 7 was confirmed by
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2
spiking the crude mixture with authentic material (purchased from Sigma-Aldrich)
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and recording the H NMR spectrum and also by comparing the C NMR
spectrum of the crude mixture with that of the authentic material.
centrifugation. The supernatant was diluted 100-fold with H
mass spectrometry (ESI, positive ion mode).
2
O and analysed using
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Chemical synthesis of N-formyl-glycylglycine (formyl-Gly-Gly) . Glycyl-glycine
15.1mmol, 2g) was stirred in formic acid (35ml) and heated at 55°C while adding
Formation of dipeptide Ac-Ala-Gly in reactions of Ac-Ala and glycine under
activation chemistry. An aqueous solution (0.5ml, H O/D
(
acetic anhydride (13.5ml) dropwise. The solution was stirred at room temperature
for 1h then ice–water (12ml) was added and the solution was concentrated under
reduced pressure. The residual solution was left in a fridge at 4°C overnight where-
upon crystals formed at the bottom of the flask. The crystals were collected by
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2
O, 9:1) of A5′P 9 (0
or 10mM), Ac-ꢁ-Ala (100mM), glycine (50mM) and DCI 7 (0, 20 or 50mM)
was adjusted to pH 5.2 or 4 with HCl (1M) and NaOH (1M) solutions. Methyl
isonitrile 1 (6μl, final concentration to 200mM) was then added and the mixture
was monitored using NMR spectroscopy at 23°C.
filtration, washed with diethyl ether and dried overnight using a desiccator to give a:
1
white solid; yield 810mg (31%); H NMR (400Hz, D
2
O): δ=8.2 (s, 1H), 4.1 (s, 2H),
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General procedure for desalting of RNA by ethanol precipitation.
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2
.0 (s, 2H) ppm; C NMR (101Hz, D O): δ=173.3, 171.6, 164.9, 41.1, 41.0 ppm.
Oligonucleotides were desalted by addition of 2M imidazole nitrate solution (pH
6.2, 1/10 the volume of the aliquot taken from the reaction), followed by a 3M
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 3h and then centrifuged for 30min at 16,000g. The
supernatant was removed and the pellets were washed with 75% (v/v) aqueous
ethanol before additional centrifugation (10min at 16,000g). The resulting pellets
were air dried before being re-dissolved in water.
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Chemical synthesis of N-acetyl-alanylglycine (Ac-Ala-Gly) . Alanyl-glycine
(
1mmol, 146mg) was dissolved in a minimum amount of saturated NaHCO
3
solution. Then, 2 equiv. of acetic anhydride were added. After 30min at room
+
+
temperature, Na was removed using an H -formed Dowex-50 column. The
1
solution was lyophilized to give a: white powder; yield 170mg (83%); H NMR
(
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1
400Hz, D O): δ=8.22 (s, 1H), 8.04 (s, 1H), 4.27 (p, J=7.1Hz, 1H), 3.84–3.67 (m,
2
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3
H), 1.97 (s, 3H), 1.32 (d, J=7.2Hz, 3H) ppm; C NMR (101Hz, D O): δ=175.59,
2
75.09, 174.17, 49.74, 42.84, 21.83, 16.80 ppm.
General procedure for oligonucleotide ligation reactions. To an aqueous solution
of 7nt primer (20μM, 0.5μl of 400μM stock solution), 8nt ligator RNA (10μM,
0.5μl of 200μM stock solution), 13nt template RNA (10μM, 0.5μl of 200μM
stock solution), DCI 7 (0 or 100mM, 0 or 4μl of 250mM stock solution at pH 6),
1-methylimidazole (0 or 100mM, 0 or 1μl of 1M stock solution at pH 6), divalent
Standard procedure for carboxylate and phosphate activation in reactions
containing A3′P 3. An aqueous solution (0.5ml, H O/D O, 9:1) of A3′P 3
2
2
(
(
(
1
10mM), carboxylic acid (100mM, if required) and heterocyclic compound
100mM, if required) was adjusted to the corresponding pH value with HCl
1M) and NaOH (1M) solutions. Methyl isonitrile 1 (3μl, final concentration to
2+
2+
2+
2+
metal ion (Mn , Mg , Fe or Zn , 0 or 10mM, 0 or 1μl of 100mM stock solution
at pH 6) was added nuclease-free water to 8μl, then methyl isonitrile 1 (400mM,
2μl of a 2M aqueous stock solution) was added and the reaction was kept at room
temperature. Aliquots of 3.0μl were taken at the indicated time points and desalted
by ethanol precipitation. For each aliquot, the resulting pellet was re-dissolved in
3.0μl of nuclease-free water and 1.0μl of the resulting solution was mixed with
4.0μl of loading dye (90% (v/v) formamide, 5% (v/v) glycerol, 24mM EDTA,
Orange G). The resulting mixture was analysed using gel electrophoresis.
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00mM) was then added and the reaction was analysed using H and P NMR
spectroscopy at 23°C. The pH had changed only slightly (<0.2) by the time the
isonitrile was totally consumed.
Standard procedure for carboxylate and phosphate activation in reactions
2 2
containing A5′P 9. An aqueous solution (0.5ml, H O/D O, 9:1) of A5′P 9 (10mM),
carboxylic acids (200mM) and heterocyclic compounds (100mM, if required) was
adjusted to the desired pH value with HCl (1M) and NaOH (1M) solutions. Methyl
isonitrile 1 (3μl, final concentration to 100mM) was then added and the reaction
Pyrophosphate formation under activation chemistry. An aqueous solution
(0.5ml, H O/D O, 9:1) of pUACUGGCA (1mM), DCI 7 (100mM), MgCl
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was analysed using H and P NMR spectroscopy at 23°C. The pH had changed
2
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2
only slightly (<0.2) by the time the isonitrile was totally consumed.
(10mM) and N-methylimidazole (100mM) was adjusted to pH 6 with HCl (1M)
and NaOH (1M) solutions. Methyl isonitrile 1 (12μl, final concentration to
400mM) was then added and the mixture was monitored using NMR spectroscopy
at 23°C.
Standard procedure for carboxylate and phosphate activation in reactions
containing A3′P 3 and A5′P 9. An aqueous solution (0.5ml, H
2 2
O/D O, 9:1) of