The origin of life in an RNA pocket
The RNA constructs: ec_P1c, tt_P1m, tt_P1n, tt_A1, tt_A2 were purchased from Dharmacon, Inc. . The DNA primers for the constructs tt_P1c, tt_P1, tt_P2, tt_P2c, tt_A1P1, tt_A2P2 were obtained from Sigma-Aldrich™ and inserted into pUC57 plasmid using restriction enzymes. Since the sequences of sa_P1c and ef_P1c had high similarity to tt_P1c, site-directed mutagenesis of the tt_P1c-pUC57 plasmid was performed to achieve sa_P1c and ef_P1c plasmids. A transfer PCR was set up using Phusion Polymerase.
To determine the role of the dimers in product formation, the above described peptide activity assay was performed also in the absence of MgCl, but in the presence of KCl, or in the absence of both salts for tt_P1c, tt_P1 and tt_A1P1. The remaining protocol was similar to that described above.5–10 mg of CHCA were dissolved in a 500 μl solution of 50% milli-Q water, 47% acetonitrile and 3% trifluoroacetic acid to provide a saturated solution, which was centrifuged.
The analytes were separated using Waters Premier Acquity UPLC system. The aqueous mobile phase A consisted of 50 mM 1,1,1,3,3,3-hexafluoro-2-propanol and 2.5 mM hexylamine . The organic mobile phase B consisted of methanol with 50 mM 1, 1,1,3,3,3-hexafluoro-2-propanol and 2.5 mM hexylamine. The separation was achieved with Waters Acquity Premier BEH C18 Column, 1.7 μm, 2.1 × 50 mm at 0.3 mlL/min flow rate, 60°C. After a one min hold at 10% B, the gradient was ramped to 50% B in 6.
Dimerization of the RNA constructs sa_P1c and ef_P1c was achieved using the slow annealing method. Samples of sa_P1c and ef_P1c were mixed with milli-Q water and DB to a final concentration of 1.8 μM. RNA samples were placed in a heat block at 90ºC for 30 s to denature them. They were then gradually denatured stepwise at the rate of 1.5ºC per minute to room temperature gradually over a period of 30–40 min. The RNA was then brought to 4ºC by keeping it on ice for a minimum of 2 h.
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