Extended Data Table 3.
PLOR recipes for the fluorescent-labeled RiboA71 samples.
| Sample* | NTP addition (NTP coefficients represent the multiples of template) | [DNA], total vol.† |
|---|---|---|
| U24Cy3-C55Cy5 | Elongation (cycle 2): ATP, GTP; (cycle 3): UTP; (cycle 4): ATP, 5-aminoallyl-UTP; (cycle 5): ATP, 6GTP, 6UTP; (cycle 6): 3ATP, 3CTP, UTP; (cycle 7): ATP, 2GTP; (cycle 8): 2CTP, 2UTP; (cycle 9, 37°C): 3ATP, Cy5-CTP; (cycle 10): CTP, GTP, 3UTP; Termination (cycle 11): 2ATP, 4CTP, 5UTP | 5 μM, 3 mL |
| U24Cy3-C55Cy5-B | Elongation (cycle 2): ATP, GTP; (cycle 3): UTP; (cycle 4): ATP, 5-aminoallyl-UTP; (cycle 5): ATP, 6GTP, 6UTP; (cycle 6): 3ATP, 3CTP, UTP; (cycle 7): ATP, 2GTP; (cycle 8): 2CTP, 2UTP; (cycle 9, 37°C): 3ATP, Cy5-CTP; (cycle 10): CTP, GTP, 3UTP; (cycle 11): 2ATP, 3UTP; (cycle 12): CTP; Termination (cycle 13): 2UTP, 3 biotin-11-CTP | 5 μM, 3 mL |
| U24Cy3-U65Cy5 | Elongation (cycle 2): ATP, GTP; (cycle 3): UTP; (cycle 4): ATP, 5-aminoallyl UTP; (cycle 5): ATP, 6GTP, 6UTP; (cycle 6): 3ATP, 3CTP, UTP; (cycle 7): ATP, 2CTP, 2GTP; (cycle 8): 3ATP, 2CTP, 5UTP; (cycle 9): ATP, GTP; (cycle 10): 2UTP; (cycle 11, 37°C): ATP, Cy5-UTP; Termination (cycle 12): 4CTP, 2UTP | 5 μM, 3 mL |
| U24Cy3-U65Cy5-B | Elongation (cycle 2): ATP, GTP; (cycle 3): UTP; (cycle 4): ATP, 5-aminoallyl UTP; (cycle 5): ATP, 6GTP, 6UTP; (cycle 6): 3ATP, 3CTP, UTP; (cycle 7): ATP, 2CTP, 2GTP; (cycle 8): 3ATP, 2CTP, 5UTP; (cycle 9): ATP, GTP; (cycle 10): 2UTP; (cycle 11, 37°C): ATP, Cy5-UTP; (cycle 12): CTP; Termination (cycle 13): 2UTP, 3 biotin-11-CTP | 5 μM, 3 mL |
| U24A555-U65A488-B | Elongation (cycle 2): ATP, GTP; (cycle 3): UTP; (cycle 4): ATP, 5-aminoallyl UTP; (cycle 5): ATP, 6GTP, 6UTP; (cycle 6): 3ATP, 3CTP, UTP; (cycle 7): ATP, 2CTP, 2GTP; (cycle 8): 3ATP, 2CTP, 5UTP; (cycle 9): ATP, GTP; (cycle 10): 2UTP; (cycle 11, 37°C): ATP, Alexa488-UTP; (cycle 12): CTP; Termination (cycle 13): 2UTP, 3 biotin-11-CTP | 5 μM, 1 mL |
*
Cy3- and Alexa555-labeled samples were generated by adding Cy3-NHS ester (GE Healthcare, Buckinghamshire, UK) or Alexa555-NHS ester (Invitrogen, Eugene, OR, USA), respectively, to a solution containing the lyophilized transcript product dissolved in ddH2O, followed by the addition of 0.5 volumes of 0.3 M sodium bicarbonate (pH 8.3). The reactions were incubated in the dark with gentle mixing for 1 hr at 37 °C, 12 hr at 25 °C, and then 1 hr at 37 °C.
†
The template concentrations and total reaction volumes were listed for NMR and FRET sample synthesis.