10.1002/anie.201916272
Angewandte Chemie International Edition
RESEARCH ARTICLE
concentrations of 0.45 mM OsO4 and 180 mM NH4Cl in a total volume of
22 µL. The mixture was incubated for 2 hours at 40 °C, then transferred
into Vivaspin 500 (MWCO 3000, PES) centrifugal concentrators
(Sartorius, Göttingen, Germany) and washed two times with 400 µL of
water. Afterwards, the RNA was lyophilized, dissolved in water (15 µL),
and hydrazine stock solution (5 µL, 1.5 M, pH = 8.98) was added to give
a final concentration of 375 mM hydrazine in a total volume of 20 µL. The
solution was incubated for 2 hours at 40 °C. The RNA solution was
transferred into centrifugal concentrators and washed four times with 400
µL of water as described before. Progress of the RNA conversion
reactions was monitored by anion exchange HPLC; 6soG and A'
modified RNA was stored at -20°C in aqueous solution or lyophilized. As
an alternative to ultrafiltration, the RNA can also be precipitated by
adding 90 µL of precipitation solution (650 µL water, 150 µL 1 M NaOAc,
pH 5.2, 10 µL glycogen (20mg/mL)) and 250 µL of cold ethanol, stored at
-20 °C for 30 minutes, followed by centrifugation (13000 rpm, 4 °C, 30
min). The supernatant was discarded and the RNA was lyophilized or
dissolved in water (15 µL) and further processed as described above. For
isolated, metabolically labeled cellular RNA, a milder hydrazine stock
solution was used. Briefly, after OsO4/NH4Cl treatment and precipitation,
the RNA pellet was resuspended in water (15 µL) and hydrazine stock
solution (5 µL; 0.5 M, pH 8.11, 5 mM EDTA, 0.5 M Tris) was added to
give a final concentration of 125 mM hydrazine.
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We thank Johannes Kremser (University of Innsbruck) for
assistance in tRNA synthesis and Vincent Oliéric for assistance
in data collection at the SLS Swiss Light Source. This work was
supported by the Austrian Science Fund FWF (P27947, P31691,
F8011-B) to R.M; P27024-BBL, F8009-B to A.L.), the Austrian
Research Promotion Agency FFG [West Austrian BioNMR
858017], and the Wiener Wissenschafts-, Forschungs- und
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