Synthesis of [7-15N]-GTPs for RNA structure and dynamics by NMR spectroscopy

Abstract

Several isotope-labeling strategies have been developed for the study of RNA by nuclear magnetic resonance (NMR) spectroscopy. Here, we report a combined chemical and enzymatic synthesis of [7-15N]-guanosine-5′-triphosphates for incorporation into RNA via T7 RNA polymerase-based in vitro transcription. We showcase the utility of these labels to probe both structure and dynamics in two biologically important RNAs. Graphical abstract

Isotope Labels Combined with Solution NMR Spectroscopy Make Visible the Invisible Conformations of Small-to-Large RNAs

“…Reduction of 41 with sodium dithionite followed by acidification by H 2 SO 4 forms 2,6-diamino-5-[amino- 15 N]­pyrimidin-4-ol 42 . In the final step, reflux with formamide (HCONH 2 ) followed by HCOOH provides the desired [7- 15 N]-guanine 43 with a total yield of 65% (Scheme ).…”

“…In addition, 16 h were added for any explicit mention of overnight procedures, and 24 h were added for any chromatographic purifications. b Number in parentheses represents the number of chromatographic purification steps. c All data for [2- 13 C]-adenine and [7- 15 N]-adenine labeling came from the same doubly labeled [2- 13 C, 7- 15 N]-adenine labeling scheme d This synthetic procedure is from Dayie and co-workers …”

“…b Number in parentheses represents the number of chromatographic purification steps. c All data for [2-13 C]-adenine and [7-15 N]-adenine labeling came from the same doubly labeled [2-13 C, 7-15 N]-adenine labeling scheme.104 d This synthetic procedure is from Dayie and co-workers 116. h i b i t e d .…”