High-Yield Spin Labeling of Long RNAs for Electron Paramagnetic Resonance Spectroscopy
| Autor: | Mark Kerzhner, Olav Schiemann, Hideto Matsuoka, Michael Famulok, Christine Wuebben |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
TPP riboswitch
010405 organic chemistry Chemistry Aptamer Electron Spin Resonance Spectroscopy RNA Resonance Pyrimidinones Site-directed spin labeling Aptamers Nucleotide 010402 general chemistry Ligand (biochemistry) 01 natural sciences Biochemistry PreQ1 riboswitch 0104 chemical sciences Riboswitch Biophysics Nucleic Acid Conformation Click Chemistry Pyrroles Spin Labels Spectroscopy |
| Zdroj: | Biochemistry. 57:2923-2931 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/acs.biochem.8b00040 |
| Popis: | Site-directed spin labeling is a powerful tool for investigating the conformation and dynamics of biomacromolecules such as RNA. Here we introduce a spin labeling strategy based on click chemistry in solution that, in combination with enzymatic ligation, allows highly efficient labeling of complex and long RNAs with short reaction times and suppressed RNA degradation. With this approach, a 34-nucleotide aptamer domain of the preQ1 riboswitch and an 81-nucleotide TPP riboswitch aptamer could be labeled with two labels in several positions. We then show that conformations of the preQ1 aptamer and its dynamics can be monitored in the absence and presence of Mg2+ and a preQ1 ligand by continuous wave electron paramagnetic resonance spectroscopy at room temperature and pulsed electron–electron double resonance spectroscopy (PELDOR or DEER) in the frozen state. |
| Databáze: | OpenAIRE |
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