Noncovalent Spin Labeling of Riboswitch RNAs To Obtain Long-Range Structural NMR Restraints

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• 作者：Christina Helmling ; Irene Bessi ; Anna Wacker ; Kai A. Schnorr ; Hendrik R. A. Jonker ; Christian Richter ; Dominic Wagner ; Michael Kreibich ; Harald Schwalbe
• 刊名：ACS Chemical Biology
• 出版年：2014
• 出版时间：June 20, 2014
• 年：2014
• 卷：9
• 期：6
• 页码：1330-1339
• 全文大小：431K
• 年卷期：v.9,no.6(June 20, 2014)
• ISSN：1554-8937

文摘

Paramagnetic relaxation enhancement (PRE) NMR is a powerful method to study structure, dynamics and function of proteins. Up to now, the application of PRE NMR on RNAs is a significant challenge due to the limited size of chemically synthesized RNA. Here, we present a noncovalent spin labeling strategy to spin label RNAs in high yields required for NMR studies. The approach requires the presence of a helix segment composed of about 10 nucleotides (nt) but is not restricted by the size of the RNA. We show successful application of this strategy on the 2鈥瞕G sensing aptamer domain of Mesoplasma florum (78 nt). The aptamer domain was prepared in two fragments. A larger fragment was obtained by biochemical means, while a short spin labeled fragment was prepared by chemical solid-phase synthesis. The two fragments were annealed noncovalently by hybridization. We performed NMR, cw-EPR experiments and gel shift assays to investigate the stability of the two-fragment complex. NMR analysis in ¹⁵N-TROSY and ¹H,¹H-NOESY spectra of both unmodified and spin labeled aptamer domain show that the fragmented system forms a stable hybridization product, is in structural agreement with the full length aptamer domain and maintains its function. Together with structure modeling, experimentally determined ¹H-螕₂ rates are in agreement with reported crystal structure data and show that distance restraints up to 25 脜 can be obtained from NMR PRE data of RNA.