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Metal Ion-RNA Interactions Studied via Multinuclear NMR

Type of publication Peer-reviewed
Publikationsform Review article (peer-reviewed)
Author Donghi Daniela, Sigel Roland K. O.,
Project Coordination Chemistry within the Core of Large RNAs: Regulating Tertiary Contacts and Function
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Review article (peer-reviewed)

Journal Methods in Molecular Biology
Volume (Issue) 848
Page(s) 253 - 274
Title of proceedings Methods in Molecular Biology
DOI 10.1007/978-1-61779-545-9_16

Abstract

Metal ions are indispensable for ribonucleic acids (RNAs) folding and activity. First they act as charge neutralization agents, allowing the RNA molecule to attain the complex active three dimensional structure. Second, metal ions are eventually directly involved in function. Nuclear magnetic resonance (NMR) spectroscopy offers several ways to study the RNA–metal ion interactions at an atomic level. Here, we first focus on special requirements for NMR sample preparation for this kind of experiments: the practical aspects of in vitro transcription and purification of small (<50 nt) RNA fragments are described, as well as the precautions that must be taken into account when a sample for metal ion titration experiments is prepared. Subsequently, we discuss the NMR techniques to accurately locate and characterize metal ion binding sites in a large RNA. For example, 2 J-[1H,15N]-HSQC (heteronuclear single quantum coherence) experiments are described to qualitatively distinguish between different modes of interaction. Finally, part of the last section is devoted to data analysis; this is how to calculate intrinsic affinity constants.
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