Preparation of large RNA oligonucleotides with complementary isotope-labeled segments for NMR structural studies

Abstract

RNA structure determination by solution NMR spectroscopy is often restricted to small RNAs (<15 kDa) owing to the problem of chemical shift degeneracy. A fruitful coupling of novel NMR techniques with segmental RNA labeling methodologies could be a powerful tool to overcome the molecular mass limitation of RNA NMR spectroscopy. Herein, we describe a time- and cost-effective procedure to prepare and purify segmentally labeled large RNAs. Two sets of RNA fragments with complementary labeling schemes, such as one fragment 13C- and the other 15N-labeled, are prepared by in vitro transcription from a single plasmid DNA. The desired RNA fragments are excised from the primary transcript by two cis-acting hammerhead ribozymes, yielding the required engineered ends for subsequent, complementary ligation. The resulting RNA oligonucleotides display NMR spectra with greatly reduced resonance overlap and thus enable NMR studies of smaller labeled RNA segments within the native context of a large RNA. The procedure is expected to take 3–4 weeks to implement.

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Figure 1: Design of linked, cis-acting hammerhead ribozymes.
Figure 2: Cloning strategy to prepare plasmid template with linked cis-acting hammerhead ribozymes and products of in vitro transcription.
Figure 3: Complementary segmental labeling yields simplified NMR spectra.

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Acknowledgements

A.G.T. is grateful for an EMBO long-term fellowship.

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Correspondence to Peter J Lukavsky.

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The authors declare no competing financial interests.

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Tzakos, A., Easton, L. & Lukavsky, P. Preparation of large RNA oligonucleotides with complementary isotope-labeled segments for NMR structural studies. Nat Protoc 2, 2139–2147 (2007) doi:10.1038/nprot.2007.306

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