Multiple segmental and selective isotope labeling of large RNA for NMR structural studies
2008
Nelissen, Frank H.T. | van Gammeren, Adriaan J. | Tessari, Marco | Girard, Frederic C. | Heus, Hans A. | Wijmenga, Sybren S.
Multiple segmental and selective isotope labeling of RNA with three segments has been demonstrated by introducing an RNA segment, selectively labeled with ¹³C₉/¹⁵N₂/²H₍₁', ₃', ₄', ₅', ₅''₎-labeled uridine residues, into the central position of the 20 kDa ε-RNA of Duck Hepatitis B Virus. The RNA molecules were produced via two efficient protocols: a two-step protocol, which uses T4 DNA ligase and T4 RNA ligase 1, and a one-pot protocol, which uses T4 RNA ligase 1 alone. With T4 RNA ligase 1 all not-to-be-ligated termini are usually protected to prevent formation of side products. We show that such labor-intensive protection of termini is not required, provided segmentation sites can be chosen such that the segments fold into the target structure or target-like structures and thus are not trapped into stable alternate structures. These sites can be reliably predicted via DINAMelt. The simplified NMR spectrum provided evidence for the presence of a U28 H₃-imino resonance, previously obscured in the fully labeled sample, and thus of the non-canonical base pair U28:C37. The demonstrated multiple segmental labeling protocols are generally applicable to large RNA molecules and can be extended to more than three segments.
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