Tzakos Andreas G, Easton Laura E, Lukavsky Peter J
MRC Laboratory of Molecular Biology, Cambridge, UK.
Nat Protoc. 2007;2(9):2139-47. doi: 10.1038/nprot.2007.306.
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 (13)C- and the other (15)N-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.
由于化学位移简并问题，通过溶液核磁共振光谱法测定RNA结构通常仅限于小RNA（<15 kDa）。将新型核磁共振技术与RNA片段标记方法有效结合，可能是克服RNA核磁共振光谱分子量限制的有力工具。在此，我们描述了一种制备和纯化片段标记大RNA的省时且经济高效的方法。通过从单个质粒DNA进行体外转录，制备两组具有互补标记方案的RNA片段，例如一个片段用（13）C标记，另一个用（15）N标记。所需的RNA片段通过两个顺式作用的锤头状核酶从初级转录本中切除，产生后续互补连接所需的工程化末端。所得的RNA寡核苷酸显示出共振重叠大大减少的核磁共振光谱，从而能够在大RNA的天然环境中对较小的标记RNA片段进行核磁共振研究。预计该方法实施需要3至4周时间。
