While uniform isotope labeling of ribonucleic acids (RNA) can simply and efficiently be achieved by in-vitro transcription, the specific introduction of nucleotides in larger constructs is non-trivial and often ineffective. Here, we demonstrate how a medium-sized (67-mer), biocatalytically relevant RNA (hammerhead ribozyme, HHRz) can be formed by spontaneous hybridization of two differently isotope-labeled strands, each individually synthesized by in-vitro transcription. This allows on the one hand for a significant reduction in the number of isotope-labeled nucleotides and thus spectral overlap particularly under magic-angle spinning (MAS) dynamic nuclear polarization (DNP) NMR conditions, on the other hand for orthogonal ¹³C/¹⁵N-labeling of complementary strands and thus for specific investigation of structurally or functionally relevant inter-strand and/or inter-stem contacts. By this method, we are able to confirm a non-canonical interaction due to single-site resolution and unique spectral assignments by two-dimensional ¹³C–¹³C (PDSD) as well as ¹⁵N–¹³C (TEDOR) correlation spectroscopy under “conventional” DNP enhancement. This contact is indicative of the ribozyme's functional conformation, and is present in frozen solution irrespective of the presence or absence of a Mg²⁺ co-factor. Finally, we use different isotope-labeling schemes in order to investigate the distance dependence of paramagnetic interactions and direct metal-ion DNP if the diamagnetic Mg²⁺ is substituted by paramagnetic Mn²⁺.

尽管可以通过体外转录简单而有效地实现核糖核酸（RNA）的统一同位素标记，但在较大的构建体中核苷酸的特异性导入并非易事，而且通常无效。在这里，我们演示了如何通过两种不同同位素标记的链的自发杂交形成中等大小（67-mer），生物催化相关的RNA（锤头状核酶，HHRz），每条链均通过体外转录合成。一方面，这可以显着减少同位素标记的核苷酸的数量，从而显着减少光谱重叠，尤其是在魔术角旋转（MAS）动态核极化（DNP）NMR条件下，另一方面，对于正交¹³ C / ¹⁵互补链的N-标记，因此用于结构或功能上相关的链间和/或茎间接触的特定研究。通过这种方法，我们能够通过二维¹³ C – ¹³ C（PDSD）以及¹⁵ N– ¹³ C（TEDOR）相关光谱法来确定由于单点分辨率和独特的光谱分配而导致的非规范相互作用。“常规” DNP增强。这种接触指示核酶的功能构象，并且无论是否存在Mg ²⁺都存在于冷冻溶液中辅助因子。最后，我们使用不同的同位素标记方案来研究顺磁性相互作用和Mg ²⁺被顺磁性Mn ²⁺取代时直接金属离子DNP的距离依赖性。