Optimized Preparation of Segmentally Labeled RNAs for NMR Structure Determination

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• RNA structures are underrepresented in public databases compared to proteins.
• Nuclear Magnetic Resonance (NMR) is crucial for RNA structure determination but limited to smaller molecules (<60 nucleotides) due to signal overlap.
• A mutated DNA polymerase (TGK) can extend primer RNAs for NMR studies of larger RNA molecules.

Purpose of the Study:

• To address challenges in preparing homogeneous RNA samples for NMR structural studies.
• To identify and overcome limitations in using TGK polymerase for extending primer RNAs.
• To enable routine production of segmentally labeled RNAs for high-resolution structural analysis.

Main Methods:

• Investigated sequence- and enzyme-dependent complications in TGK extension of primer RNAs.
• Identified wild-type T7-RNA polymerase (RNAP^WT) non-templated run-on as a source of heterogeneity.
• Utilized a T7 RNAP mutant for improved primer RNA homogeneity and 2'-O-methylated DNA templates.
• Employed extended DNA templates to shift heterogeneity from the region of interest.
• Developed a method for large-scale synthesis of homogeneous template DNA.

Main Results:

• Wild-type T7-RNA polymerase (RNAP^WT) run-on caused significant product heterogeneity.
• A T7 RNAP mutant and optimized template design yielded homogeneous primer RNAs in high yield.
• Minor heterogeneity at 3' ends was managed by extending DNA templates.
• A scalable method for homogeneous template DNA synthesis was established.

Conclusions:

• Overcame heterogeneity issues in TGK-mediated RNA extension for NMR studies.
• Enabled routine production of homogeneous, segmentally labeled RNAs for high-resolution structural analysis.
• Expanded the utility of NMR for studying larger and more complex RNA structures.