RNA ligation

The assembly of complex RNA molecules, harboring site-specific modifications, is a multistep procedure that can be subdivided into three stages (Fig. 2.1) (i) preparation of unmodified RNA ligation precursor fragments, (ii) preparation of modified RNA ligation precursor fragments, and... 

Protocol 2 Targeted RNase H cleavage of unmodified RNA ligation precursor fragment... 

Preparation of Modified (Dye Labeled) RNA Ligation Precursor Molecules... 

Despite these clear advantages, the efficiency of splinted RNA ligation reactions catalyzed by T4 DNA ligase is typically limited by the amount of enzyme one can introduce into the reaction. This problem is derived from the poor turnover of T4 DNA ligase on DNA/RNA heteroduplexes. More recently, a variant of T4 RNA ligase, called T4 RNA ligase 2 (Nandakumar et al, 2004), has been identified. This enzyme has a pronounced preference... 

Protocol 4 Splinted RNA ligation method for producing FRET-labeled telomerase RNA... 

We have developed the splinted RNA ligation procedure outlined in this chapter to generate site-specifically dye-labeled telomerase RNA constructs. These modified telomerase RNA constructs may be used to characterize dynamic RNA structural properties using Forster resonance energy transfer (FRET) (Stone et al., 2007). Our laboratory specializes in single molecule FRET measurements, which facilitates the direct observation of transient RNA structural states. The details of single molecule FRET... 

Figure 2.6 Application of splinted RNA ligation procedure single molecule FRET. (A) Diagram of prism-type total internal reflection fluorescence microscope (TIRFM) for single molecule FRET measurements. (B) Distribution of single-molecule FRET values for dye-labeled telomerase RNA molecules generated by splinted RNA ligation. (C) Dye intensity and FRET traces of a single telomerase RNA molecule Cy3 emission (green), Cy5 emission (red), FRET ratio (blue).

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