Telomerase RNA

Figure 2.1 General procedure for preparation of dye-labeled Telomerase RNA.

Protocol 1 In vitro transcription of full-length telomerase RNA... 

Figure 2.2 Schematic diagram of in vitro transcription reaction. The telomerase RNA gene is cloned behind a T7 RNA promoter. PCR is used to generate linear DNA and the PCR amplicons are in vitro transcribed with T7 RNA polymerase. The RNA is purified away from free nucleotide on a desalting column.

Full-length telomerase RNA prepared by in vitro transcription (see above)... 

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).

F. Bachand and C. Autexier. 2001. Functional regions of human telomerase reverse transcriptase and human telomerase RNA required for telomerase activity and RNA-protein interactions Mo/. Cell Biol. 21 1888-1897. (PuhMed) (Full Text in PMC)... 

The telomerase RNA pairs with the telomere sequence and the enzyme then extends the telomere by RNA-templated DNA synthesis. DNA polymerase can then make (all but the end) double stranded. 

Soder, A., Hoare, S., Muir, S., Going, J., Parkinson, E., and Keith, W. (1997) Amplification, increased dosage and in situ expression of the telomerase RNA gene in human cancer. Oncogene 14,1013-1021. 

Kushner DM, Paranjape JM, Bandyopad-hyay B, Cramer H, Leaman DW, et al. 2000. 2-5A antisense directed against telomerase RNA produces apoptosis in ovarian cancer cells. Gynecol. Oncol. 76 183-92... 

Treatment of prostate cancer in vitro and in vivo with 2-5A-anti-telomerase RNA component. Oncogene 19 2205-11... 

---