Figure 5.

Co-transcriptional production of RNA-DNA hybrids by yeast RNA Pol II. (a) Schematics of R/DNA hybrid production. (b) Optimization of the full-length RNA synthesis. The RNA primers used for analytical transcription reactions were labeled at the 5′-end by phosphorylation with γ-[³²P] ATP. RNA was annealed to DNA, and the elongation complexes were formed as described in Kireeva et al. (18). Transcription was initiated by addition of 1 mM NTPs unless indicated otherwise. Lower bands correspond to intermediate transcription stops. (c and d) Transcription products eluted from the Ni-NTA agarose cartridge containing immobilized Pol II were concentrated by ethanol precipitation, re-dissolved in standard buffer solution and MG2 aptamer (c) and DS siRNA (d) releases were monitored as described above. For (d), the lower relative efficiencies of the functionalities release can be explained by the lower concentrations of the initial hybrids and are consistent with published data (1).