Abstract
Two new methods of detecting nascent transcripts in vitro in single molecule fluorescence experiments were developed in this study. The first method is to utilize the PP7 protein-PP7 target hairpin system. Templates containing PP7 target hairpin encoding region were constructed for single molecule fluorescence experiments. When dye-labeled PP7 protein specifically binds to the PP7 target hairpin in RNA, we should be able to detect transcripts by the fluorescence of dye-labeled PP7. Experiments on the templates showed the insertion of PP7 target hairpin system had little influence on transcription. Nonspecific binding of PP7 protein to the templates was also observed, which could be a problem at low transcription efficiencies. The second method is to detect transcription by dissociation of a DNA triplex. The triplex assay has been designed and the necessary DNAs have been constructed. This work provides solid basis of needed materials and understanding for these two new methods. With future study based on this work, a powerful tool for single-molecule studies of initiation may be developed.