Using molecular beacons to study dispersal of mRNPs from the gene locus.
Using molecular beacons to study dispersal of mRNPs from the gene locus.
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DOI：
10.1007/978-1-60327-461-6_6
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发表时间：
2009
影响因子：
--
通讯作者：
P. V. D. van den Bogaard;Sanjay Tyagi
中科院分区：
文献类型：
--
作者：
P. V. D. van den Bogaard;Sanjay Tyagi
Before leaving the site of transcription, newborn messenger RNAs (mRNAs) become associated with a number of different proteins. How these large messenger ribonucleoprotein (mRNP) complexes then move through the dense nucleoplasm to reach the nuclear periphery has been a fascinating question for the last few years. We have studied the mechanism of this process by tracking individual mRNPs in real time. We were able to track mRNPs at single-molecule resolution because we utilized mRNAs that were engineered to have a sequence motif repeated 96 times in their untranslated region. These mRNAs were visualized with the help of molecular beacons that were specific for the repeated sequence; the binding of 96 molecular beacons to each mRNA molecule rendered them so intensely fluorescent that they were visible as fine fluorescent spots that could be tracked by high-speed video microscopy. In this chapter, we describe the details of the construction of genes containing the tandem repeats, the integration of such genes into the genome of a cell line, the design and testing of molecular beacons, time-lapse imaging of mRNPs, and computer-aided generation and analysis of the tracks of the individual mRNPs. These methods will be useful for studies of other dynamic processes such as mRNA export, splicing, and decay.
