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Probing messenger RNA conformational heterogeneity using single-molecule fluorescence anisotropy
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7.A transcription reaction was carried out using T7 Luciferase control DNA (Bangalore genei) and T7 transcription kit (Promega). The mRNA was labeled by mixing Alexa488-labelled rUTP (molecular probes) with unlabeled rUTP supplied with the kit in a 1:20 ratio. The labeled mRNA transcripts were purified using RNeasy kit (Qiagen). The transcripts were then diluted 50-fold in nuclease-free de-ionized water for subsequent measurement of anisotropy time series. Critical dimensional experiments confirmed that introduction of the fluorescent label did not perturb mRNA structure.
8.J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Kluwer Academic/Plenum, New York, 1999).
9.Translation diffusion related measurements using fluorescence correlation spectroscopy (FCS) on mRNA as a function of salt concentration proved to be less sensitive to conformational heterogeneity than rotation related measurements outlined in this letter.
10.Data analysis: Two different but synchronized counters are used to acquire the fluorescence intensity of the parallel and perpendicular components of fluorescence time series and is passed through a filter (computer program written in LabView) to separate the mRNA signal from the background fluorescence. The filtered data yields a series of fluorescence peaks, which is obtained from different labeled mRNA molecules. Each fluorescence peak is averaged to find the mean and and are subsequently used to calculate the fluorescence anisotropy of the given mRNA molecule.
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