Improving the sensitivity of aptamer-driven fluorescent protein complementation for RNA labeling and detection
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In eukaryotic cells, some mRNAs localize to distinct areas of the cell where RNA is translated and the encoded protein is specifically localized. Recent studies have suggested that even though prokaryotic cells lack internal compartmentalization, different RNAs can localize to distinct regions of the bacterial cell. Our lab is developing methods for labeling and detecting RNA with the goal of determining localization of endogenous RNAs within single cells. We currently employ an eIF4a protein-specific aptamer for RNA labeling using one of two methods. (1) Target RNA is tagged with the aptamer sequence at the 3' end and the aptamer triggers protein complementation of two fusion proteins, each containing split EGFP and split eIF4A proteins. (2) Two RNA probes, each containing a half of a split eIF4a-specific aptamer and an antisense sequence complementary to the target RNA, bind the unmodified transcript through complementary interactions. This binding brings the two fragments of the split aptamer in close proximity and allows proper folding of a split aptamer. A fluorescent signal is generated by the aptamer-driven reassociation of the fusion proteins. In this work, we investigate the sensitivity of the first method for detecting transcripts expressed from their natural chromosomal loci, and describe attempts to increase the sensitivity of the method by using multiple aptamer tagging. We also present results suggesting that the second method, combining protein complementation and split aptamer approach, provides high sensitivity enabling detection of endogenous bacterial RNAs expressed at low level.
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