Published online 4 April 2005
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tRNA-linked molecular beacons for imaging mRNAs in the cytoplasm of living cells
Department of Molecular Genetics, Public Health Research Institute 225 Warren Street, Newark, NJ 07103, USA 1Department of Cell Biology, New York University School of Medicine New York, NY 10016, USA
*To whom correspondence should be addressed. Tel: +1 973 854 3372; Fax: +1 973 854 3374; Email: sanjay{at}phri.org
Received January 17, 2005. Accepted February 24, 2005.
When oligonucleotide probes are microinjected into cells to image the distribution of RNAs, they are rapidly sequestered into the nucleus. As a result, it is difficult to detect mRNAs in the cytoplasm of living cells. We were able to overcome this process by attaching tRNA transcripts to the probes. We show that when fluorescently labeled tRNAs, tRNAs with extensions at their 5' end, or chimeric molecules in which a molecular beacon possessing a 2'-O-methylribonucleotide backbone is linked to a tRNA, are injected into the nucleus of HeLa cells, they are exported into the cytoplasm. When these constructs are introduced into the cytoplasm, they remain cytoplasmic. These constructs allow the distribution of both the general mRNA population and specific mRNAs to be imaged in living cells. This strategy should also be useful for enhancing the efficacy of antisense oligonucleotides by keeping them in the cytoplasm. Our observations show that the fidelity of the tRNA export system is relaxed for unnatural tRNA variants when they are introduced into the nucleus in large amounts.
Present addresses: M. M. Mhlanga, Institut Pasteur, Unité de Biologie Cellulaire du Noyau, Paris, France 75015
C. W. Fung, Integrated Program in Cellular, Molecular and Biophysical Studies, Columbia University, New York, NY 10032, USA
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