tRNA-linked molecular beacons for imaging mRNAs in the cytoplasm of living cells.
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.
Mhlanga
Musa M
MM
Department of Molecular Genetics, Public Health Research Institute 225 Warren Street, Newark, NJ 07103, USA.
Vargas
Diana Y
DY
Fung
Cindy W
CW
Kramer
Fred Russell
FR
Tyagi
Sanjay
S
D021581:Active Transport, Cell Nucleus; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism; D002642:Chick Embryo; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000235:genetics*; D005456:Fluorescent Dyes / Q000737:chemistry; D005818:Genetic Engineering; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D015347:RNA Probes / Q000032:analysis / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D005456:Fluorescent Dyes; D015347:RNA Probes; D012333:RNA, Messenger; D012343:RNA, Transfer