Translation inhibition reveals interaction of 2'-deoxy and 2'-O-methyl molecular beacons with mRNA targets in living cells.
Understanding the interaction between oligonucleotide probes and RNA targets in living cells is important for biological and clinical studies of gene expression in vivo. Here, we demonstrate that starvation of cells and translation inhibition by blocking the mTOR or PI-3 kinase pathway could significantly reduce the fluorescence signal from 2'-deoxy molecular beacons (MBs) targeting K-ras and GAPDH mRNAs in living cells. However, the intensity and localization of fluorescence signal from MBs targeting nontranslated 28S rRNA remained the same in normal and translation-inhibited cells. We also found that, in targeting K-ras and GAPDH mRNAs, the signal level from MBs with 2'-O-methyl backbone did not change when translation was repressed. Taken together, our findings suggest that MBs with DNA backbone hybridize preferentially with mRNAs in their translational state in living cells, whereas those with 2'-O-methyl chemistry tend to hybridize to mRNA targets in both translational and nontranslational states. This work may thus provide a significant insight into probe design for detection of RNA molecules in living cells and RNA biology.
Nitin
Nitin
N
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Rhee
Won Jong
WJ
Bao
Gang
G
D000730:Androstadienes / Q000494:pharmacology; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000737:chemistry*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D007700:Kinetics; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D014176:Protein Biosynthesis / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012339:RNA, Ribosomal, 28S / Q000032:analysis / Q000737:chemistry; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D013696:Temperature; D000077191:Wortmannin; D018631:ras Proteins / Q000235:genetics
D000730:Androstadienes; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D012339:RNA, Ribosomal, 28S; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D011494:Protein Kinases; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D018631:ras Proteins; D020123:Sirolimus; D000077191:Wortmannin