title: Brightness through local constraint--LNA-enhanced FIT hybridization probes for in vivo ribonucleotide particle tracking.
abstract: Imaging the dynamics of RNA in living cells is usually performed by means of transgenic approaches that require modification of RNA targets and cells. Fluorogenic hybridization probes would also allow the analysis of wild-type organisms. We developed nuclease-resistant DNA forced intercalation (FIT) probes that combine the high enhancement of fluorescence upon hybridization with the high brightness required to allow tracking of individual ribonucleotide particles (RNPs). In our design, a single thiazole orange (TO) intercalator dye is linked as a nucleobase surrogate and an adjacent locked nucleic acid (LNA) unit serves to introduce a local constraint. This closes fluorescence decay channels and thereby increases the brightness of the probe-target duplexes. As few as two probes were sufficient to enable the tracking of oskar mRNPs in wild-type living Drosophila melanogaster oocytes.
authors.affiliation: Department of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin (Germany).
authors.forename: Felix
authors.initials: F
authors.lastname: Hövelmann
authors.forename: Imre
authors.initials: I
authors.lastname: Gaspar
authors.forename: Simon
authors.initials: S
authors.lastname: Loibl
authors.forename: Eugeny A
authors.initials: EA
authors.lastname: Ermilov
authors.forename: Beate
authors.initials: B
authors.lastname: Röder
authors.forename: Jesper
authors.initials: J
authors.lastname: Wengel
authors.forename: Anne
authors.initials: A
authors.lastname: Ephrussi
authors.forename: Oliver
authors.initials: O
authors.lastname: Seitz
mesh_terms: D000818:Animals; D001483:Base Sequence; D052160:Benzothiazoles / Q000737:chemistry*; D029721:Drosophila Proteins / Q000235:genetics; D004331:Drosophila melanogaster / Q000737:chemistry / Q000235:genetics; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D007364:Intercalating Agents / Q000737:chemistry*; D008958:Models, Molecular; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry*; D011804:Quinolines / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis; D012265:Ribonucleotides / Q000032:analysis*
chemical_list: D052160:Benzothiazoles; D029721:Drosophila Proteins; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D009841:Oligonucleotides; D011804:Quinolines; D012333:RNA, Messenger; D012265:Ribonucleotides; C477371:locked nucleic acid; C091675:osk protein, Drosophila; C051000:thiazole orange
keywords: fluorescent probes; mRNA; microscopy; oligonucleotides; ribonucleoprotein particles