Fluorescence Detection of Intron Lariat RNA with Reduction‐Triggered Fluorescent Probes

Abstract

Fluoreszenz durch Nähe: Reduktionsinduzierte Fluoreszenzsonden, die ein Fluorescein‐Derivat oder Triphenylphosphan tragen, binden sequenzspezifisch am Verzweigungspunkt von Lariat‐RNA. Die chemische Reaktion der beiden Substrate führt zur Bildung von nativem Fluorescein und zur Emission eines Fluoreszenzsignals (siehe Bild). Prälariat‐ und Lariat‐Strukturen können mit dieser Methode unterschieden werden.

Identification of a novel component C2ORF3 in the lariat–intron complex: lack of C2ORF3 interferes with pre‐mRNA splicing via intron turnover pathway

“…If this is the case, the C2ORF3 foci must contain lariat introns. We have established specific detection of branched RNA using reduction-triggered fluorescent probes (Furukawa et al 2011), and this technique will be available to determine whether lariat introns are localized in the C2ORF3 foci. The unexpected detection of C2ORF3 protein also in the nucleoli suggests a function for C2ORF3 in rRNA processing.…”

Reactions Templated by Nucleic Acids: More Ways to Translate Oligonucleotide‐Based Instructions into Emerging Function

“…The orthogonality and biocompatibility of the Staudinger reaction [107] have inspired several groups to explore the utility of this reaction in templated processes (Figure 11.9). [79,[108][109][110][111][112][113][114][115][116][117][118][119][120][121] The reaction has been found to be highly efficient using both DNA and PNA strands and generally proceeds within 30 min with high conversion. A major focus has been the use of this reaction in nucleic acid sensing.…”

“…Alternatively, several groups have reported new profluorophores that are activated by an azide reduction (Figure 15.1, see discussion below). [109][110][111][118][119][120][121] The major advantage of this strategy is that an excess of phosphine can be employed to overcome the partial oxidation of phosphine under aerobic conditions. Analogously, azide-based immolative linkers have been used to dissociate a fluorophore and its quencher or release a profluorophore or other bioactive molecules (Figure 15.2).…”

“…In these first examples, the fluorescence of coumarin was greatly reduced by converting the electron-donating group, the amino or phenol, into an azide or azidomethyl ether group, respectively. The same logic was applied to fluorophores with red-shifted emission: azidorhodamine, [110,118] azidomethylfluorescein, [119] bis-(azido)rhodamine, [111] bis(azidomethyl)fluorescein, [121] and bis(azido)naphthorhodamine (Figure 15.1). [120] The reaction of azidorhodamine with triphenylphosphine was found to generate a surprisingly stable aza-ylide that did not hydrolyze over the time course of the templated reaction (templated reaction was complete within 30 min and the half-life of azaylide decomposition was approximately 24 h).…”

“…The same chemistry was used to detect Lariat RNA (LaRNA), a 2',5' branched linkage implicated in Group II introns and spliceosomes. [121] The branching geometry brings into proximity nucleic acid segments that would otherwise be distant. Using probes targeting these segments in conjunction with templated Staudinger reduction, LaRNA could be detected in vitro with higher accuracy than using the analogous approach with fluorophores yielding a FRET signal; detection at levels as low as 50 pm was achieved because of template turnover-induced signal amplification.…”