Europium cryptate-tethered ribonucleotide for the labeling of RNA and its detection by time-resolved amplification of cryptate emission

TRACE (time-resolved amplification of cryptate emission), also called HTRF for pharmaceutical applications, is a homogeneous time-resolved fluorescenc e technique well adapted for the study of molecular interactions. It is bas ed on fluorescence resonance energy transfer (FRET) between europium trisbi pyridine cryptate (TBPEu3+) as energy donor and crosslinked allophycocyanin , symbolized by XL665, as acceptor, leading to a long-lived FRET signal. TB PEu3+-labeled uridine triphosphate (UTP), referred to as K-11-UTP in the te xt, was obtained by coupling TBPEu3+ moiety to a C-5 functionalized UTP ana log. K-11-UTP can be directly incorporated in RNA strands during enzymatic synthesis. This was demonstrated in an in vitro transcription reaction prom oted by T-7 RNA polymerase. The reaction was performed in the presence of K -11-UTP and biotin-labeled cytidine triphosphate (biotin-16-CTP) in admixtu re with natural ribonucleotides. After the addition of streptavidin-XL665 c onjugate (SA-XL665), which binds on biotinylated cytidine residues, a long- lived FRET signal was obtained. This proved that both europium cryptate and biotin were incorporated into the same RNA strand and are close enough to generate a FRET signal. The study of this FRET detection assay format showe d that such doubly labeled RNA can be easily detected even when a very low percentage of K-11-UTP is used (less than 1% of total UTP concentration). E uropium-cryptate-labeled RNA can also be monitored using a homogeneous hybr idization assay format involving a biotinylated probe. After the addition o f SA-XL665, the FRET signal generated demonstrates the formation of RNA: DN A hybrids. Europium-cryptate-labeled nucleotide thus gives access to a new type of RNA nonisotopic labeling and homogeneous detection assays. (C) Acad emic Press.