B. Alpha-bazin et al., Europium cryptate-tethered ribonucleotide for the labeling of RNA and its detection by time-resolved amplification of cryptate emission, ANALYT BIOC, 286(1), 2000, pp. 17-25
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.