ends of the probe places the quencher in close proximity to the
reporter fluorophore to quench its fluorescence. Hybridization of
the target RNA to the loop domain disrupts the stem, opening the
probe and thus separating the quencher from the reporter, which
can then emit a detectable fluorescence upon excitation.
Although many studies have used MBs for RNA detection,
increasing evidence has indicated that MBs have limited accuracy
and sensitivity for detecting intracellular RNAs, owing to their
tendency to be sequestered in the nucleus and generate false-
positive signals [2–4]. Additionally, because heterogeneous deliv-
ery can result in large variations in intracellular MB fluorescence, it
could be difficult to determine whether high MB fluorescence
reflects high RNA expression or uneven MB delivery. To overcome
these drawbacks, we developed a novel probe called ratiometric
bimolecular beacons (RBMBs) (Fig.1)[5]. RBMBs are synthesized
by hybridizing a stem-loop forming oligo and a linear oligo. The
stem-loop forming oligo has a short and a long stem arm, and is
singly labeled with a reporter dye at its short-arm terminus (5^0 -
end). The longer arm is perfectly complementary to the linear
oligo, which is labeled with a quencher and a reference dye at the
two termini. Hybridization of the two oligos brings the quencher
into close proximity to the reporter dye to quench its fluorescence,
while the reference dye is situated at the far end of the hybrid and
thus remains unquenched. Additionally, hybridization results in a
2-base (UU) single-stranded overhang at the 3^0 -end of the duplex,
which resembles an siRNA molecule that is efficiently exported
from the nucleus by exportin [5].
We have shown that, similar to MBs, RBMBs can emit a distin-
guishable fluorescence signal when hybridized to target RNA.
Additionally, the siRNA-like domain functions to facilitate nuclear
export and the unquenched reference dye allows for measurements
of reporter fluorescence to adjust for cell-to-cell variability in
RBMB delivery. Furthermore, we have shown that these attributes
render RBMBs more effective for sensitive and accurate intracellu-
lar measurements of RNA (Fig.1), as compared with conventional
MBs [5, 6]. In this chapter, we describe synthesis of RBMBs and
the assessment of their utility for quantifying single engineered
mRNA transcripts in cells.2 Materials
2.1 Plasmids 1. pLenti-d2EGFP-96mer encodes a transcript harboring 96 tan-
dem repeats of the 50-base sequence: 5^0 - CAG-
GAGTTGTGTTTGTGGACGAAGAGCACCAGCCAGCT-
GATCGACCTCGA-3^0 downstream of the d2EGFP coding
sequence. The underlined sequence is the MB target sequence.
When cotransfected with viral packaging vectors into a cell
232 Yantao Yang et al.