suitable for virus packaging, such as the HEK-293FT cell, the
resulting lentiviral particles containing the pLenti-d2EGFP-
96mer gene can be efficiently introduced into host cells (see
Notes 1and 2 ).2.2 Cell Culture 1. HEK-293FT.
- HT1080 cells.
- HT1080-d2EGFP-96mer cells that stably express engineered
d2EGFP mRNA transcripts with 96 tandem repeats in the 3^0 -
untranslated region (UTR), generated by infecting HT1080
cells with pLenti-d2EGFP-96mer. - Minimum essential media (MEM) supplemented with 1% Pen/
Strep and 10% FBS. - Phenol red-free solution of 0.25% trypsin and 1 mM EDTA.
- 1phosphate-buffered saline (PBS), free of Mg+2and Ca+2.
2.3 RBMB Design
and Synthesis
- Oligonucleotides: RBMBs are composed of two 2^0 -O-methyl
RNA oligonucleotides, denoted as Oligo 1 and Oligo 2. Oligo
Fig. 1Design of RBMBs and strategies for assessing RBMB performance for imaging single RNA transcripts in
cells. (a) RBMBs are stem-loop-forming oligo probes that possess a reporter dye, quencher, and reference
dye. In the absence of target, the reporter dye is held in close proximity to the quencher, leading to a low-
fluorescent state. Hybridization of the loop-domain to a complementary target separates the reporter dye and
quencher, resulting in the restoration of fluorescence. The reference dye remains unquenched independent of
the RBMB configuration. The 3^0 -UU overhang drives nuclear export. (b) To assess the ability of RBMBs to
quantify RNA copy numbers in single cells, smFISH is performed following microporation of RBMBs. In this
figure, smFISH probes aregreen, while RBMB reporter dye and reference dye areredandmagenta,
respectively. Hybridization of the RBMBs or smFISH probes to each RNA transcript results inbright punctate
spotswithin cells when imaged by conventional fluorescence microscopy. Colocalization of RBMB signal with
the smFISH signal indicates successful detection of the RNA transcript by RBMBs (Reproduced from [6] with
permission from Oxford University Press)
Ratiometric Bimolecular Beacons 233