by separating the fluorophore from the quencher. This simple yet
effective design has led to widespread use of MBs to study the
intracellular behavior of various RNA molecules including mRNA
[2–11], viral RNA [12–15], and noncoding RNA [16, 17].
Although MBs are considered useful tools for RNA analysis in
various cellular contexts, it has become increasingly evident that in
the cellular environment, MBs—particularly when synthesized with
a backbone composed of DNA or 2^0 -O-methyl RNA (2Me)—are
frequently sequestered in the nucleus where they are degraded by
nucleases and/or bound nonspecifically by stem-loop binding pro-
teins. Either of these activities can cause separation of the fluoro-
phore and the quencher, leading to generation of background
fluorescence that can hamper accurate and sensitive detection of
target RNA.
One strategy to reduce MB nonspecific opening has been to
chemically modify the backbone to confer greater biostability. We
have recently shown the feasibility of this approach by incorporat-
ing 2Me MBs with phosphorothioate (PS) internucleotide linkages
in place of conventional phosphodiester bonds. By optimizing the
number and location of PS modifications within the MB backbone,
we found that the design with full PS modification of the loop and
no PS modification of the stem (2Me/PSLOOPMB) exhibits the
least false-positive signals in cells. In this chapter, we describe the
ratiometric imaging methods that were utilized to evaluate the
extent of nonspecific interactions of MB architectures with different
PS configurations in living cells (seeNote 1). We expect the meth-
ods introduced here can be easily adapted for analysis of intracellu-
lar performance of MB architectures with other chemistries, and
should benefit the design of more advanced MBs or MB-based
probes for in vivo applications.2 Materials
2.1 Cell Culture 1. HeLa cells.
- Dulbecco’s Modified Eagle’s Medium (DMEM) without phe-
nol red and without antibiotics, supplemented with 10% FBS
and 1GlutaMAX (Thermo Fisher). - Phenol red-free solution of 0.25% trypsin and 1 mM EDTA.
2.2 Oligonucleotides 1.2Me
50 - mGmUmCmAmCmCmUmCmAmGmCmGmUmAmAm-
GmUmGmAmUmGmUmCmGmUmGmAmC-3^0.
2.2Me/PSSTEM
50 - mG*mU*mC*mA*mC*mCmUmCmAmGmCmGmUm-
AmAmGmUmGmAmUmGmUmC*mG*mU*mG*mA*mC-
30.244 Mingming Chen et al.