Communications
Biosensors
Genetically Encoded Ratiometric RNA-Based Sensors for
Quantitative Imaging of Small Molecules in Living Cells
Rigumula Wu, Aruni P. K. K. Karunanayake Mudiyanselage, Fatemeh Shafiei, Bin Zhao,
Yousef Bagheri, Qikun Yu, Kathleen McAuliffe, Kewei Ren, and Mingxu You*
Abstract: Precisely determining the intracellular concentra-
tions of metabolites and signaling molecules is critical in
studying cell biology. Fluorogenic RNA-based sensors have
emerged to detect various targets in living cells. However, it is
still challenging to apply these genetically encoded sensors to
quantify the cellular concentrations and distributions of targets.
Herein, using a pair of orthogonal fluorogenic RNA aptamers,
DNB and Broccoli, we engineered a modular sensor system to
apply the DNB-to-Broccoli fluorescence ratio to quantify the
cell-to-cell variations of target concentrations. These ratiomet-
ric sensors can be broadly applied for live-cell imaging and
quantification of metabolites, signaling molecules, and other
synthetic compounds.
readout, artifacts can easily arise from variations in the
cellular RNA distributions. For quantitative and multiplexed
imaging of cellular analytes,[8] it is critical to develop new
RNA sensor pairs that have little spectra overlap and that can
be orthogonally imaged.
Here, we develop ratiometric RNA sensors to quantify
the cellular concentrations and distributions of small mole-
cules. The sensor comprises Broccoli and a dinitroaniline
(DN)-binding aptamer, DNB.[6b,9] We have engineered novel
red-colored RNA sensors by fusing target-binding aptamers
into DNB. Using Broccoli as the reference, we can quantita-
tively image various small molecules in living cells.
We first wondered if it is possible to develop DNB-based
metabolite sensors. Dinitroaniline is a general contact
quencher for fluorophores including sulforhodamine B
(SR). The conjugation of DN and SR generates a non-
fluorescent complex, SR-DN. The binding of DNB isolates
DN, which activates the SR fluorescence.[9] After analyzing
the DNB structure, we realized that its P4/L4 hairpin could be
potentially used to fuse with target-binding aptamers (Fig-
ure 1a).[9] We replaced this domain with three sequences that
maintained a similar hairpin structure (Supporting Informa-
tion, Figure S1). Similar to DNB (83.1-fold), all three
mutations activated the SR-DN fluorescence (62.8- to 80.4-
fold). In contrast, RNA with a mismatched P4 stem com-
pletely lost its binding with SR-DN (Supporting Information,
Figure S1). Indeed, DNB functions depend on the structure,
but not the sequence, of P4/L4.
F
luorescent probes that allow live-cell imaging of small
molecules have enabled us to better understand cellular
signaling and metabolite flux. Various fluorescent small-
molecule probes and genetically encoded fluorescent protein
(FP)-based sensors have been developed to image metabo-
lites and signaling molecules.[1] The function of FP sensors
requires a target-binding domain that can both selectively
recognize the target and result in sufficient conformational
change to refold the FP or change the orientation between
two FPs.[2] However, for many physiologically important
analytes, these adequate target-binding domains are not
easily identified. The limited signal-to-noise ratio has further
prevented their wide applications.[3]
We and others have developed a new class of genetically
encoded sensors based on fluorogenic RNA aptamers.[4]
Aptamers are short single-stranded oligonucleotides that
can bind to their targets with high affinity and specificity.[5]
Fluorogenic RNA aptamers, for example, Spinach or Broc-
coli, can bind and activate the fluorescence of dyes such as
3,5-difluoro-4-hydroxybenzylidene-1-trifluoroethyl-imidazo-
linone (DFHBI-1T).[6] By fusing a target-binding aptamer
into Spinach/Broccoli, genetically encoded RNA-based sen-
sors have been developed for live-cell imaging of metabolites,
signaling molecules, proteins, and metal ions.[4,7]
We inserted an adenine-binding aptamer[10] into P4/L4
(Figure 1a). In silico structural predication guided our design
of five adenine-targeting sensors with different transducer
sequences. An optimal sensor (Transducer 2, Supporting
Information, Table S1) exhibited a 3.6-fold fluorescence
enhancement in the presence of 10 mm adenine (Figure 1b).
Similarly, we developed DNB-based sensors for an antibiotic,
tetracycline, and a signaling molecule, c-di-GMP.[4d,11] A 1.5-
to 11.9-fold fluorescence increase was observed after adding
200 mm tetracycline (Figure 1b). Interestingly, the optimal
transducer sequence, Transducer 3, has been previously used
in a ribozyme-based tetracycline sensor.[11a] An optimal sensor
for c-di-GMP was achieved with a 10.6-fold fluorescence
enhancement after adding 10 mm c-di-GMP. Transducer 1,
whose sequence was previously used in a Spinach-based
sensor c-di-GMP,[4d] also exhibited similar fold enhancement
(Figure 1b). Previously identified transducers may be directly
applicable in these modular DNB-based sensors.
Almost all these fluorogenic RNA sensors were devel-
oped based on a Spinach/Broccoli-dye complex (lex/lem
,
approximately 480 nm/ 503 nm). With a single-wavelength
[*] R. Wu, A. P. K. K. Karunanayake Mudiyanselage, F. Shafiei,
Dr. B. Zhao, Y. Bagheri, Q. Yu, K. McAuliffe, Dr. K. Ren, Prof. M. You
University of Massachusetts, Amherst, MA 01003 (USA)
E-mail: mingxuyou@umass.edu
These sensors also preserve the high selectivity towards
their targets (Figure 1c). After demonstrating the robust
sensor performance under different temperature and Mg2+
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
Angew. Chem. Int. Ed. 2019, 58, 1 – 6
ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers!