Communication
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ABSTRACT: We report the development of a new strategy for the chemical analysis of live cells based on protein spherical nucleic
acids (ProSNAs). The ProSNA architecture enables analyte detection via the highly programmable nucleic acid shell or a functional
protein core. As a proof-of-concept, we use an i-motif as the nucleic acid recognition element to probe pH in living cells. By
interfacing the i-motif with a forced-intercalation readout, we introduce a quencher-free approach that is resistant to false-positive
signals, overcoming limitations associated with conventional fluorophore/quencher-based gold NanoFlares. Using glucose oxidase as
a functional protein core, we show activity-based, amplified sensing of glucose. This enzymatic system affords greater than 100-fold
fluorescence turn on in buffer, is selective for glucose in the presence of close analogs (i.e., glucose-6-phosphate), and can detect
glucose above a threshold concentration of ∼5 μM, which enables the study of relative changes in intracellular glucose
concentrations.
he chemical analysis of live cells at the molecular level
provides fundamental insight into dynamic cellular
The first examples of SNA-based intracellular probes were
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NanoFlares (NFs).
The NF construct consists of a gold
processes, informs about the role of intracellular analytes in
disease progression, and has guided the development of new
nanoparticle core that acts as a quencher. Oligonucleotide
duplexes comprising a recognition strand and a shorter
fluorophore-labeled reporter strand are immobilized onto the
gold nanoparticle through a gold−thiol linkage. Inside the cell,
the target of interest displaces the reporter strand, as it binds to
the recognition sequence and results in fluorescence turn on
due to separation of the fluorophore and quencher. By
designing the recognition strand to be complementary to
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medical diagnostic tools. Although fluorescent probes based
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on both molecular recognition (binding-based sensing) and
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molecular reactivity (activity-based sensing) have led to
significant new capabilities, the majority of techniques
necessitate the fixing or lysis of the cells, the use of cytotoxic
transfection reagents, or the genetic encoding of the cells.
Specifically, protein- and nucleic acid-based approaches, such
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nucleic acids in cells, genetic content can be measured.
On the other hand, using aptamer and DNAzyme sequences,
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as enzyme-linked immunosorbent assays, genetically encoded-
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ions, small molecules, and proteins can be detected.
allow for live-cell genetic and metabolic analyses,
NFs
the
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fluorescent proteins and RNA sensors, polymerase chain
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reaction, and fluorescence in situ hybridization, are
routinely used to detect a wide variety of biological analytes.
However, exogenous proteins and nucleic acids are not
efficiently internalized by cells; thus, their development into
live-cell intracellular probes is challenging.
sorting and isolation of circulating tumor cells based on
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variations in genetic profiles, and the identification of
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diseased tissue in vivo.
However, NFs suffer from several limitations. Since their
fluorescence is solely dependent on the fluorophore’s distance
from the gold core, NFs are susceptible to false-positive signals
arising from nuclease degradation of the oligonucleotides,
dehybridization of the reporter strands, or cleavage at the
To overcome these limitations, we have developed a
powerful new class of intracellular probes based on protein
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spherical nucleic acids (ProSNAs).
This design allows
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analyte detection via a quencher-free approach using either the
nucleic acid or protein component. Additionally, this platform
allows for the detection of intracellular analytes through
binding or activity-based sensing. ProSNAs are based on the
SNA architecture and consist of a protein core functionalized
with a dense shell of radially oriented oligonucleotides. The
SNA architecture is ideally suited for making intracellular
measurements, as it is nontoxic to cells, elicits minimal
immune response, can be taken up by cells without the need
for transfection reagents, and is more resistant to nuclease
degradation compared to traditionally used linear oligonucleo-
gold−thiol linkage. In addition, NFs rely on a displacement
event for signal generation which retards probe−target binding
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kinetics. Finally, NFs can only be designed for targets with
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known nucleic acid-based recognition sequences.
We hypothesized that the use of a quencher-free strategy
coupled to an SNA architecture could overcome many of these
challenges. To test this hypothesis, we first designed SNAs in
Received: June 25, 2020
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tide probes. Additionally, it enables the intracellular delivery
of functional proteins and confers stability against protease
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degradation.
©
XXXX American Chemical Society
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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