Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells

Article abstract of DOI:10.1021/ja307689w

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.

Full text of DOI:10.1021/ja307689w

Communication
pubs.acs.org/JACS
Photocleavable DNA Barcode−Antibody Conjugates Allow Sensitive
and Multiplexed Protein Analysis in Single Cells
Sarit S. Agasti,†,§ Monty Liong,†,§ Vanessa M. Peterson,† Hakho Lee,† and Ralph Weissleder*,†,‡
†Center for Systems Biology, Massachusetts General Hospital/Harvard Medical School, 185 Cambridge Street, CPZN 5206, Boston,
Massachusetts 02114, United States
‡Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, United States
S
* Supporting Information
describe here the synthesis and validation of a DNA-barcoding-
ABSTRACT: DNA barcoding is an attractive technology,
as it allows sensitive and multiplexed target analysis.
However, DNA barcoding of cellular proteins remains
challenging, primarily because barcode amplification and
readout techniques are often incompatible with the cellular
microenvironment. Here we describe the development and
validation of a photocleavable DNA barcode−antibody
conjugate method for rapid, quantitative, and multiplexed
detection of proteins in single live cells. Following target
binding, this method allows DNA barcodes to be
photoreleased in solution, enabling easy isolation,
amplification, and readout. As a proof of principle, we
demonstrate sensitive and multiplexed detection of protein
biomarkers in a variety of cancer cells.
based cellular protein detection method, which we term “light-
mediated cellular barcoding” (LMCB). The LMCB method
relies on the use of antibodies conjugated to specific DNA
barcodes through a photocleavable linker molecule for initial
target recognition and subsequent barcode amplification
following cleavage of the DNA barcode−antibody conjugate
(DNA−Ab). The generic concept of the LMCB method is
shown in Scheme 1. Cells are first labeled with DNA−Abs
Scheme 1. Schematic Illustration of the Light-Mediated
a
Cellular Barcoding Strategy
he ability to detect scant proteins and antigens in single
Tcells is becoming increasingly important in biological
research, forensic science, and clinical diagnostics. Analyzing
protein signatures at single-cell resolution would aid studies of
the role of cellular heterogeneity in disease progression, stem
cell differentiation, response to drugs, and other cellular
signaling processes.1 Clinically, accurate molecular profiling
and proteomic analysis of rare cells (e.g., circulating tumor
cells) holds considerable promise for early disease detection
and monitoring of treatment response.2 Thus, sensitive,
reliable, and multiplexable protein detection technologies are
currently in great demand.3
To date, several platforms for analyzing cellular proteins have
been described.4 Although some recently developed methods
have shown promise for single-cell analysis,5 the majority of
current methods are limited either by their need for large
numbers of cells or by their ability to detect only few proteins
simultaneously. One enticing approach is DNA barcoding, since
a single DNA barcode can be detected through PCR
amplification; infinite numbers of DNA barcodes can be easily
discriminated on the basis of their sequence and/or size.6,7
Although DNA barcoding technology has been applied to the
detection of soluble proteins via several different formats,6,8,9
the successful application of this technology to live cells has
been rare.10
a
Protein targets are labeled with DNA−Abs and then photocleaved to
release DNA barcodes. Amplified barcodes are analyzed using gel
electrophoresis for multiplexed detection of protein biomarkers from
single cells.
targeted to specific protein biomarkers. Irradiation of the
labeled cells with light (∼365 nm) cleaves the linker between
the antibodies and the barcodes, causing the barcodes to be
released into the solution for easy isolation. Barcode
amplification by polymerase chain reaction (PCR) and
subsequent gel electrophoresis analysis of the amplified
barcodes allows simultaneous detection and quantification of
multiple protein analytes from single cells.
Scheme 2 summarizes the synthetic approach used for the
preparation of DNA−Abs. Figure S1 in the Supporting
Information shows the characterization of the photocleavage
We hypothesized that DNA barcoding could be applied to
live-cell analysis by using a light-mediated barcode releasing
technique, which would enable barcode amplification and
readout to be readily carried out following target binding. We
Received: August 9, 2012
Published: October 23, 2012
© 2012 American Chemical Society
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HER2 antibody (85bDNA−HER2). Cells were labeled with
85bDNA−HER2 against HER2/neu and then photocleaved for
15 min. Released barcodes were separated from the cells via
centrifugation (300g, 3 min). PCR was then performed to
amplify the 85-base DNA barcodes. The target marker (HER2/
neu), which was previously undetectable, could be readily
detected from single SK-BR-3 cells after ∼25 cycles of PCR; as
shown in Figure 2a, a band corresponding to the 85-base DNA
Scheme 2. Synthetic Scheme of the Antibody−DNA
Conjugation and the Photocleavage Reaction Leading to
Barcode Release
reaction of the bifunctional linker by UV−vis spectroscopy. For
cancer cell analysis, antibodies against epidermal growth factor
receptor (EGFR), epithelial cell adhesion molecule (EpCAM),
and human epidermal growth factor receptor 2 (HER2/neu)
were conjugated with 55-, 70-, and 85-base DNA barcodes,
respectively (Figure S2). We verified that after barcode
conjugation, the barcode-modified antibodies still efficiently
recognize their specific targets in various cell lines (Figure S3).
To demonstrate that the barcodes are indeed released upon
light irradiation in intact live cells, we initially used fluorescent
dye (FAM)-labeled, DNA-barcode-conjugated anti-HER2 anti-
body (FAM−DNA−HER2). A strong fluorescence signal
emanated from the cell surface following incubation of SK-
BR-3 cells (overexpressing the surface marker HER2/neu) with
FAM−DNA−HER2 (Figure 1 left), while our control experi-
Figure 2. (a) Detection of HER2/neu in SK-BR-3 cells. After 25 cycles
of PCR, a DNA band corresponding to the 85-base DNA barcode was
visible. Control 3T3 cells, consistent with their low expression of
HER2/neu, had a minimal 85-base DNA band even after PCR
amplification. (b) No DNA band was detected in the absence of light
irradiation, demonstrating the critical role of light in the assay method.
(c, d) Expression of (c) HER2/neu and (d) EGFR (both relative to the
control 3T3 cells) from qLMCB correlated well with results from
standard flow cytometry-based detection. Error bars represent
variations between duplicate measurements.
barcode was seen on the gel. In contrast, even after 25 cycles of
PCR amplification, 3T3 cells failed to show a significant band
for the 85-base DNA barcode, which is consistent with the very
low expression of HER2/neu in these cells. Another control
experiment with HER2/neu-negative MDA-MB-231 cells
likewise failed to show a significant barcode band following
PCR amplification (Figure S6). Omitting the light-irradiation
step resulted in no detectable signal (Figure 2b), confirming the
importance of photocleavage in the LMCB method. To
determine the expression level of different markers quantita-
tively, we next performed SYBR Green-based quantitative
LMCB (or qLMCB) on the DNA barcodes. Figure 2c shows
the HER2/neu expression level in both SK-BR-3 and 3T3 cells,
whereas Figure 2d shows the EGFR expression level in human
epithelial carcinoma A431 and 3T3 cells. Expression levels
determined using the qLMCB method were consistent with
those obtained by flow cytometry, with the difference being
that flow cytometry required much larger cell numbers for
analysis (∼105 cells for flow cytometry vs ∼1 cell for qLMCB).
Overall, these results demonstrate that the protein signatures of
single cells can easily be transformed into a detectable,
quantifiable, and reliable signal using the LMCB method.
To determine the detection threshold of the LMCB method,
we performed dilution experiments with SK-BR-3 cells. Cells
were targeted using 85bDNA−HER2 and photocleaved as
previously described. Figure 3a shows the gel analysis following
25 cycles of PCR amplification of DNA barcodes from different
numbers of cells. A clear 85-base DNA band (varying in
intensity depending on cell concentration) was observed for all
samples, whose sizes ranged from 104 cells to single cells. To
Figure 1. Fluorescently labeled DNA barcodes conjugated to anti-
HER2 antibodies were used to stain SK-BR-3 cells. A decreased
fluorescence signal after light irradiation demonstrated that barcodes
were released from the labeled cells. See Figure S4 for additional
images, including bright-field images.
ment using HER2/neu-negative MDA-MB-231 cells showed
negligible fluorescence (Figure S4), indicating specific binding
of the barcode-conjugated antibodies to the target molecular
markers. A microscope image taken after 10 min of light
exposure of the stained SK-BR-3 cells showed a significant
decrease in the fluorescence signal from the cells (Figure 1
right) resulting from release of the fluorescent barcodes from
the cells following photocleavage. DNA barcode release was
complete within 15 min of light exposure, as measured by flow
cytometry (Figure S5a). A control experiment performed with a
non-photocleavable anti-HER2 antibody showed a minimal
decrease in fluorescence over time (Figure S5b), indicating that
the barcode release was a consequence of the photolytic
cleavage of the linker molecule.
We subsequently applied the LMCB method to both SK-BR-
3 (HER2/neuhigh) and control fibroblast 3T3 (HER2/neulow
)
single cells using the 85-base DNA-barcode-conjugated anti-
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of size. Figure 4a shows that the signals from individual
biomarkers can be clearly distinguished from each other on the
Figure 3. Detection sensitivity of the LMCB method. (a) After 25
cycles of PCR, the DNA barcodes from samples containing varying
numbers of SK-BR-3 cells were detected. (b) Image showing a single
SK-BR-3 cell inside a microplate well for digital analysis (scale bar 50
μm). (c) Analysis of a single SK-BR-3 cell using the LMCB method in
digital format. Following PCR amplification, an 85-base DNA barcode
could be detected in individual wells containing single cells (“single-
cell wells”). In contrast, wells in which cells were absent (“no-cell
wells”) failed to produce a significant band following amplification. (d)
Gel electrophoresis results showing the detection sensitivity of the
LMCB method as a function of PCR cycle number.
Figure 4. (a) Multiplexed protein detection using the LMCB method.
Individual biomarker signals (corresponding to their expression levels)
can be clearly distinguished from one other on the basis of barcode
size. (b) Comparison of the LMCB results from (a) and data acquired
using flow cytometry. For each biomarker, band intensities
(normalized to control 3T3 cells) from the gel were plotted against
fluorescence intensities (normalized to control 3T3 cells) from flow
cytometry (R2 = 0.90).
basis of barcode size. Varying band intensities, corresponding to
the expression levels of biomarkers in different cells, are clearly
evident in the gel. To verify the reliability of the multiplexed
LMCB method, we compared the results to standard flow
cytometry data obtained using 105 cells/experiment. As shown
in Figure 4b, there was a good correlation between the LMCB
and flow cytometry results.
In summary, we have developed a novel method, light-
mediated cellular barcoding (LMCB), that enables rapid,
quantitative, multiplexed detection of protein expression in
single live cells. The approach is robust and could be adapted to
the analysis of other targets of interest, such as soluble proteins
and pathogens. It is also a relatively simple technique that does
not require complex purification steps, during which analytes
are often lost. In this work, differently sized DNA barcodes
were separated using size chromatography. While this provided
a clean and simple model system for validating the technology,
we ultimately anticipate either sequencing of the DNA barcodes
to enable greater diversity or the use of imaging approaches
with digital color-coded barcodes for multiplexing.7a
verify further the single-cell sensitivity of the LMCB method,
we performed cellular analysis in digital format. A dilute SK-
BR-3 cell solution was distributed between many wells of a 384-
well microplate in such a way that each well contained on
average either a single cell or no cells. The wells were then
imaged to identify the ones containing a single cell (Figure 3b).
Barcodes isolated from the wells containing single SK-BR-3
cells produced a barcode band on the gel, whereas wells
containing either no cells or control MDA-MB-231 cells
(HER2/neu-negative) failed to show a significant band
following amplification (Figure 3c and Figure S6). This result
indicates that the LMCB method provides analysis of individual
cells to characterize heterogeneities among a cell type (Figure
S7). We also determined the effect of the number of PCR
cycles on the detection sensitivity. As shown in Figure 3d,
HER2/neu expression from 102 cells could be easily detected
after 20 cycles of PCR, whereas for samples containing only
single cells, ∼30 cycles were required to obtain a detectable
signal. This suggests that adjusting the number of PCR
amplification cycles could increase the detection sensitivity,
allowing even relatively low abundance biomarkers to be
detected in cells.
Commonly used multiplexing methods are largely reliant on
the use of fluorescently labeled antibodies; however, this allows
only limited numbers of labels to be discriminated. In contrast,
the use of DNA barcodes as labels for multiplexing represents
an ideal platform because infinite numbers of DNA barcodes
can easily be discriminated on the basis of their sequences and/
or sizes. To test the LMCB method in a multiplexed format, we
used barcode-conjugated antibodies for the simultaneous
detection of EGFR, EpCAM, and HER2/neu in four different
cell lines. Cells were incubated with a cocktail of antibodies for
respective target binding and then photocleaved for barcode
release and isolation. Since all of the barcodes have similar
sequences toward the 5′ and 3′ ends, the barcodes could then
be simultaneously amplified by PCR using a single set of primer
pairs (Figure S2).9b Following amplification, individual
barcodes were separated by gel electrophoresis on the basis
ASSOCIATED CONTENT
* Supporting Information
Synthesis of materials, experimental methods, and supporting
figures. This material is available free of charge via the Internet
at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
rweissleder@mgh.harvard.edu
■
Author Contributions
§S.S.A. and M.L. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank H. J. Chung, K. S. Yang, K. Tran, and J. A. Hendricks
for helpful discussions and Y. Fisher-Jeffes for reviewing the
manuscript. This work was supported in part by the National
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Communication
Institutes of Health (Grants 2P50CA086355-12 and
R01EB010011 and TPEN Contract HHSN268201000044C).
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