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Supplementary Information
Target-induced proximity ligation triggers recombinase polymerase
amplification and transcription-mediated amplification to detect
tumor-derived exosomes in nasopharyngeal carcinoma with high
sensitivity
Wanli Liu 1#, Jianpei Li 1#, Yixian Wu 2, Shan Xing 1, Yanzhen Lai 1, Ge Zhang 2*
1 State Key Laboratory of Oncology in Southern China, Department of Clinical
Laboratory Medicine, Sun Yat-sen University cancer center; 2 School of
Pharmaceutical Sciences, Sun Yat-sen University, China
Corresponding Author: [email protected] ; Tel: 86-20-39943027
List of Contents:
1. Sequence
2. Plasma and patients’ character
3. Experimental section
3.1 Exosome isolation and characterization
3.2 Preparation of DNA-Ab conjugates and DNA-GNP conjugates
3.3 Experimental optimization of the PLA-RPA-TMA assay
3.4 Evaluation of the amplification ratio of the PLA-RPA-TMA assay
3.5 PLA-RPA-TMA assay for the detection of EGFR+ exosomes
3.6 Specificity testing for the PLA-RPA-TMA assay1
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1. Sequence
Table S1:Sequence of primer and probe
Name Sequence (5'-3' )
DNA1SH-TGC TTT CAT ACG TTT AGC CCA ATC TTG GAT TTC GAA
ATA GAT TAA TAC
DNA2SH-CC AAC ATT GGG CTA AAC GTA TGA AAG TCC CTA TAG
TGA GTC GTA TTA
DNA control 1
TGC TTT CAT ACG TTT AGC CCA ATC TTG GAT TTC GAA ATA
GAT TAA TAC GAC TCA CTA TAG GGA CTT TCA TAC GTT TAG
CCC AAT GTT GG
DNA control 2
CCA ACA TGG GCT AAA CGT ATG AAA GTC CCT ATA GTG AGT
CGT ATT AAT CTA TTT CGA AAT CCA AGA TTG GGC TAA ACG TAT
GAA AGC A
Forward primer TGC TTT CAT ACG TTT AGC CCA ATC TTG GAT
Reverse primer CTT TAT CTA TCC AAG ATT GGG CTA AAC GTA
Linker GGG ACU UUC AUA CGU UUA GCC CAA UGU UGG
DNA3 SH-AAA AAA AAA A CCA ACA TTG GGC TAA
DNA4 SH-AAA AAA AAA A ACG TAT GAA AGT CCC
Note: The red part of DNA1 is complementary to the red part of DNA2
The underlined part of DNA1 and DNA2 is the T7 promoter sequence.
2. Plasma and patient characteristics
Plasma samples were collected from 50 patients with NPC at the time of diagnosis
before any treatment at Sun Yat-sen University Cancer Center (SYSUCC) from 2015
to 2016. Based on an epipharyngoscopic examination, 100 healthy controls, including
50 Epstein-Barr virus (EBV)-viral capsid antigen (VCA)-IgA antibody-negative
donors and 50 EBV-VCA-IgA antibody-positive donors without NPC, all volunteered
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to undergo routine health examinations at SYSUCC. All blood samples were obtained
via peripheral venipuncture and plasma (EDTA anticoagulant) was extracted after
centrifugation at 3500 g for 15 min and subsequent centrifugation at 10,000 g for 30
min to remove the cell debris. Plasma samples were stored at -80°C until analysis.
The characteristics of patients with NPC and healthy controls are described in Table
S2. Prior to using plasma samples, informed consent was obtained from each
participant. This experiment was approved by the Institute Research Ethics
Committee of SYSUCC.
Table S2:Character of the nasopharyngeal carcinoma patients
Charateristics No. of NPC
patients
No. of healthy controls
EBV-VCA-IgA– EBV-VCA-IgA+
Age
≥45 41 40 38
<45 9 10 12
Gender
Female 8 10 11
Male 42 40 39
Epipharyngoscope examination
Positve 50 0 0
Negative 0 50 50
EBV-VCA-IgA
Positive 50 0 50
Negative 0 50 0
Clincal stage
Stage I 11
Stage II 39
3. Experimental Section
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3.1 Exosome isolation and characterization
3.1.1 Cell lines
The NPC cell line C666-1 (EBV-positive) was grown in RPMI 1640 medium
(Invitrogen, USA), and the nasopharyngeal epithelium cell line NPEC1 was cultured
in Keratinocyte-SFM medium (10744–019, Thermo Fisher Scientific) as previously
described (Feng et al., 2017). Both media were supplemented with 10% fetal bovine
serum (FBS, HyClone), and both cell cultures were maintained in a completely
humidified atmosphere with 5% CO2 in air at 37°C.
3.1.2 Exosome isolation and characterization
Exosomes were isolated using the methods described by Théry et al. (Théry et al.,
2001), with some modifications. Briefly, cells were grown to 75% confluence, rinsed
three times with PBS, and cultured in RPMI 1640 without FBS for 48 h. Cells were
pelleted by centrifugation at 300 × g for 10 min. An additional centrifugation at 2000
× g for 10 min was used to pellet dead cells. The supernatant was collected and
centrifuged at 10,000 g for 30 min and then passed through a 0.22 μm filter. Then, the
filtered supernatant was subjected to ultracentrifugation at 100,000 g for 75 min two
times to pellet the vesicular components. The resulting exosomal pellet was
resuspended in PBS. All spins were done at 4 °C.
The exosome size and number were measured by a nanoparticle tracking analysis
(NTA) using a NanoSight 300 (Malvern Instruments Ltd., UK). The particle
suspensions were diluted with PBS to a concentration of 1–8 × 108 particles/mL for
analysis. Data for each sample was collected for 60 sec and analyzed using NanoSight
NTA 2.3 software. All samples were analyzed in triplicate.
For transmission electron microscope (TEM) analysis, purified exosomes were
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adsorbed onto 150 mesh Formvar®-coated grids, stabilized with evaporated carbon
film (Ted Pella Inc., Redding, CA, USA), and fixed in 4% glutaraldehyde (Sigma) at
4 °C for 5 min. After being rinsed four times with autoclaved deionized water, fixed
samples were stained for 2 min with uranium acetate (Sigma), dried for 20 min, and
observed with a JEM-1400 TEM which operated at 200 kV (JEOL, Japan).
Photographs were captured with a Canon A650 digital camera.
3.1.3 Western blotting analysis and immunofluorescent staining
Cellular and exosome proteins were separated by 12% SDS-PAGE and transferred
onto polyvinylidene difluoride (PVDF) membrane. After being blocked with 5% non-
fat dry milk in PBS containing 0.2% Tween-20 (PBST), the membrane were
incubated with antibodies against CD9 (1:1,000), CD81 (1:500), CD63 (1:1,000),
LMP1 (1:500), EGFR (1:1,000) and α-tubulin (1:3,000) overnight at 4°C. After
washing for several times, the PVDF membrane was incubated in horseradish
peroxidase-labeled goat anti-mouse antibody (1:5000, Santa Cruz Biotechnology,
Dallas, USA) for 2 h at room temperatures. The bands were detected by Pierce ECL
Plus Western Blotting Substrate (Thermo Scientific, USA) according to the
manufacturer’s protocols.
For immunofluorescent staining, after the samples were fixed with formalin and
blocked, anti-LMP1 (1:50) or anti-EGFR (1:100) antibodies were applied overnight at
4°C. Slides were washed 3 times for 15 min each in PBST and then incubated with
FITC-labeled secondary antibodies for 30 min at RT (1:500, eBioscience, USA). After
3 washes, the slides were incubated with diluted DAPI for 5 min at RT, mounted with
an anti-fade mounting media (Neuromics, USA) and then visualized using a
fluorescence microscope (Olympus, Japan).
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3.1. 5 Results
We isolated exosomes from the supernatant of C666-1 cancer cells that overexpress
two NPC-specific biomarkers, LMP1 (an EBV-encoded primary oncogene) and
EGFR (a target for treatment) (Liu et al., 2016;Meckes et al., 2010;Verweij et al.,
2011), by ultracentrifugation to generate NPC TEXs. Meanwhile, exosomes were also
isolated from NPEC1 cells, an immortalized nasopharyngeal epithelial cell line, and
served as negative controls (LMP1– and EGFR– exosomes). The TEM analysis showed
purified vesicles in an oval and globular form (Fig. S1A-B). The size of the isolated
vesicles mainly ranged from 30–300 nm, and the average vesicle size was 112±3.7 nm
for NPCE1 cells and 114±4.8nm forC666-1 cells (Fig. S1C-D). The total numbers of
exosomes from C666-1 and NPCE1 cells were 4.12±0.28×109 particles/mL and
4.87±0.45×109/mL, respectively. The presence of the exosome biomarkers, including
CD9, CD63 and CD81, as well as the tumor markers LMP1 and EGFR in the
exosomes isolated from C666-1 cells were verified by western blotting. However,
LMP1 and EGFR were not expressed in either exosomes or lysates from NPCE1 cells
(Fig. S2A-B). Additionally, immunofluorescence staining confirmed that LMP1 and
EGFR were overexpressed in C666-1 cells (Fig. S2C). Based on these data, the
isolated vesicular population has an exosomal origin and thus is suitable for use as a
model to establish a PLA-RPA-TMA assay for the detection of NPC TEXs.
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Fig. S1. TEM images of purified exosomes from the supernatant of NPEC1 (A) and
C666-1 cells (B). Dynamic light scattering (DLS) was used to determine the absolute
exosome size distribution and concentration of exosomes (particles/mL) in NPEC1
(C) and C666-1 cells (D), as indicated in the figure. The standard deviations for the
measurements from three tests are presented as ±SD.
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Fig. S2. (A) Western blot analyzed the expression of exosomal markers (CD63, CD9
and CD81), LMP1 and EGFR in 20μg exosomal protein purified from NPEC1 or
C666-1 cell supernatants. (B) Western blot analyzed the expression of LMP1 and
EGFR in 50 μg protein from whole-cell lysates of NPEC1 or C666-1 cells. β-actin
served as an internal control. (C) Immunofluorescence staining for LMP1 and EGFR
proteins on the C666-1 cell membrane. C666-1 cells were stained with either anti-
LMP1 or anti-EGFR antibodies, followed by FITC-conjugated secondary antibodies
(green). Nuclear counterstaining was performed using DAPI (blue).
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3.2 Preparation of DNA-Ab conjugates and DNA-GNP conjugates
In this study, two pairs of oligonucleotide-antibody conjugates (Ab1-DNA1 and
Ab2-DNA2 for LMP1; Ab3-DNA1 and Ab4-DNA2 for EGFR) were synthesized
using Sulfo-LC-SPDP cross-linkers. Ab1/Ab2 and Ab3/Ab4 recognize the two
different epitopes of the LMP1 and EGFR targets, respectively. The Ab-DNA
conjugates obtained for LMP1 detection were characterized using UV-Vis
spectroscopy. An absorption peak for the purified Ab-DNA conjugates was detected
at 260 nm, similar to the absorption peak for DNA, and a mild shoulder at
approximately 280 nm originated from the Ab protein, suggesting that the DNA was
indeed linked to the Ab (Fig. S3).
Fig. S3. UV absorption spectra of the Ab-DNA probes used for LMP1 detection. (A)
Comparison of Ab1-DNA1 with Ab1 and DNA1. (B) Comparison of Ab2-DNA2 with
Ab2 and DNA2.
In addition, DNA-GNP conjugates were also confirmed using a linker cross-linking
GNP colorimetric assay. Because the synthesized RNA linker sequence is the same as
the TMA product, it cross-links GNP1 and GNP2 if the GNPs are coupled with the
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corresponding complementary oligonucleotides. As shown in Fig. S4, when linkers
(30 nM or 50 nM) were added to DNA-GNP conjugate solutions (GNP1 and GNP2),
GNP-1 and GNP-2 were cross-linked with a concomitant red-to-blue color change,
whereas no color change was observed in samples without linkers (Fig. S4A). UV-Vis
absorption spectroscopy also detected a 20 nm shift in the peak for aggregated GNPs
(530-550 nm) in linker-added samples compared to the characteristic absorption peak
for the dispersed GNPs in no-linker samples (Fig. S4B). Thus, GNP1 and GNP2 were
successfully prepared and detected the linker RNA.
Fig. S4. UV absorption spectra (A), corresponding TEM images (B), and
corresponding color images (C) of the mixture of GNP1 and GNP2 probes after
incubation with the test solution containing 30 nM or 50 nM linkers or no linkers
(PBS). The absorbance peak is located at 530 nm for dispersed GNP1 and GNP2 (no
linkers) and 550 nm for aggregated GNP1 and GNP2 (30 nM or 50 nM linkers).
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3.3 Experimental optimization of the PLA-RPA-TMA assay
We optimized the experimental conditions for the PLA-RPA-TMA assay to obtain a
better signal response. Some critical factors influence the assay, such as the amount of
Ab-DNA and T7 polymerase; the length of the complementary sequences in the two
PLA probes; and the reaction times for antigen-antibody binding, the RPA-TMA
assay and the GNP-based colorimetric assay. RPA and TMA assays were performed
under the conditions required for the commercial kits. Because the working
temperature of the RPA and TMA assays is 37-42°C and the antigen-antibody reaction
also works well at 37°C, a constant working temperature of 37°C was used for the
PLA-RPA-TMA
Fig. S5. Optimization of the length of Ab1-DNA1 probe at the 3’ end complementary
to the 3’ end of the Ab2-DNA2 probe. Corresponding test fluorescence signal to
background ratio (F/F0) of different number of bases in the complementary DNA
sequence in an experimental group (105 particles/mL LMP1+, F) and control group
(PBS, F0) calculated by monitoring the fluorescence of the PLA-RPA reaction.
Experimental conditions: 2.0 μg/mL Ab1-DNA1 and Ab2-DNA2; the time allowed
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for binding to the LMP1 antigen was 90 min. Data are based on three replicates; error
bars = standard error.
reaction. In addition, the manufacturer recommends an RPA reaction time of 20 min,
and a nonspecific RPA signal was observed at times exceeding 30 min. Therefore, a
20 min of RPA-TMA reaction time was used in this experiment to obtain a better
signal-to-background ratio. First, as shown in Fig. S5, the length of complementary
sequencesin two PLA probes was examined to ensure that the assay is useful for
target detection, and a 6-nt sequence provided the best signal-to-background ratio.
Second, 2μg/mL each of Ab1-DNA1 and Ab2-DNA2 and 20 U of T7 RNA
polymerase were appropriate concentrations for the PLA-RPA-TMA assay (Fig. S6).
Third, the decrease in absorbance increased as the time of incubation with the Ab-
DNA probes increased and reached the maximum value at 90 min; thus, 90 min was
the optimal time for the antigen-antibody binding reaction (Fig. S7A). The decrease in
absorbance at 530 nm reached a plateau at 10 min for the GNP-based colorimetric
assay, and thus 10 min was set as the GNPs colorimetric assay time (Fig. S7B).
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Fig. S6. (A) Optimization of the concentration of Ab1-DNA1 and Ab2-DNA2 probes
for the PLA-RPA-TMA assay (length of the complementary DNA sequence, 6-nt; the
time allowed for binding to the LMP1 antigen, 90 min; the colorimetric assay time, 10
min; and 20 U of T7 RNA polymerase). Decrease in the absorbance at 530 nm (A0-
Ax) in the PLA-RPA-TMA assay was used to detect 105 particles/mL LMP1+ in
samples with different concentrations of Ab1-DNA1 and Ab2-DNA2. (B)
Optimization of the concentration of T7 RNA polymerase for the PLA-RPA-TMA
assay (105 particles/mL LMP1+; the length of complementary DNA sequence, 6-nt;
the time allowed for binding to the LMP1 antigen, 90 min; the colorimetric assay
time, 10 min; and 2.0 μg/mL Ab1-DNA1 and Ab2-DNA2 each). Data are based on
three replicates; error bars = standard error.
Fig. S7. (A) Effects of the time allowed for the Ab-DNA probes to bind to the LMP1
antigen on the PLA-RPA-TMA assay (105 particles/mL LMP1+; the length of
complementary DNA sequence, 6-nt; 2.0 μg/mL Ab1-DNA1 and Ab2-DNA2 each;
20 U of T7 RNA polymerase; the colorimetric assay time, 10 min). (B) Effects of the
GNP-based colorimetric assay time on the PLA-RPA-TMA assay (105 particles/mL
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LMP1+; length of the complementary DNA sequence, 6-nt; 2.0 μg/mL Ab1-DNA1
and Ab2-DNA2 each; 20 U of T7 RNA polymerase; the time allowed for binding to
the LMP1 antigen, 90 min). Data are based on three replicates; error bars = standard
error.
3.4 Evaluation of the amplification ratio of the PLA-RPA-TMA assay.
To evaluate the amplification ratio of the RPA-TMA assay, an 89 bp dsDNA was
synthesized as the control DNA template in the following manner: the DNA control 1
and the DNA control 2 (Table S1) were mixed at the mole ratio of 1:1, heated to 90°C
for 1 min, and then cooled down to room temperature. The control DNA was detected
by the RPA-TMA assay. 102 or 103 copies of the control DNA were added to the
RPA-TMA reaction solution (47.5 μL) contained 0.48 μM primer pair, 1× RPA
rehydration buffer, 20 U of T7 RNA polymerase, 4 μL of murine RNase inhibitor, 2
mM NTPs, 1 μL of T7 RNA polymerase mix, and 5 mM MgCl2. The reaction was
started by adding 2.5 μL of 280 mM MgAc and was incubated for 20 min at 37°C
with rotation. After stopping the RPA-TMA reaction by heating to 80°C for 1 min, the
concentration of RNA was quantified by Quant-iT™ RiboGreen® assay (Invitrogen,
USA) according to the manufacture instructions. To eliminate the DNA in the sample,
1 μL 10 × DNase digestion buffer was added to 9 μL products of RPA-TMA assay.
Then 5 units of RNase-free DNase I were added and incubated the sample at 37°C for
90 min. The sample was diluted at 1000-fold into TE buffer and added 1.0 mL of the
aqueous working solution of the Quant-iT™ Ribo Green® reagent. After mixed and
incubated for 5 min at room temperature, the fluorescence of the sample was
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measured using a PerkinElmer LS55 fluorescence spectrometer (PerkinElmer, USA).
The RNA concentration of the sample was determined from the standard curve
generated in RNA standard curves. The RNA sequence of the RPA-TMA assay is 5′-
GGG ACU UUC AUA CGU UUA GCC CUU UGU UGG-3′ (30 bp) and its
molecular weight is 9743.
In our study, under the condition similar to the PLA-RPA-TMA assay, 102 or 103
copies of 89 bp templates were analyzed by RPA-TMA and the RNA products were
quantified by Quant-iT™ RiboGreen® assay. The amplification ratio of RPA-TMA
assay was calculated according to the following formula: amplification ratio= output
of RNA (copies)/ input of DNA (copies). As shown in Table S3, the amplification
ratio RPA-TMA assay was about 5.08×1010 for 102 copies of input DNA and
6.54×1010 for 103 copies of input DNA.
Table S3:Evaluation of the amplification ratio of RPA-TMA assay
input
(copies)output
amplification
ratioRNA (ng) RNA (pM) RNA (copies)
102 84.35 8.66 5.21×1012 5.21×1010
102 80.13 8.22 4.95×1012 4.95×1010
102 82.68 8.48 5.10×1012 5.10×1010
103 1005.56 103.21 6.21×1013 6.21×1010
103 1060.02 108.79 6.55×1013 6.55×1010
103 1112.74 114.21 6.87×1013 6.87×1010
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3.5 PLA-RPA-TMA assay for the detection of EGFR+ exosomes
Fig. S8. (A) Typical UV absorption spectra obtained from the PLA-RPA-TMA assay
for the detection of EGFR+ exosomes (0–109 particles/mL). (B) Corresponding color
images of the PLA-RPA-TMA assay. (C) Decrease in absorbance at 530 nm with
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increasing concentration of EGFR+ exosomes. (D) The linear range of the PLA-RPA-
TMA assay for detection of EGFR+ exosomes was 102 to 108 particles/mL. Data are
based on three replicates; error bars = standard error.
3.6 Specificity testing for the PLA-RPA-TMA assay
Fig. S9. Specificity testing for the PLA-RPA-TMA assay. Comparing the decrease in
absorbance at 530 nm for the target(A) LMP1+ exosomes (105 particles/mL)or
(B)EGFR+ exosomes (105 particles/mL) with different Ab-DNA combinations
(indicated in the figure) and other non-target proteins (10%BSA, 105 particles/mL
LMP1–exosomes or EGFR–exosomes from NPEC1 cells). Data are based on three
replicates; error bars = standard error.17
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