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Supporting Information

Spinach-Based Fluorescent Light-up Biosensors for Multiplexed and Label-Free Detection of microRNAs

Zhan-Ming Ying, Bin Tu, Lan Liu, Hao Tang*, Li-Juan Tang, Jian-Hui Jiang*

Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (P. R. China)

Email: haotang@hnu.edu.cn; jianhuijiang@hnu.edu.cn Tel: 86-731-88821916; Fax: 86-731-88821916

Table of Contents:

S-2. Reagents and Materials, DNA-RNA splinted ligation assay, T7 In vitro trancription process

S-3 Gel electrophoresis analysis of RNA sensor, Fluorescence measurement setup

S-4. RT-qPCR quantification

S-5. Table S1. Sequences of miRNAs, DNA probes and PCR primer

S-6. Figure S1. Agarose gel electrophoresis images of in vitro transcription products

S-7. Figure S2. Selection gel imaging of miR-21and miR-141 initiated T7 in vitro transcription products.

S-8. Table S2 Comparison of detection limit of Spinach-based light-up biosensors for nucleuic acid

dtection

S-9 Figure S3. Selectivity investigation of the Spinach-based fluorescent light-up biosensor

S-10. Figure S4. Stability invesitigation of the Spinach-based fluorescent light-up biosensor

S-11. Figure S5. Agarose gel electrophoresis images of extracted total cellular mircoRNA

S-12. Figure S6. Agarose gel electrophoresis images of qPCR products

S-13. Figure S7. Expression analysis of miR-21 and miR-141 for different cell lines

S-14. Table S3 and S4. Average Ct values in q-PCR assay of miR-21 and miR-141

Reagents and materials. AmpliScribe T7-Flash Transcription Kit was purchased from Epicenter

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2018

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(Madison, WI, USA). Malachit green chloride was purchased from Sigma-Aldrich Co. (St. Louis, MO,

USA). DFHBI-1T was purchased from Lucerna Technologies(New York, USA). Precast 6% TBE

PAGE gel, RiboRuler Low Range RNA ladder, SYBR Green II fluorophore, Quant-iT RiboGreen RNA

assay kit were purchased from Life Technologies Corporation (New York, USA). SplintR DNA ligase

was purchased from New England BioLabs (Ipswich, MA, USA). RPMI 1640 medium, DMEM high

glucose medium, penicillin, streptomycin and 10% heat-inactivated fetal bovine serum were purchased

from Thermo Scientific HyClone (MA, USA). HeLa cells (human cervical carcinoma cell line), L-02

cells (mouse endothelial cell line) and 22RV1 cells(human prostate cancer cell line) were obtained from

the cell bank of Central Laboratory at Xiangya Hospital (Changsha, China). MCF-7(human breast

adenocarcinoma cell line) and MCF-10A(Non-cancerous human breast epithelial cell line) were

purchased from the cell bank of Chinese academy of sciences (Beijing, China). SanPrep column

microRNA mini-preps kit, TBE buffer (5×225 mM Tris-Boric Acid, 50 mM EDTA, pH 8.0), phosphate

buffered saline (1×PBS) (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4), All oligonucleotides

sequence were purchased from Sangon Biotech Co.Ltd.(Shanghai, China), with purity and yield

confirmed by high performance liquid chromatography (HPLC). All solutions were prepared with an

electric resistance >18.3 MΩ. Sequences of the synthesized oligonucleotides are given in Table S1.

DNA-RNA splinted ligation assay. Splinted ligation was preformed according to previously reported

method.S1 Briefly, 200 nM probe A1/A2 and probe B1/B2 with a given concentrations of miRNA were

adjust to 7 μL reaction buffer containing 10 mM Tris-HCl, 50 mM KCl, 0.1 mM EDTA in water and

heated to 95 °C for 3 minutes and placed on ice immediately. Then 1μL 10x SplintR buffer and 2 μL 10

units of SplintR DNA ligase were add to reaction buffer and incubated at 37 °C for 10 minutes, the

reaction was terminated by heated to 95 ℃ for 10 min.

T7 In vitro trancription process. Ligation product was used for in vitro transcription based on the

AmpliScribe T7-flash transcription kit with some modifications. A transcription reaction containing 2

μL ligation product, 1 μL 2nd T7 promoter sequence (500 nM), 2 μL 10x AmpliScribe T7 reaction

buffer, 2 μL DTT(10 mM), 0.5 μL 20U RNase inhibitor, 2 μL 12U T7 transcription enzyme, 1.5 μL

NTPs (10 mM for each NTP) and 4.5 μL RNase-free water. The reaction mixture was incubated at 37

°C for 2 h, afterward, the DNA template was degraded by 1 μL of RNase-free DNaseI incubated for 15

min at 37 °C. The transcript RNA products were purified using Bio-Spin columns, and quantified using

both absorbance values and the Quant-iT RiboGreen RNA assay kit. The purified RNA was directly for

downstream reaction or store at -80 °C.

Gel electrophoresis analysis of RNA sensor. The experiment sample obtained from different steps of

the reaction were collected and analyzed using 1.5% agarose gel electrophoresis in 1×TBE buffer at

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room temperature. The gel was stained by 0.5 μg/mL GoldView and 0.5 μg/mL ethidium bromide.

Electrophoresis was performed at a constant voltage of 100 V for 90 min with a load of 10 μL of sample

in each lane. After electrophoresis, the gel was visualized using a Tocan 240 gel imaging system

(Shanghai Tocan Biotechnology Company, China).

About 100 ng of in vitro T7 transcription prodcuts utlizing miR-21 and miR-141 were loaded into

different wells of precast 6% TBE PAGE gel, respectively and ran at 100 V in 1X TBE buffer for 80

minutes. Experiments was preformed according to previously reported method.S2 RiboRuler low range

RNA ladder was used as a molecular weight standard. After electrophoresis, the gel was washed with

water 3 times and the first was stained for 30 min with 10 μM DFHBI-1T in buffer containing 40 mM

Tris-HCl (pH 7.4), 100 mM KCl, 10 mM MgCl2. Then the gel was imaged under 365 nm UV light.

Next,the gel was stained for 30 min with 10 μM MG in buffer containing 40 mM Tris-HCl (pH 7.4),

100 mM KCl, 10 mM MgCl2. Then the gel was imaged with 365 nm UV light to see all the RNA in the

sample. Finally, the gel was stained for 30 min with SYBR Green II fluorophore diluted 1/10,000 in

TBE buffer and imaged under the same instrument using the 365 nm UV light.

Fluorescence measurement of the Spianch-base biosensor for miRNA detecion. The RNA

transcripts with different concentrations of target miR-21 and miR-141 were mixed with a solution of

fluorophore DFHBI-1T/MG (final concentration: 10 μM) in a buffer (100 μL, 20 mM Tris-HCl pH 7.6,

100 mM KCl, 10 mM MgCl2) and incubated at room temperature for 30 min, and fluorescence was

measured at respective excitation and emission wavelengths. The fluorescence spectra were collected at

room temperature using a quartz cuvette on an F-7000 fluorescence spectrophotometer (Hitachi, Japan).

For DFHBI-1T, excitation wavelength was 440 nm and the emission wavelengths were in the range

from 460 nm to 580 nm with a slit width of 5 nm for both excitation and emission. For MG, excitation

wavelength was 610 nm and the emission wavelengths were in the range from 630 nm to 700 nm with a

slit width of 5 nm for both excitation and emission.

To evaluate its selectivity, the RNA sensor was also used for detecting other co-existing cellular

components including 50 μM miR-143, 50 μM let-7f, 50 μM miR-222, 50 μM miR-141, L-02 cells

lysate and MCF-10a cells lysate. The cell lysate was prepared according to the following procedure: L-

02 Cells or MCF-10A(1× 106) were dispensed in an RNase-free 1.5 mL centrifuge tube, washed three

times with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM phosphate, pH 7.4), centrifuged at 2000 rpm for

2min, and then suspended in 100 μL RIPA lysis buffer containing 1% glycerol and 0.1 mM 4-(2-

aminoethyl) benzenesulfonyl fluoride hydrochloride). The resulting lysate (50 μL) was used

immediately in the assay. Other components were directly used in the assay as received without

purification.

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Cell culture and total miRNA extraction. The five human cancer cell lines included human breast

cancer cell line (MCF-7), Non-cancerous human breast epithelial cell line(MCF-10A), mouse

endothelial cell line(L-02), human prostate cancer cell line(22RV1), and human cervical carcinoma cell

line (HeLa). 22RV1, L-02 and HeLa cells were cultured in RPMI 1640 medium supplemented with

10% heat-inactivated fetal bovine serum and 100 U/mL penicillin and 100 g/mL streptomycin. MCF-7

and MCF-10A cells line were cultured in DMEM high glucose medium. These cells were maintained at

37 °C in a humidified atmosphere containing 5% CO2. Total mircoRNA samples were isolated from

MCF-7, 22RV1, HeLa cell line by using SanPrep column microRNA mini-preps kit. An 100 ng aliquot

of the total mircoRNA extracts was taken from each cell line and was analyzed in parallel using in this

study.

RT-qPCR quantification of the miRNA expression level in cells. Total microRNA were extracted

from MCF-7, 22RV1 and Hela cells using Sanprep column microRNA mini-preps kit (Sangon Biotech

Co., Ltd, Shanghai, China) following the manufacture’s instructions. The integrity of microRNA was

assessed by gel electrophoresis. The cDNA samples were then prepared using AMV first strand cDNA

synthesis kit (BBI, Toronto, Canada). Briefly, a total volume of 11 μL solution containing 5 μL of the

total RNA, 1 μL specific primer (0.2 μg/μL), 5 μL nuclease-free water was incubated at 65 °C for 5 min

followed by ice bath for 2 min. Then total volume of 20 μL reaction solution was obtained by adding 1

μL RNase inhibitor (20U/μL), 2 μL dNTPs (10 mM for each of dATP, dGTP, dCTP and dTTP), 4 μL

5× AMV reverse transcriptase buffer and 2 μL AMV reverse transcriptase (10 U/μL, Sangon Biotech

Co., Ltd, Shanghai, China). The reaction solution was incubated at 25 °C for 10 min and 50°C for 30

min followed by heat inactivation of reverse transcriptase for 5 min at 85 °C. The cDNA samples were

store at -20 °C for future use. The qPCR analysis of mRNA was performed with SYBRGreen PCR

master mix (ABI, CA, USA) according to the manufacturer’s instructions on an ABI Stepone plus

qPCR instrument (CA, USA). The 20 μL reaction solution contained 2 μL of cDNA sample, 10 μL

SYBRGreen qPCR Master Mix, 0.4 μL upstream primer (10 μM), 0.4 μL downstream primer (10 μM)

and 7.2 μL nuclease-free water. Primers used for miRNA21 and miRNA141 were given in Table S1.

The qPCR conditions were as follows: staying at 95 °C for 3 min, then followed by 40 cycles of 95 °C

for 7s, 57 °C for 10 s and 72 °C for 15 s.

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Table S1. Sequences of miRNAs and DNA probes in this work.a

Name Sequences (5'-3')

miRNA21 UAG CUU AUC AGA CUG AUG UUG A

miRNA141 UAA CAC UGU CUG GUA AAG AUG G

2nd T7 promoter sequence TAA TAC GAC TCA CTA TAG GG

A1 p-CTG ATA AGC TAG CGA TAC CCT ATA GTG AGT CGT ATT A

B1GTG TGG GAG CCC ACA CTC TAC TCG ACA GAT ACG AAT ATC TGG ACC CGA CCG TCT CCC ACA CTC AAC ATC AGT

A2 p-AGA CAG TGT TAG CGA TAC CCT ATA GTG AGT CGT ATT A

B2GGG GAT CCA TTC GTT ACC TGG CTC TCG CCA GTC GGG ATC CCC CCA TCT TTA CC

U6-F CTC GCT TCG GCA GCA CA

U6-R AAC GCT TCA CGA ATT TGC GT

miR-21 F ACA CTC CAG CTG GGT AGC TTA TCA GAC TG

miR-21 R CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG TCA ACA TCA

miR-141 F ACA CTC CAG CTG GGT AAC ACT GTC TGG T

miR-141 R CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG CCA TCT TT

a The yellow letter indicates complementary regions of the target mircoRNA, blue letter indicates

aptamer template and the italic green letter indicates the 1st T7 promoter sequence.

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Figure S1. Agarose gel (4%) electrophoresis images of in vitro transcription product with various

hybrid DNA mixture as transcript template. (A) The DNA template formed using 1 μM A1 , 1 μM B1

and 200 nM miR-21. Lane 1: without splintR ligase and 2nd T7 promoter sequence (correponding to

lane 4 in Figure 1A). Lane 2: without 2nd T7 promoter sequence (correponding to lane 5 in Figure 1A).

Lane 3: with splintR ligase and 2nd T7 promoter sequence (correponding to lane 6 in Figure 1A), the

92nt size of transcript products were obtained. (B) The DNA template formed using 1 μM A2 and 1μ M

B2 plus 200 nM miR-141. without splintR ligase and 2nd T7 promoter sequence (correponding to lane

11 in Figure 1A). Lane 2: without 2nd T7 promoter sequence (correponding to lane 12 in Figure 1A).

Lane 3: with splintR ligase and 2nd T7 promoter sequence (correponding to lane 13 in Figure 1A), the

73nt size of transcript products were obtained.

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Figure S2. Selection gel imaging of miR-21 and miR-141 initiated T7 in vitro transcription products.

Lane 1 was miR-21 initiated RNA transcripts products (MG RNA aptamer), lane 2 was RNA ladder,

and lane 3 was miR-141 initiated RNA transcript products (Broccoli). MG dye, DFHBI-1T, and SYBR

Green II were then used to stain all three lanes in sequence. Because no washing process was used, the

staining of each step was preserved. A distinct band appeared lane 1 after staining by MG dye while the

other two lanes showed no change. After stained by DFHBI-1T dye, a distinct band appeared in

Broccoli aptamer lane. SYBR Green II was then used to detect all RNA and stain RNA ladder. The

products in MG aptamer lane and Broccoli aptamer lane were around 73nt and 92nt, respectively, which

was consistent with sequences of probe B1 and B2.

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Table S2. Comparison of detection limit of Spinach-based light-up biosensors for nucleuic acid dtection

Method Target Detection limit Ref

Universal split spinach aptamer probe

Split spinach aptamer probes

Conformation switching method

tRNA scaffold based structure switching

Modified Spinach relies on structurally interactingRNA switch

Target triggered ligation and T7 in vitro transcription amplification

miRNA/miDNA

DNA/RNA

miRNA

miRNA

miRNA

miRNA

1.5 nM /4.2 nM

1.5 nM/1.8 nM

1 μM

1 nM

10 μM

3 pM

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This work

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Figure S3. The selectivity investigation of the Spinach-based fluorescent light-up biosensor. (A) miR-

21 detecion. 50 μM miR-222, 50 μM miR-143, 50 μM miR-141, 50 μM let-7f, 50 μL L-02 cells lysate.

(B) miR-141 detecion. 50 μM miR-143, 50 μM miR-21, 50 μM miR-222, 50 μM let-7f, 50 μL MCF-

10A cells lysate.

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Figure S4. The stability investigation of Spinach-based fluorescent light-up biosensor. (A) miR-21

detection. (B) miR-141 detection.

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Figure S5. Agarose gel (1.5%) electrophoresis images of extracted total cellular mircoRNA. Each cell

line was extracted in two repetitive experiments. Lane 1: MCF-7 cell. Lane 2: HeLa cell. Lane 3:

22RV1 cell. M: DNA size marker.

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Figure S6. Agarose gel (1.5%) electrophoresis image of qPCR products for miRNA and U6 gene

analysis. (A) Lane 1: amplification product for miR-21 analysis. Lane 2: DNA Marker. Lane 3:

amplification product for U6 gene. (B) Lane 1: amplification product for U6 gene. Lane 2: DNA

Marker. Lane 3: amplification product for miR-141 analysis

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Figure S7. Expression analysis of miR-21 and miR-141 for different cell lines. Each sample was

analyzed in three repetitive assays. (A) Left: RT-qPCR curves for miR-21 analysis in MCF-7(blue) and

Hela (red) cells. Right: Relative expression levels for miR-21. (B) Left: RT-qPCR curves for miR-141

analysis in 22RV1 (blue) and MCF-7(red) cells. Right: Relative expression levels for miR-141.

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Table S3. Average Ct values in q-PCR assay of miR-21a

Cell line miR-21 U6 ∆Ct ∆∆Ct 2-(∆∆Ct)

MCF-7 16.94 10.80 6.14 0 1

HeLa 18.66 10.89 7.77 1.64 0.32

Table S4. Average Ct values in q-PCR assay of miR-141 a

Cell line miR-141 U6 ∆Ct ∆∆Ct 2-(∆∆Ct)

22RV1 16.47 10.77 5.69 0 1

MCF-7 18.93 10.69 8.24 2.54 0.17

a. The relative expression level was estimated by the values of 2-(∆∆Ct) and U6 gene was used as reference. From the data, the expression level of miR-21 in MCF-7 was estimated to be 3.1 fold of that in Hela cell line, the expression level of miR-141 in 22RV1was estimated to be 5.8 fold of that in MCF-7 cell line.

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