Supporting Information
Amplification-free detection of circulating microRNA biomarkers from body fluids based on fluorogenic oligonucleotide-templated reaction between engineered Peptide-Nucleic Acid probes: application to prostate cancer diagnosis. Gavin A.D. Metcalf1,2, Akifumi Shibakawa2, Hinesh Patel1, Ailsa Sita-Lumsden2, Andrea Zivi2, Nona Rama2, Charlotte L. Bevan2 and Sylvain Ladame1*.
Supporting Information
Table of Contents: 1. DNA and RNA sequences 2. PNA probe structure and characterization via MALDI-TOF 3. Characterization of the adduct between cysteine and coumarin by MALDI-TOF and 1 H NMR 4. Biocompatibility tests 5. In vitro quantitative detection by fluorogenic OTR 6. Sequence specificity and SNP analysis 7. Detection of pre-miRNA sequences with PNA probes 8. qPCR internal controls
S1
S2 S2 S8 S9 S9 S10 S12 S13
1. DNA and RNA sequences HPLC purified DNA and RNA oligonucleotides were purchased from either Invitrogen (USA) or Eurogentec (Belgium). microRNA miR-141 miR-375 miR-132 miR-200a
RNA sequence (5’ to 3’) UAA CAC UGU CUG GUA AAG AUG G UUU GUU CGU UCG GCU CGC GUG A ACC AUG GCU GUA GAC UGU UA UAA CAC UGU CUG GUA ACG AUG U
DNA sequence (5’ to 3’) TAA CAC TGT CTG GTA AAG ATG G TTT GTT CGT TCG GCT CGC GTG A ACC ATG GCT GTA GAC TGT TA TAA CAC TGT CTG GTA ACG ATG T
2. PNA probe structure and characterization via MALDI-TOF Figure S1. PNA-miR141-Coum:
S2
Figure S2. PNA-miR-141-SH:
S3
Figure S3. PNA-miR-132-Coum:
S4
Figure S4. PNA-miR-132-SH:
S5
Figure S5. PNA-miR-375-Coum:
S6
Figure S6. PNA-miR-375-SH:
S7
3. Characterization of the cysteine-coumarin covalent adduct formed between by MALDI-TOF and 1H NMR. Reaction between Coumarin 3 (10 mM) and L-Cysteine (40 mM, 4 equivalents) in a mixture of d6-DMSOD2O (9-1) was monitored by 1H NMR and MALDI-TOF. 1H NMR spectrum after 1hour shows near-total disappearance of the α,β-unsaturated system (Figure S7). MALDI-TOF analysis of the crude reaction mixture confirms the formation of the cysteine-coumarin covalent adduct (Figure S8). Figure S7. 1H NMR spectrum of the covalent adduct formed between Coumarin 3 and L-cysteine (after 1h at room temperature).
Figure S8. MALDI-TOF spectrum of the covalent adduct formed between Coumarin 3 and L-cysteine. Coum Coum + Cys complex
S8
4. Biocompatibility tests Biocompatibility tests were performed to investigate the integrity and function of PNA probes within biofluids. A reaction mixture was prepared with a final concentration of 10mM TRIS-HCl buffer solution (pH=7.4, Sigma), 5µM PNA-Thiol, 5µM PNA-Cou1 and 1µM miR-132 (DNA), in DNase- RNase-free water to a volume of 100µl in eppendorf tubes. 10% (v/v) of thoroughly thawed biofluid sample (serum, plasma, urine, or saliva) was introduced to this solution. Eppendorfs were incubated at 37°C for 3h (QBD digital dry block heater, Grant). After which samples were transferred to 384-well microtitre plates and fluorescence emission spectra recorded using a Fluorostar fluorescence plate reader (Omega, UK) (λexc = 485nm, λem = 520nm). Results obtained displayed a good sensing ability within these fluids, with background fluorescence being minimal. Figure S9. miR-141 DNA/RNA sensing in buffer and various biofluids from healthy donors.
5. In vitro quantitative detection by fluorogenic OTR Sensing reactions were performed using a fixed stoichiometric concentration of PNA probes (5μM each) and varying the concentration of RNA (1μM to 50nM). Results obtained showed near-linear correlation between fluorescence intensity and RNA concentrations ranging between 50nM and 1μM, with R values presenting as 0.974 and 0.993 for miR-375 and miR-132 respectively. Figure S10. Fluorescence intensity as a function of miR-375 (Left) and miR-132 (Right) RNA concentration.
S9
6. Sequence specificity and SNP analysis Figure S11. Sequences of oligonucleotides used for the SNP analysis. Name
Sequence (5’ to 3’)
miR-141
TAACACTGTCTGGTAAAGATGG
SNP1
TATCACTGTCTGGTAAAGATGG
Mutated
SNP2
TAACACTGTCTGGTACAGATGG
(SNP)
SNP3
TAACACGGTCTGGTAAAGATGG
SNP4
TAACACTGTCTGGTAATGCTGG
miR-132
ACCGTGGCTTTCGATTGTTACT
SNP1
ACTGTGGCTTTCGATTGTTACT
Mutated
SNP2
ACCGTAGCTTTCGATTGTTACT
(SNP)
SNP3
ACCGTGGCTTTCGAATGTTACT
SNP4
ACCGTGGCTTTCGATTGTCACT
Non-Mutated
miR-375
UUUGUUCGUUCGGCUCGCGUGA
Mutated (SNP)
SNP3
UUUGUACGUUCGGCUCGCGUGA
SNP4
UUAGUUCGUUCGGCUCGCGUGA
Non-Mutated
Non-Mutated
Figure S12. SNP array analysis for miR-141 sensing. N o r m a lis e d R F U ( f o ld )
1 .5
1 .0
0 .5
4 S
N
P
3 S
N
P
2 N
S
S
N
P
1 P
C T N
T
C
0 .0
Figure S13. SNP array analysis for miR-132 sensing.
1 .0
0 .5
S10
4 P
3 N S
N S
N S
P
2 P
1 N S
N
T
P
C
C
0 .0 T
N o r m a lis e d R F U ( f o ld )
1 .5
Figure S14. SNP array analysis for miR-375 sensing.
N o r m a lis e d R F U ( f o ld )
1 .5
1 .0
0 .5
4 S
N
P
3 P S
N
N
T
T
C
C
0 .0
Figure S15. Specificity test exploring the ability of miR-141 probes to discern a single base difference in miR-200a target sequence. T-test; miR-141/miR200a expression (p=0.5947).
N o r m a lis e d R F U ( f o ld )
1 .5
1 .0
0 .5
) (1 M
m
m
iR
iR
-2
0
-1
0
4
a
1
R
R
N
N
A
A
N
T
(1 M
C
)
0 .0
miR-141 sequence (5’ to 3’): UAA CAC UGU CUG GUA AAG AUG G miR-200a sequence (5’ to 3’): UAA CAC UGU CUG GUA ACG AUG U
S11
7. Detection of pre-miRNA sequences with PNA probes Initial tests were performed to investigate if our technology could detect pre-miRNA sequences as well as mature miRNAs. A commercially available stem loop sequence (Sigma) was designed to simulate pre-miR141 and this was tested in combination with the mature miR-141 DNA sequence. Results displayed heightened fluorescence in the presence of both pre- and mature-miR-141 sequences, in comparison to mature-miR-141 alone, thus suggesting that PNA probes have an ability to disrupt the stem loop structure and anneal to target sequences. a) Sequence and structure of pre-miR141:
Stem Loop
b) Sequence of the DNA analogue of pre-miR141 used for this study : CGGCCG GCCCTG GGTCCA TCTTCC AGTACA GTGTTG GATGGT CTAATT GTGAAG CTCCTA ACACTG TCTGGT AAAGAT GGCTCC CGGGTG GGTTC
Figure S16. Fluorescent signal of mature miR-141 and pre-miR-141 (Stem loop) sequences.
200000
100000
p
re
) p
-m
iR
-1
m
4
a
1
tu
(S
re
te
m
m
iR
lo
T
-1
o
4
C
1
0
N
R F U ( a .u .)
300000
S12
8. qPCR internal controls Figure S17. Ct values of cel-miR-39 and UniSp6, used as internal controls for qPCR experiments.
UniSp6 15
20
20
30 35
40
40
N R
AL
R Y
R
AM
35
miR-141-3p
AM
30
25
AL
25
R Y
Raw Ct value
15
N
Raw Ct value
cel-miR-39-3p
miR-375
20 25
Raw Ct value
30 35 40
25 30 35
miR-141-3p (PNA probe) 4.5
Signal value (RFU)
Signal value (RFU)
AM
miR-375 (PNA probe)
4.5 4.0 3.5 3.0 2.5
4.0 3.5 3.0 2.5
AL
RY
RN
AL
RY
RN
AM
miR-141-3p
AM
miR-375
4.5
4.5
4.0
4.0
PNA (RFU)
PNA (RFU)
AL
R
N
AM
R
N
R Y
AL
40
R Y
Raw Ct value
20
3.5 3.0
3.5 3.0
S13 2.5 20
25
30
qPCR (Ct)
35
40
2.5 20
25
30
qPCR (Ct)
35
40
Supporting Information
Table of Contents: 1. DNA and RNA sequences 2. PNA probe structure and characterization via MALDI-TOF 3. Characterization of the adduct between cysteine and coumarin by MALDI-TOF and 1 H NMR 4. Biocompatibility tests 5. In vitro quantitative detection by fluorogenic OTR 6. Sequence specificity and SNP analysis 7. Detection of pre-miRNA sequences with PNA probes 8. qPCR internal controls
S1
S2 S2 S8 S9 S9 S10 S12 S13
1. DNA and RNA sequences HPLC purified DNA and RNA oligonucleotides were purchased from either Invitrogen (USA) or Eurogentec (Belgium). microRNA miR-141 miR-375 miR-132 miR-200a
RNA sequence (5’ to 3’) UAA CAC UGU CUG GUA AAG AUG G UUU GUU CGU UCG GCU CGC GUG A ACC AUG GCU GUA GAC UGU UA UAA CAC UGU CUG GUA ACG AUG U
DNA sequence (5’ to 3’) TAA CAC TGT CTG GTA AAG ATG G TTT GTT CGT TCG GCT CGC GTG A ACC ATG GCT GTA GAC TGT TA TAA CAC TGT CTG GTA ACG ATG T
2. PNA probe structure and characterization via MALDI-TOF Figure S1. PNA-miR141-Coum:
S2
Figure S2. PNA-miR-141-SH:
S3
Figure S3. PNA-miR-132-Coum:
S4
Figure S4. PNA-miR-132-SH:
S5
Figure S5. PNA-miR-375-Coum:
S6
Figure S6. PNA-miR-375-SH:
S7
3. Characterization of the cysteine-coumarin covalent adduct formed between by MALDI-TOF and 1H NMR. Reaction between Coumarin 3 (10 mM) and L-Cysteine (40 mM, 4 equivalents) in a mixture of d6-DMSOD2O (9-1) was monitored by 1H NMR and MALDI-TOF. 1H NMR spectrum after 1hour shows near-total disappearance of the α,β-unsaturated system (Figure S7). MALDI-TOF analysis of the crude reaction mixture confirms the formation of the cysteine-coumarin covalent adduct (Figure S8). Figure S7. 1H NMR spectrum of the covalent adduct formed between Coumarin 3 and L-cysteine (after 1h at room temperature).
Figure S8. MALDI-TOF spectrum of the covalent adduct formed between Coumarin 3 and L-cysteine. Coum Coum + Cys complex
S8
4. Biocompatibility tests Biocompatibility tests were performed to investigate the integrity and function of PNA probes within biofluids. A reaction mixture was prepared with a final concentration of 10mM TRIS-HCl buffer solution (pH=7.4, Sigma), 5µM PNA-Thiol, 5µM PNA-Cou1 and 1µM miR-132 (DNA), in DNase- RNase-free water to a volume of 100µl in eppendorf tubes. 10% (v/v) of thoroughly thawed biofluid sample (serum, plasma, urine, or saliva) was introduced to this solution. Eppendorfs were incubated at 37°C for 3h (QBD digital dry block heater, Grant). After which samples were transferred to 384-well microtitre plates and fluorescence emission spectra recorded using a Fluorostar fluorescence plate reader (Omega, UK) (λexc = 485nm, λem = 520nm). Results obtained displayed a good sensing ability within these fluids, with background fluorescence being minimal. Figure S9. miR-141 DNA/RNA sensing in buffer and various biofluids from healthy donors.
5. In vitro quantitative detection by fluorogenic OTR Sensing reactions were performed using a fixed stoichiometric concentration of PNA probes (5μM each) and varying the concentration of RNA (1μM to 50nM). Results obtained showed near-linear correlation between fluorescence intensity and RNA concentrations ranging between 50nM and 1μM, with R values presenting as 0.974 and 0.993 for miR-375 and miR-132 respectively. Figure S10. Fluorescence intensity as a function of miR-375 (Left) and miR-132 (Right) RNA concentration.
S9
6. Sequence specificity and SNP analysis Figure S11. Sequences of oligonucleotides used for the SNP analysis. Name
Sequence (5’ to 3’)
miR-141
TAACACTGTCTGGTAAAGATGG
SNP1
TATCACTGTCTGGTAAAGATGG
Mutated
SNP2
TAACACTGTCTGGTACAGATGG
(SNP)
SNP3
TAACACGGTCTGGTAAAGATGG
SNP4
TAACACTGTCTGGTAATGCTGG
miR-132
ACCGTGGCTTTCGATTGTTACT
SNP1
ACTGTGGCTTTCGATTGTTACT
Mutated
SNP2
ACCGTAGCTTTCGATTGTTACT
(SNP)
SNP3
ACCGTGGCTTTCGAATGTTACT
SNP4
ACCGTGGCTTTCGATTGTCACT
Non-Mutated
miR-375
UUUGUUCGUUCGGCUCGCGUGA
Mutated (SNP)
SNP3
UUUGUACGUUCGGCUCGCGUGA
SNP4
UUAGUUCGUUCGGCUCGCGUGA
Non-Mutated
Non-Mutated
Figure S12. SNP array analysis for miR-141 sensing. N o r m a lis e d R F U ( f o ld )
1 .5
1 .0
0 .5
4 S
N
P
3 S
N
P
2 N
S
S
N
P
1 P
C T N
T
C
0 .0
Figure S13. SNP array analysis for miR-132 sensing.
1 .0
0 .5
S10
4 P
3 N S
N S
N S
P
2 P
1 N S
N
T
P
C
C
0 .0 T
N o r m a lis e d R F U ( f o ld )
1 .5
Figure S14. SNP array analysis for miR-375 sensing.
N o r m a lis e d R F U ( f o ld )
1 .5
1 .0
0 .5
4 S
N
P
3 P S
N
N
T
T
C
C
0 .0
Figure S15. Specificity test exploring the ability of miR-141 probes to discern a single base difference in miR-200a target sequence. T-test; miR-141/miR200a expression (p=0.5947).
N o r m a lis e d R F U ( f o ld )
1 .5
1 .0
0 .5
) (1 M
m
m
iR
iR
-2
0
-1
0
4
a
1
R
R
N
N
A
A
N
T
(1 M
C
)
0 .0
miR-141 sequence (5’ to 3’): UAA CAC UGU CUG GUA AAG AUG G miR-200a sequence (5’ to 3’): UAA CAC UGU CUG GUA ACG AUG U
S11
7. Detection of pre-miRNA sequences with PNA probes Initial tests were performed to investigate if our technology could detect pre-miRNA sequences as well as mature miRNAs. A commercially available stem loop sequence (Sigma) was designed to simulate pre-miR141 and this was tested in combination with the mature miR-141 DNA sequence. Results displayed heightened fluorescence in the presence of both pre- and mature-miR-141 sequences, in comparison to mature-miR-141 alone, thus suggesting that PNA probes have an ability to disrupt the stem loop structure and anneal to target sequences. a) Sequence and structure of pre-miR141:
Stem Loop
b) Sequence of the DNA analogue of pre-miR141 used for this study : CGGCCG GCCCTG GGTCCA TCTTCC AGTACA GTGTTG GATGGT CTAATT GTGAAG CTCCTA ACACTG TCTGGT AAAGAT GGCTCC CGGGTG GGTTC
Figure S16. Fluorescent signal of mature miR-141 and pre-miR-141 (Stem loop) sequences.
200000
100000
p
re
) p
-m
iR
-1
m
4
a
1
tu
(S
re
te
m
m
iR
lo
T
-1
o
4
C
1
0
N
R F U ( a .u .)
300000
S12
8. qPCR internal controls Figure S17. Ct values of cel-miR-39 and UniSp6, used as internal controls for qPCR experiments.
UniSp6 15
20
20
30 35
40
40
N R
AL
R Y
R
AM
35
miR-141-3p
AM
30
25
AL
25
R Y
Raw Ct value
15
N
Raw Ct value
cel-miR-39-3p
miR-375
20 25
Raw Ct value
30 35 40
25 30 35
miR-141-3p (PNA probe) 4.5
Signal value (RFU)
Signal value (RFU)
AM
miR-375 (PNA probe)
4.5 4.0 3.5 3.0 2.5
4.0 3.5 3.0 2.5
AL
RY
RN
AL
RY
RN
AM
miR-141-3p
AM
miR-375
4.5
4.5
4.0
4.0
PNA (RFU)
PNA (RFU)
AL
R
N
AM
R
N
R Y
AL
40
R Y
Raw Ct value
20
3.5 3.0
3.5 3.0
S13 2.5 20
25
30
qPCR (Ct)
35
40
2.5 20
25
30
qPCR (Ct)
35
40
