Supplementary information
Table S1. Oligonucleotides used
Primer Name
Sequence 5’ to 3’*
pf AIM1
CACCTCTCAGTGATAAAGGAT
pr AIM1
TGCACCACTCAGTCTTTCA
pfAIM1 SNP
CACCTCTCAGTGATAAAGGATAAGTT
G4AIM1
CGGGGGGGATGGGGGGTGGTAGGG
BSpf AIM1 all
AAAGTTATTTGAAAGTTAATGTTATGG
BSpr AIM1 all
AAAAGTAACTAATAACAACCAACATCT
BSpf2 AIM1 CG
ATAAAGGATAGGTTTTTAGGTCG
pfBLCAP
TTGCAGGATGAGACAGGCAG
prBLCAP
TCAGCCTATCACCCCAGACA
pfBLCAP SNP
TTGCAGGATGAGACAGGCAGAACC
G4BLCAP
GGGGAGGTGGGGGGACGTGGGGGTGAGGGG
BSpf BLCAP all
TTGTAGGATGAGATAGGTAGAGT
BSpr BLCAP all
ATACTTCCTTTCCAAACCATTA
BSpf2 BLCAP CG
TTGTAGGATGAGATAGGTAGAGTCG
pfBLCAP (B)
CAACGTGTCTCTGGGGCATA
Experimental use
Template Mixing
Template Mixing and Sequencing
Primer Mutagenesis
CD Spectroscopy
Bisulfite PCR amplification
Bisulfite PCR amplification
Bisulfite methylation specific
PCR amplification
Template Mixing
Template Mixing and Sequencing
Primer Mutagenesis
CD Spectroscopy
Bisulfite PCR amplification
Bisulfite PCR amplification
Bisulfite methylation specific
PCR amplification
Template Mixing
pr BLCAP (B)
TCGGGGAGCAGGTTTGCTGT
Template Mixing and Sequencing
pfBLCAP (B) SNP
CAACGTGTCTCTGGGGCATATAAAGGA
Primer Mutagenesis
G4BLCAPB
GGGGGGTGGGGGCGGTGGGGGGGGG
CD Spectroscopy
BSpf BLCAP (B)
GGGTATATAGAGGAAGTTAGAGGTTAT
Bisulfite PCR amplification
all
CCATTTACTAGATATCTATTTTGAAAT
Bisulfite PCR amplification
BSpr BLCAP (B)
GTATATAGAGGAAGTTAGAGGTTATCG
Bisulfite methylation specific
all
PCR amplification
BSpf2 BLCAP (B)
CG
Template Mixing
pfDNMT1
GGCAGAAGTCCTTCCTTCCC
prDNMT1
AGCCTCATTCCCATCAAGTAGC
pfDNMT1 SNP**
GGCAGAAGTCCTTCCTTCCCAAAT
G4DNMT1
CGGGGGCTGGGGGCTGAGGGCCGGTTGGG
BSpfDNMT1all
GTAGGTGTTTGAGATTTATGTT
BSprDNMT1all
AACATTTCTATCACTCAAATATTA
BSpf2DNMT1CG
TATTTGGATAGTGGGGTCG
pfDNMT1 (B)
ACAGTGGGTCGTTTCTTCCC
Template Mixing
prDNMT1 (B)
TTCAGTAAGGGATGGCTGGC
Template Mixing and Sequencing
pfDNMT1 (B) SNP
ACAGTGGGTCGTTTCTTCCCTATGTCT
Primer Mutagenesis
G4DNMT1B
GGGCCCTGGGGCTGGGGCGGG
CD Spectroscopy
Template Mixing and Sequencing
Primer Mutagenesis
CD Spectroscopy
Bisulfite PCR amplification
Bisulfite PCR amplification
Bisulfite methylation specific
PCR amplification
BSpfDNMT1 (B) all
TGATAATATTTGTGAGGGGTTTT
Bisulfite PCR amplification
BSprDNMT1 (B) all
CTATACCATTAAACCCTACCTTCA
Bisulfite PCR amplification
BSpf2DNMT1 (B)
TGATAATATTTGTGAGGGGTTTTCG
Bisulfite methylation specific
CG
PCR amplification
pfGRB10
GTTTGGGAAACTGCCTGCAG
prGRB10
GCAACAGATGAGAACAGTGG
pfGRB10 SNP
GTTTGGGAAACTGCCTGCAGAAAG
G4GRB10
GGGTGGGGGAGGGCTGGG
BSpf GRB10all
TAGTTTATATGTTTATTTGTAGATTTAG
BSpr GRB10all
ATCAACTTTAAACATTACAAATATAC
BSpf2 GRB10CG
TAGTTTATATGTTTATTTGTAGATTTAGCG
pfKCNQ1
CCTGGCTTCGGTCCCCTGGA
prKCNQ1
GCAGTGCCCAGCACAGGGAG
pfKCNQ1 SNP
CCTGGCTTCGGTCCCCTGGACTGG
G4KCNQ1
CGGGGACTGGGGCTGGGGCTGGGG
BSpf KCNQ1all
TGTTTTTGGTGTAGGTGG
BSpr KCNQ1all
CCTAAACACCCACAAACCT
BSpf2 KCNQ1CG
TTTTTGGTGTAGGTGGCG
pfPLAGL1
AGAGCAAACTCGGCAGGCGG
Template Mixing
Template Mixing and Sequencing
Primer Mutagenesis
CD Spectroscopy
Bisulfite PCR amplification
Bisulfite PCR amplification
Bisulfite methylation specific
PCR amplification
Template Mixing
Template Mixing and Sequencing
Primer Mutagenesis
CD Spectroscopy
Bisulfite PCR amplification
Bisulfite PCR amplification
Bisulfite methylation specific
PCR amplification
Template Mixing
prPLAGL1
AGAGCAAACTCGGCAGGCGG
Template Mixing and Sequencing
pfPLAGL1 SNP
AGAGCAAACTCGGCAGGCGGATTA
Primer Mutagenesis
G4PLAGL1
GGGGTTGGGGGAAAGGGGTTTGGG
CD Spectroscopy
PLAGL1Fa
CCATAAGAGACGAAAGTGCAAACA
PCR amplification genomic DNA
PLAGL1Ra
CGAGGAGGGTGTGCCTTTG
PCR amplification genomic DNA
G4PLAGL1a
GGGGCTGCCTGGGTTGCGGGTGACGATCTGCGGGG
CD Spectroscopy of genomic G4a
G4PLAGL1b
CGGGTCCGGGCTCCGCGGGGCCGGGTGCGGG
CD Spectroscopy of genomic G4b
BSpf PLAGL1all
ATAGATTATGATATTTAGTAGAGTAAACT
Bisulfite PCR amplification
BSpr PLAGL1all
CAAATACCCCTCACCATCCT
Bisulfite PCR amplification
BSpf2 PLAGL1
ATAGATTATGATATTTAGTAGAGTAAACTCG
Bisulfite methylation specific
PCR amplification
*Bold nucleotides indicate guanines predicted by QGRS Mapper to contribute towards G4 formation.
**Underlined nucleotides denote the position of the artificial SNP introduced by primer mutagenesis
Figure S1. DNA sequences of PCR amplicons used in template mixing experiments.
Horizontal black arrows represent the forward and reverse primers used for amplification. The
longer forward arrow represents the primer used during primer mutagenesis.
The single
nucleotide above the vertical black arrows represents the nucleotide substituted during primer
mutagenesis. Underlined bases represent CpG dinucleotides which can be potentially
methylated using M. SssI. Bold bases represent the G4 forming motifs. Black box indicates
the recognition sequence for HpaII/MspI , used for assessing successful methylation.
23
Figure S2. Example of primer combinations used for primer mutagenesis and
amplification.
The pfAIM1 SNP primer (purple) was used to generate a synthetic template containing an “A”
allele (yellow) at the 23rd nt in place of a “G”. The alternative template was generated using
pfAIM1 primer (green), and consisted of the wild-type “G” containing sequence. Reamplification during the subsequent mixing experiments was performed using the pfAIM1
primer, and a common reverse primer (Supplementary Table 1.). Genotyping of products was
performed by Sanger sequencing with the reverse primer.
Figure S3. Genomic DNA sequences of PLAGL1 used to demonstrate ADO in genomic
DNA
Horizontal black arrows represent the forward and reverse primers used for amplification. Bold
bases represent the G4 forming motifs.
A
B
Figure S4. CD spectra of PLAGL1 G4 from genomic DNA at 20oC and 95oC.
(A) G4PLAGL1a (B) G4PLAGL1b. Molar ellipticity (x105 deg.cm2.dmol-1) is on the vertical
axis and wavelength (nm) is on the horizontal axis. Solid lines represent CD spectra in the
presence of 10 mM Tris-HCl 50 mM KCl and 1.5 mM MgCl2, and the dashed lines represent
CD spectra in 10 mM Tris-HCl and 1.5 mM MgCl2. DNA sequences of G4PLAGL1a and
G4PLAGL1b are shown in Supplementary Figure 2 and Supplementary Table 1.
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10
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6
4
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-2
-4
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-6
Figure S5. CD spectra of AIM1 G4 at 20oC and 95oC.
Spectral profiles obtained at 20oC (grey lines) and 95oC (black lines), in PCR buffer. Samples
analysed in 10 mM Tris-HCl, 50 mM KCl and 1.5 mM MgCl2 are represented by solid lines
and samples analysed in 10 mM Tris-HCl and 1.5 mM MgCl2 are represented by dashed lines.
Molar ellipticity (x105 deg.cm2.dmol-1) is on the vertical axis and wavelength (nm) is on the
horizontal axis.
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-4
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-6
Figure S6. CD spectra of BLCAP G4 at 20oC and 95oC.
Spectral profiles obtained at 20oC (grey lines) and 95oC (black lines), in PCR buffer. Samples
analysed in 10 mM Tris-HCl, 50 mM KCl and 1.5 mM MgCl2 are represented by solid lines
and samples analysed in 10 mM Tris-HCl and 1.5 mM MgCl2 are represented by dashed lines.
Molar ellipticity (x105 deg.cm2.dmol-1) is on the vertical axis and wavelength (nm) is on the
horizontal axis.
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-4
Figure S7. CD spectra of BLCAP (B) G4 at 20oC and 95oC
Spectral profiles obtained at 20oC (grey lines) and 95oC (black lines), in PCR buffer. Samples
analysed in 10 mM Tris-HCl, 50 mM KCl and 1.5 mM MgCl2 are represented by solid lines
and samples analysed in 10 mM Tris-HCl and 1.5 mM MgCl2 are represented by dashed lines.
Molar ellipticity (x105 deg.cm2.dmol-1) is on the vertical axis and wavelength (nm) is on the
horizontal axis.
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6
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-2
-4
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-6
Figure S8. CD spectra of DNMT1 G4 at 20oC and 95oC.
Spectral profiles obtained at 20oC (grey lines) and 95oC (black lines), in PCR buffer. Samples
analysed in 10 mM Tris-HCl, 50 mM KCl and 1.5 mM MgCl2 are represented by solid lines
and samples analysed in 10 mM Tris-HCl and 1.5 mM MgCl2 are represented by dashed lines.
Molar ellipticity (x105 deg.cm2.dmol-1) is on the vertical axis and wavelength (nm) is on the
horizontal axis.
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6
4
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-2
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315
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-4
Figure S9. CD spectra of DNMT1 (B) G4 at 20oC and 95oC.
Spectral profiles obtained at 20oC (grey lines) and 95oC (black lines), in PCR buffer. Samples
analysed in 10 mM Tris-HCl, 50 mM KCl and 1.5 mM MgCl2 are represented by solid lines
and samples analysed in 10 mM Tris-HCl and 1.5 mM MgCl2 are represented by dashed lines.
Molar ellipticity (x105 deg.cm2.dmol-1) is on the vertical axis and wavelength (nm) is on the
horizontal axis.
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10
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6
4
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-4
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315
310
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295
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-6
Figure S10. CD spectra of GRB10 G4 at 20oC and 95oC.
Spectral profiles obtained at 20oC (grey lines) and 95oC (black lines), in PCR buffer. Samples
analysed in 10 mM Tris-HCl, 50 mM KCl and 1.5 mM MgCl2 are represented by solid lines
and samples analysed in 10 mM Tris-HCl and 1.5 mM MgCl2 are represented by dashed lines.
Molar ellipticity (x105 deg.cm2.dmol-1) is on the vertical axis and wavelength (nm) is on the
horizontal axis.
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-2
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-3
Figure S11. CD spectra of KCNQ1 G4 at 20oC and 95oC.
Spectral profiles obtained at 20oC (grey lines) and 95oC (black lines), in PCR buffer. Samples
analysed in 10 mM Tris-HCl, 50 mM KCl and 1.5 mM MgCl2 are represented by solid lines
and samples analysed in 10 mM Tris-HCl and 1.5 mM MgCl2 are represented by dashed lines.
Molar ellipticity (x105 deg.cm2.dmol-1) is on the vertical axis and wavelength (nm) is on the
horizontal axis.
10
8
6
4
2
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-2
-4
320
315
310
305
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Figure S12. CD spectra of PLAGL1 G4 at 20oC and 95oC.
Spectral profiles obtained at 20oC (grey lines) and 95oC (black lines), in PCR buffer. Samples
analysed in 10 mM Tris-HCl, 50 mM KCl and 1.5 mM MgCl2 are represented by solid lines
and samples analysed in 10 mM Tris-HCl and 1.5 mM MgCl2 are represented by dashed lines.
Molar ellipticity (x105 deg.cm2.dmol-1) is on the vertical axis and wavelength (nm) is on the
horizontal axis.