Electronic Supplementary Material (ESI) for Analyst
This journal is © The Royal Society of Chemistry 2012
Self reporting RNA probes as an alternative to cleavable small
molecule mass tags
Jo-Anne Riley,a Tom Brown,a Nittaya Gale,b Julie Hernimana and G. John Langley*a
a
Chemistry, FNES, University of Southampton, Southampton, SO17 1BJ, UK. Tel: (0) 23 8059 2182; E-mail: [email protected]
ATDBio LTD, University of Southampton, Southampton, SO17 1BJ, UK. Fax: (0)23 8059 2991; Tel: (0)23 8059 6778; E-mail:
[email protected]
b
Electronic Supplementary Information
(a)
TIC
UV chromatogram
(b)
Intens.
[%]
100
[2HFIP - H]-
Res2099_t=0_2_RE2_01_5421.d: -MS, 7.1-7.2min #(422-428)
334.9860
80
[M – 4H]4-
[M – 3H]3-
1542.1182
60
2056.4926
40
20
0
500
(c)
1000
1500
Intens.
[%]
2000
Intens.
[%]
2500
3000
3500 m/z
Res2099_t=0_2_RE2_01_5421.d: -MS, 7.1-7.2min, Deconvoluted (MaxEnt)
Res2099_t=0_2_RE2_01_5421.d: -MS, 7.1-7.2min, Deconvoluted (MaxEnt)
6172.5053
6172.5053
6171.5131
100
6170.5050
6173.5146
100
80
6174.5146
80
6169.5069
60
6175.5132
60
6168.4924
40
6176.5108
20
6167.5105
6177.5272
40
6166.5185
6178.5153
6179.5336
0
6164
6166
6168
6170
6172
6174
6176
6178
6180
m/z
20
0
2000
4000
6000
8000
10000
12000
14000
m/z
Figure S1 – HPLC-ESI MS analysis of ONT 1 prior to addition of RNase A, showing (a) TIC and UV
chromatogram, (b) negative ion ESI mass spectrum and (c) MaxEnt deconvoluted spectrum
Electronic Supplementary Material (ESI) for Analyst
This journal is © The Royal Society of Chemistry 2012
(a)
TIC
UV chromatogram
[M – 4H]4-
(b)
Intens.
[%]
100
Res2093_1_t=0_RD2_01_4128.d: -MS, 7.1-7.2min #(423-431)
1557.8294
[2HFIP - H]334.9879
[M – 3H]3-
80
2077.4358
60
40
20
1246.2706
1860.7707
0
500
1000
1500
Intens.
[%]
(c)
6235.3350
2000
2500
3000
3500 m/z
Res2093_1_t=0_RD2_01_4128.d: -MS, 7.1-7.2min, Deconvoluted (MaxEnt)
Intens.
[%]
-MS, 7.1-7.2min, Deconvoluted (MaxEnt)
6235.3347
150
6236.3289
150
6237.3252
6234.3273
6238.3176
100
6233.3255
100
6239.3320
6232.3226
50
6240.3305
50
6231.3107
6241.3155
6230.3167
0
6228
6230
6242.3259
6232
6234
6236
6238
6240
6242
6244
m/z
2112.1856
0
2000
4000
6000
8000
10000
12000
14000
m/z
Figure S2 – HPLC-ESI MS analysis of ONT 2 prior to addition of RNase A, showing (a) TIC and UV
chromatogram, (b) negative ion ESI mass spectrum and (c) MaxEnt deconvoluted spectrum.
Electronic Supplementary Material (ESI) for Analyst
This journal is © The Royal Society of Chemistry 2012
(a)
TIC
UV chromatogram
(b)
[M – 4H]4-
Intens.
[%]
[M – 3H]3-
1523.8551
Res2622_t=0_3_RA2_01_8913.d: -MS, 8.5-8.6min #(509-515), Background Subtracted
2032.4853
6
4
2
342.1640
2479.4249
0
(c)
500
Intens.
[%]
1000
1500
6100.4867
2000
Intens.
[%]
2500
3000
3500 m/z
Res2622_t=0_3_RA2_01_8913.d: -MS, 8.5-8.6min, Background Subtracted, Deconvoluted (MaxEnt)
Res2622_t=0_3_RA2_01_8913.d: -MS, 8.5-8.6min, Background Subtracted, Deconvoluted (MaxEnt)
6100.4867
6099.4648
15
15
6098.4899
6101.4792
6102.4903
6097.4618
10
10
6103.5044
6096.4659
5
6104.4864
6095.4762
5
6105.4841
6106.5104
6094.4139
0
6092
6094
6096
6098
6100
6102
6104
6106
6108
6110 m/z
2072.8784
0
2000
4000
6000
8000
10000
12000
14000
m/z
Figure S3 – HPLC-ESI MS analysis of ONT 3 prior to addition of RNase A, showing (a) TIC and UV
chromatogram, (b) negative ion ESI mass spectrum and (c) MaxEnt deconvoluted spectrum.
Electronic Supplementary Material (ESI) for Analyst
This journal is © The Royal Society of Chemistry 2012
(a)
TIC
UV chromatogram
(b)
[M – 3H]3-
Intens.
Res2621_t=0_2_RA2_01_8400.d: -MS, 8.7-8.8min #(517-523), Background Subtracted
2011.1432
1000
800
600
[M – 4H]4-
[2HFIP - H]-
1508.3521
334.9875
400
200
0
500
(c)
1000
1500
2000
2500
Intens.
3000
3500 m/z
Res2621_t=0_2_RA2_01_8400.d: -MS, 8.7-8.8min, Background Subtracted, Deconvoluted (MaxEnt)
Intens.
6036.4534
Res2621_t=0_2_RA2_01_8400.d: -MS, 8.7-8.8min, Background Subtracted, Deconvoluted (MaxEnt)
[%]
6036.4534
20
2000
6037.4748
6034.4693
6035.4433
15
1500
6033.4646
6038.4371
10
6039.4545
1000
6032.4400
6040.4827
5
6031.4617
500
6041.4740
6030.4609
0
6028
6030
6032
6034
6036
6038
6040
6042
6044
6046 m/z
0
2000
4000
6000
8000
10000
12000
14000
m/z
Figure S4 – HPLC-ESI MS analysis of ONT 4 prior to addition of RNase A, showing (a) TIC and UV
chromatogram, (b) negative ion ESI mass spectrum and (c) MaxEnt deconvoluted spectrum.
Electronic Supplementary Material (ESI) for Analyst
This journal is © The Royal Society of Chemistry 2012
(a)
TIC
UV chromatogram
(b)
Intens.
[%]
[2HFIP - H]-
Res2622_t=0_2_RA2_01_8546.d: -MS, 8.5-8.7min #(508-517), Background Subtracted
334.9625
4
[M 2032.1308
– 3H]3-
[M – 4H]41523.8535
3
2
1
1122.0509
1283.4081
608.0582
3369.7831
0
500
(c)
1000
1500
Intens.
[%]
2000
2500
3000
3500 m/z
Res2622_t=0_2_RA2_01_8546.d: -MS, 8.5-8.7min, Background Subtracted, Deconvoluted (MaxEnt)
6100.4296
8
Intens.
[%]
Res2622_t=0_2_RA2_01_8546.d: -MS, 8.5-8.7min, Background Subtracted, Deconvoluted (MaxEnt)
6100.4296
8
6098.4496
6101.4643
6
6
6102.4237
6097.4218
4
6103.4722
4
6096.4596
2
6104.5033
6095.4378
6105.4276
2
0
6092
6094
6096
6098
6100
6102
6104
6106
6108
6110 m/z
0
2000
4000
6000
8000
10000
12000
14000
m/z
Figure S5 – HPLC-ESI MS analysis of ONT 5 prior to addition of RNase A, showing (a) TIC and UV
chromatogram, (b) negative ion ESI mass spectrum and (c) MaxEnt deconvoluted spectrum.
Electronic Supplementary Material (ESI) for Analyst
This journal is © The Royal Society of Chemistry 2012
(a)
TIC
UV chromatogram
[M – 4H]4-
Intens.
(b)
Res2623_t=0_1_RA2_01_8692.d: -MS, 8.9-9.0min #(530-536), Background Subtracted
1540.0940
600
[M – 3H]32053.4541
400
200
[2HFIP - H]334.9393
608.0536
1130.0628
2525.5460
1854.7771
3160.51763310.9617
0
500
1000
1500
2000
2500
Intens.
(c)
3000
3500 m/z
Res2623_t=0_1_RA2_01_8692.d: -MS, 8.9-9.0min, Background Subtracted, Deconvoluted (MaxEnt)
Intens.
Res2623_t=0_1_RA2_01_8692.d: -MS, 8.9-9.0min, Background Subtracted, Deconvoluted (MaxEnt)
6163.4182
1500
6163.4182
1500
6165.4037
6162.4128
1250
1250
6166.4173
1000
6161.3859
1000
750
6167.4376
6160.4066
750
500
6168.4251
6159.3930
500
250
6169.4301
6158.4431
6170.3706
0
250
6156
6158
6160
6162
6164
6166
6168
11134.8110
6170
6172
6174
m/z
12330.7985
0
2000
4000
6000
8000
10000
12000
14000
m/z
Figure S6 – HPLC-ESI MS analysis of ONT 6 prior to addition of RNase A, showing (a) TIC and UV
chromatogram, (b) negative ion ESI mass spectrum and (c) MaxEnt deconvoluted spectrum.
Electronic Supplementary Material (ESI) for Analyst
This journal is © The Royal Society of Chemistry 2012
(a)
Intens.
[%]
100
[2HFIP – H]-
Res2622_t=5min_IS_2_RA3_01_8551.d: -MS, 1.3-1.4min #(80-86)
334.9804
80
[dGdA – H]579.1193
60
40
20
[2dGdA – H]1159.2478
0
500
(b)
1500
2000
2500
[dTCp – H]-
Intens.
[%]
100
80
1000
3000
3500 m/z
Res2622_t=5min_IS_2_RA3_01_8551.d: -MS, 5.4-5.6min #(319-331)
626.0628
[2HFIP – H]334.9812
60
40
20
[2dTCp – H]1253.1334
0
500
(c)
Intens.
[%]
100
1000
1500
2000
2500
[2HFIP – H]-
3000
3500 m/z
Res2622_t=5min_IS_2_RA3_01_8551.d: -MS, 6.7-6.8min #(401-406)
334.9810
80
[dGdAUBrp – H]1045.0014
60
40
[dGdAUBrp – 2H]2521.9966
20
0
500
1000
1500
2000
2500
3000
3500 m/z
Figure S7 – Negative ion ESI mass spectra of observed digestion products of ONT 5 sampled at 5 minutes
eluting at tR (a) 1.4 min., (b) 5.4 min. and (c) 6.7 min. UBr = 5-Bromouridine, HFIP =
hexafluoroisopropanol.
Electronic Supplementary Material (ESI) for Analyst
This journal is © The Royal Society of Chemistry 2012
(a)
Intens.
Intens.
Res2099_t=5min_1_RE3_01_5425.d: -MS, 1.4-1.5min #(81-87)
120
1.8
729.9802
731.9725
100
80
1.0
60
40
730.9874
732.9773
20
733.9826
0
727
728
729
730
731
732
733
734
735
736
m/z
1.4
Intens.
Intens.
x1044
Res2099_t=24h_1_RD2_01_5468.d: -MS, 1.3-1.4min #(80-84)
731.9703
729.9704
(b)
1.0
0.8
0.6
0.4
730.9744
732.9702
0.2
1.8
1.0
1.4
733.9720
0.0
727
728
729
730
731
732
733
734
735
736
m/z
Figure S 8 – HPLC-ESI MS analysis of ONT 1 sampled at (a) 5 minutes and (b) 24 hours showing UV
chromatograms and negative ion ESI mass spectra of the brominated overdigestion product AUBrp. UBr =
5-bromouridine and p = 3ʹ-phosphate. N.B. mobile phase gradient normally used for analysis of intact
RNA was used for this analysis.
Electronic Supplementary Material (ESI) for Analyst
This journal is © The Royal Society of Chemistry 2012
% BPC Peak Area of
Expected/Overdigested
(arbitrary units)
100.0
10.0
ONT 1, 0.1
Kunitz units
RNase A
1.0
ONT 1, 0.01
Kunitz Units
of RNase A
0.1
0.0
1
10
100
1000
Reaction Time (min)
10000
Figure S 9 – A graph showing the peak area of AUBrp compared to AAUBrp for ONT 1 under different
assay conditions. Error bars show ± 1 standard deviation from triplicate results. d = deoxynucleotide, p =
3ʹ-phosphate
(a)
(b)
Figure S10 – RNase A expected cleavage sites (green solid line) and unexpected, non-specific cleavage
sites (red dashed line) for (a) ONT 1 and (b) the n-3 failure sequence of ONT 4, highlighting the proposed
origins of the unexpected dinucleotide products. UBr = 5-bromouridine