Supporting Information

Transcription

1 Supporting Information n Ultrasensitive Light-up u 2+ Biosensor Using a New DNzyme leaving a Phosphorothioate Modified Substrate Po-Jung Jimmy Huang and Juewen Liu* Department of hemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario, anada, N2L 31. * liujw@uwaterloo.ca S 1

2 able S1. DN sequences related to in vitro selection in this work. he cleavage site ribo adenine is denoted by r, FM = carboxyfluorescein, isp18 is an 18 atom hexa ethyleneglycol spacer. he 5 of the Lib r* DN is phosphorylated (denoted by the p) for the ligation reaction. he asterisk (*) denotes for phosphorothioate modification. DN Name Sequence and modifications (from 5 to 3 ) Lib FM pn 50 FM Lib r* 5' Splint DN 5 P1 primer 5 P2 primer 5 P3 primer 5 FM P4 primer 5 isp18 Blocking DN1 Blocking DN2 able S2. he other DN sequences used in this study. BHQ denotes for Black Hole Quencher 1, and p denotes for phosphorylation. Most of these sequences are to test the activity of individual clones from the in vitro selection experiment. DN Name Sequence and modifications (from 5 to 3 ) PSu1 PSu3 PSu4 PSu5 PSu6 PSu7 PSu9 PSu10 PSu11 PSu13 PSu18 PSu20 PS Sub FM PO Sub r FM 17E FM Sub FM Q PSu10 BHQ S 2

3 able S3. Sequence alignment. Sequence alignment of the direct selection using the PS library and u 2+. he cleavage site adenine is marked in red (only marked in the first sequence, all the rest are aligned at this position). he percentage of each sequence in the final library is also shown. PSu2(5.17%) ---- PSu8(1.34%) ---- PSu9(1.08%) --- PSu24(0.35%) ---- PSu6(1.37%) --- PSu12(0.88%) - PSu23(0.37%) --- PSu11(0.94%) --- PSu5(1.69%) --- PSu18(0.46%) --- PSu4(1.89%) - PSu15(0.77%) --- PSu19(0.44%) --- PSu25(0.34%) -- PSu22(0.38%) -- PSu21(0.40%) -- PSu1(10.3%) --- PSu14(0.87%) --- PSu17(0.49%) --- PSu16(0.55%) --- PSu10(0.96%) --- PSu20(0.41%) PSu3(3.18%) --- PSu13(0.87%) --- PSu7(1.37%) ******************************************* *** * PSu2 PSu8 PSu9 PSu24 PSu6 PSu12 PSu23 PSu11 PSu5 PSu18 PSu4 PSu15 PSu19 PSu25 PSu22 PSu21 PSu1 PSu14 PSu17 PSu16 PSu10 PSu20 PSu3 PSu13 PSu Y R PSu2 PSu8 PSu9 PSu S 3

4 PSu6 PSu12 PSu23 PSu11 PSu5 PSu18 PSu4 PSu15 PSu19 PSu25 PSu22 PSu21 PSu1 PSu14 PSu17 PSu16 PSu10 PSu20 PSu3 PSu13 PSu Figure S1. Scheme of in vitro selection with negative selections. he blocking DNs are intended to eliminate the e13 sequences. mixture of these two blocking DNs were used and they differ only by one nucleotide (underlined). Steps 1, 2 and 3 are a completely blocked selection cycle. he sequences that can cleave in the presence of u 2+ are amplified by PR. From round 8, negative selections were carried out with a metal mixture (Pb 2+, d 2+, and Zn 2+ ). In this case, the uncleaved oligonucleotides were collected (steps 4, 5, 6) and then reacted with u 2+ for the positive selection (steps 2, 3). he same blocking and negative selection method was previously used to isolate a DNzyme that works in the presence of d 2+. S1 S 4

5 PSu1 PSu3 PSu4 PSu5 PSu6 PSu7 PSu9 PSu10 PSu11 PSu13 PSu18 PSu20 Figure S2. he secondary structures of all the tested DNzymes after truncation into the trans cleaving form. hey all share the same substrate sequence containing a PS modification (denoted by the asterisk). he full sequence of each DNzyme is in able S2, and here only the structures of the enzyme loop regions are shown. leavage (%) uM u 10uM u Figure S3. el based activity assay of the trans cleaving DNzymes with the PS modified and FM labeled substrate in the presence of u 2+ (1 and 10 M) for 1 h. S 5

6 leavage (%) PSu3 1uM PSu6 1uM PSu9 1uM PSu10 1uM PSu18 1uM 0 Ref u d Pb Hg Figure S4. el based activity assay of the u 2+ dependent DNzymes in the presence of various thiophilic metal ions (1 μm each) for 10 min. Figure S5. gel image of the PSu10 DNzyme complex reacting with 1 M of u + or u 2+ in the presence of various concentrations of ascorbate for 20 min. S 6

7 24 leavage (%) t (min) Figure S6. Kinetics of S p substrate cleavage by PSu10. he data were fit to two rates. he initial faster rate (0.095 min 1 ) is due to the R p impurity in the sample. he slow phase ( min 1 ) is due to the S p u2+ Pb2+ Blank F (a.u.) ime (min) Figure S7. he sensor response at ph 7.5 to u 2+ and Pb 2+. S 7

8 Figure S8. Sensing u 2+ in Lake Ontario with 50 mm MES ph 6. he final lake water was 50% of the total volume. () Sensor response kinetics to various concentrations of u 2+ that was spiked into the Lake Ontario sample. (B) he slope of sensor signal (from 10.3 to 20 min) increase as a function of u 2+ concentration. Inset: the linear response at low u 2+ concentration. able S4. Representative literature examples of u 2+ optical sensors and their detection limits. ype of Sensor Detection method u 2+ Detection Limit Reference (nm) oumarin based small molecule probe Fluorescence / UV vis 500 J. m. hem. Soc. 2009, 131, Org. Lett. 2008, 10, Rhodamine based small molecule probe Fluorescence ~1000 Org. Lett. 2006, 8, Org. Lett. 2007, 9, hem. Eur. J. 2008, 14, nal. hem. 2009, 81, Fluorescence 7900 Org. Lett. 2006, 8, yclam based small molecule probe Spiropyran derivative UV vis 150 nal. hem. 2005,77, ,6 dihydroy 2 tomic absorption 45 J. Hazard Mater. 2007, mercaptopyrimidine spectrometry 142, (DHMP) DN cleaving DNzyme biosensor Peptide coated ds quantum dots Fluorescence 35 J. m. hem. Soc. 2007, 129, photoluminescence ~500 hem. ommun. 2003, 21, S 8

9 Dansylated peptide Fluorescence ~5000 hem. ommun. 2002, 20, Polyamine functionalized carbon quantum dots Fluorescence 6 nal. hem. 2012, 84, dse ZnS quantum dots Luminescence 10 Spectrochimica cta Part 2004, 60, DN functionalized gold nanoparticles UV vis 290; 5000; 20,000 Nanotechnology 2010, 21, hem. ommun. 2007, 46, Small 2010, 6, Silver nanocluster Fluorescence 8 J. Mater. hem. 2008, 18, mercaptopropionic acid (MP) & DN u/g nanoclusters Fluoresceindipicolylamine (DP) conjugate lkyne unps/zide unps lutathione (SH) functionalized unps Papain functionalized unps Perylene bisimide chromophore (PB) unps Polyethyleneimine functionalized silica nanoparticles DNzyme for PS RN cleavage Fluorescence 2.7 nal. hem. 2010, 82, Fluorescence 420 hem. ommun. 2009, 47, UV vis 20,000 ngew. hem. Int. Ed. 2008, 47, Fluorescence 3.6 hem. ommun. 2009, 13, UV vis 200 Biosens. Bioelectron. 2011, 26, Fluorescence 1000 dv. Mater. 2005, 17, Fluorescence 10 nal. hem. 2011, 83, Fluorescence 1.6 his work. dditional References (S1) Huang, P. J. J.; Liu, J. Nucleic cids Res. 2015, 43, S 9