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1 Supprting Inrmatin Ultraspeciic Multiplexed Detectin Lw-Abundance Single-Nucletide Variants by Cmbining a Masking Tactic with Flurescent Nanparticle Cunting Xiajing Pei, Tiancheng Lai, Guangyu Ta, Hu Hng, Feng Liu, and Na Li * Beijing Natinal Labratry r Mlecular Sciences (BNLMS), Key Labratry Birganic Chemistry and Mlecular Engineering Ministry Educatin, Institute Analytical Chemistry, Cllege Chemistry and Mlecular Engineering, Peking University, Beijing, 87, P. R. China Crrespndence shuld be addressed t Dr. Na Li. Tel: ; lina@pku.edu.cn. S-

2 Table cntents. Experimental sectin... S-3. Apparatus... S-3.2 Cnjugatin amin-dna n FNPs... S-3.3 Mdiicatin MBs with bitinylated DNA... S-3.4 Flurescence micrscpic imaging FNPs... S-4.5 Clr image prcessing... S-4.6 The sequences used in this study... S-6 2 Supplemental results... S-8 2. Optimizatin results the assay... S The Thermdynamic Penalties r Discriminatin Single-nucletide Variants... S The lw-abundance results artiicial SNVs synthetic targets.... S Flurescence micrscpic images r SNV detectin... S Cmparisn with appraches using cmpetitive r masking systems reprted since S-8 3 Reerences... S-2 S-2

3 . Experimental sectin. Apparatus Flurescence spectra were recrded with a Hitachi F-45 spectrlurmeter (Hitachi, Japan). The lurescence images were captured using an Olympus IX73 inverted lurescence micrscpe with an Olympus DP8 true clr CCD and Olympus cellsense stware (Olympus, Japan). PCR was carried ut with S Thermal Cyclers rm Bi-Rad Labratries, Inc. (Hercules, USA). The extracted genmic samples were quantiied by the NanDrp 2 Spectrphtmeter rm Therm Fisher Scientiic (Massachusetts, USA). The temperature were cntrlled by Mixer-K cnstant temperature mixer scillating cnstant temperature metal bath rm Shanghai Ba Jiu Industrial C., Ltd. (Shang Hai, China)..2 Cnjugatin amin-dna n FNPs This prcedure has been published in Analytical Chemistry, 28, 9, Herein, we prvide again r the cnvenience readers. The preparatin the capture DNA cnjugated lurescent nanparticle (DNA-FNP) was carried ut by -Ethyl-3-[3-(dimethylamin)prpyl] carbdiimide hydrchlride (EDC) cupling chemistry. Speciically, μl mg/ml FNP suspensin was washed three times with μl 5 mm MES buer (ph 5.). Then, μl 5 mg/ml reshly prepared EDC slutin was added and mixed, an aliqut (5 μl) μm amin-dna was added and mixed by vrtexing. The slutin with ttal vlume μl was incubated r 3 min at rm temperature with slw agitatin, and μl 5 mg/ml reshly prepared EDC in ice-cld MES buer was added and mixed well by vrtexing. The mixture was stirred vernight and llwed centriugatin. Aterward, FNPs were washed three times with μl mm PBS t remve excess amin-dna, re-suspended in ml PBS, and stred at 4 C r urther use..3 Mdiicatin MBs with bitinylated DNA This prcedure has been published in Analytical Chemistry, 28, 9, Herein, we prvide again r the cnvenience readers. The capture DNA unctinalized magnetic beads (DNA-MBs) were prepared via streptavidin-bitin S-3

4 cnjugatin. Speciically, 2 μl Dynabeads TM magnetic beads were transerred t ml PBS and washed three times with PBS buer. Then, 25 μl μm bitinylated DNA (bitin-dna) was added at rm temperature by gentle rtatin. Ater 5 min, the resulting DNA-MBs were washed ive times with PBS cntaining.% Tritn X t remve the excess bitin-dna, and re-suspended in ml PBS cntaining.% Tritn X and stred at 4 C r urther use..4 Flurescence micrscpic imaging FNPs This prcedure has been published in Analytical Chemistry, 28, 9, Herein, we prvide again r the cnvenience readers. All lurescence micrscpic images were acquired using the pixel shiting mde the Olympus CellSense stware with a reslutin pixels. The bjective lens was used r the DF determinatin and assay lw-abundance single SNV target, and the 2 bjective lens was used r multiplexed SNV detectin. The number nanparticles was acquired thrugh clr recgnitin the dts in the images based n the clr characteristics. Images were acquired with Olympus IX73 under the llwing imaging cnditin: r green nanparticles ( ex/em: 48/52 nm), exciter 472 nm, emitter 52 nm and dichric 495 nm; r the blue nanparticles ( ex/em: 36/45 nm), wide band UV excitatin, exciter ilter with excitatin band 34 nm-39 nm, dichric beams litter with dichric mirrr at 4 nm, barrier emissin ilter 42 nm; r red nanparticles ( ex/em: 525/6 nm), exciter 562 nm, emitter 64 nm, dichric mirrr at 593 nm. The imaging cnditin was kept cnsistent during the experimental prcess each reactin system unless indicated, and each cunting result was the average six images..5 Clr image prcessing This prcedure has been published in Analytical Chemistry, 28, 9, Herein, we prvide again r the cnvenience readers. The 3 clrs FNPs were recgnized and enumerated autmatically using the stware develped in C# prgramming language based n ur previus wrk. The general idea the autmatic cunting was t recgnize the FNPs reerred sequentially by shape and clr characteristics. Speciically, at irst, Gaussian blur was applied t smth the image, and, sharpen edges was used t enhance the edge the FNPs. The high-pass iltering step described in ur previus wrks was then applied t eliminate -cus FNPs signals interering with the S-4

5 recgnitin. The shape-based segmentatin was then used t divide the image int subimages with each cntaining a single bject t be identiied. The shape (area and axial rati) and clr judgments were sequentially applied t each subimage t identiy FNPs, and last the number FNPs was cunted. The three clrs FNPs were und t be best separated in the CIELCh clr space, with C* (chrma) and h (hue angle) cmpnents. T simpliy the identiicatin, an average clr was generated r each subimage. The average clr had clr dierences (calculated with CIEDE2 algrithm) less than a speciied threshld with mre than a hal the pixels the bject in a subimage. Average clrs btained rm the images each single type FNPs were cnsidered as reerence clrs. Linear bundaries the reerence clr in C* h chart were then calculated and used as the criteria r clr judgments. S-5

6 .6 The sequences used in this study Table S Sequences lignucletides used in study. Name Sequences Mdiicatin FNP-4C A2 ACG CCA CCG GCT 5'NH2 C2 FNP-4G A2 ACG CCA CCC GCT 5'NH2 C2 FNP-4D A2 ACG CCA CC GCT 5'NH2 C2 FNP-4IT A2 ACG CCA CCAA GCT 5'NH2 C2 FNP-6IA A2 ACG CCA CTCA GCT 5'NH2 C2 FNP-6IT A2 ACG CCA CACA GCT 5'NH2 C2 FNP-6IC A2 ACG CCA CGCA GCT 5'NH2 C2 FNP-6IG A2 ACG CCA CCCA GCT 5'NH2 C2 FNP-6D A2 ACG CCA CA GCT 5'NH2 C2 FNP-6A A2 ACGCCATCAGCT 5'NH2 C2 FNP-6T A2 ACGCCAACAGCT 5'NH2 C2 FNP-6C A2 ACGCCAGCAGCT 5'NH2 C2 FNP-8T A2 ACG CAA CCA GCT 5'NH2 C2 FNP-9A A2 ACG TCA CCA GCT 5'NH2 C2 FNP-9T A2 ACG ACA CCA GCT 5'NH2 C2 FNP-9D A2 ACG CA CCA GCT 5'NH2 C2 FNP-9IA A2 ACG CTCA CCA GCT 5'NH2 C2 FNP-RNA 6A FNP-RNA 6U MB A2 ACG CCA TCA GCT A2 ACG CCA ACA GCT CCA ACT ACC ACA AGT A 5'NH2 C2 5'NH2 C2 3'-Bitin-TEG FAM-6A A2 ACGCCATCAGCT 5'FAM SNV-4C ACT TGT GGT AGT TGG AGC CGG TGG CGT SNV-4G ACT TGT GGT AGT TGG AGC GGG TGG CGT SNV-4D ACT TGT GGT AGT TGG AGC GG TGG CGT SNV-4IT ACT TGT GGT AGT TGG AGC TTGG TGG CGT SNV-6IA ACT TGT GGT AGT TGG AGC TGAG TGG CGT SNV-6IT ACT TGT GGT AGT TGG AGC TGTG TGG CGT SNV-6IC ACT TGT GGT AGT TGG AGC TGCG TGG CGT SNV-6IG ACT TGT GGT AGT TGG AGC TGGG TGG CGT SNV-6D ACT TGT GGT AGT TGG AGC TG TGG CGT SNV-6A ACT TGT GGT AGT TGG AGC TGA TGG CGT SNV-6C ACT TGT GGT AGT TGG AGC TGC TGG CGT SNV-6T ACT TGT GGT AGT TGG AGC TGT TGG CGT SNV-8T ACT TGT GGT AGT TGG AGC TGG TTG CGT SNV-9A ACT TGT GGT AGT TGG AGC TGG TGA CGT SNV-9T ACT TGT GGT AGT TGG AGC TGG TGT CGT SNV-9D ACT TGT GGT AGT TGG AGC TGG TG CGT SNV-9IA ACT TGT GGT AGT TGG AGC TGG TGAG CGT SNV-RNA-6A ACT TGT GGT AGT TGG AGC UGA UGG CGU SNV-RNA-6U ACT TGT GGT AGT TGG AGC UGU UGG CGU WT ACT TGT GGT AGT TGG AGC TGG TGG CGT MH TCT TAC GCC ACC AGC TAA GA S-6

7 Table S2 Sequences lignucletides used in PCR ampliicatin. Name Sequences WT GCC TGC TGA AAA TGA CTG AAT ATA AAC TTG TGG TAG TTG GAG CTG GTG GCG TAG GCA AGA GTG CCT TGA CGA TAC AGC TAA TTC AGA ATC ATT TTG TGG ACG AAT SNV GCC TGC TGA AAA TGA CTG AAT ATA AAC TTG TGG TAG TTG GAG CTG ATG GCG TAG GCA AGA GTG CCT TGA CGA TAC AGC TAA TTC AGA ATC ATT TTG TGG ACG AAT Frward primer GCC TGC TGA AAA TGA CTG Reverse primer 5 -PO 4-ATT CGT CCA CAA AAT GAT TCT G S-7

8 2 Supplemental results 2. Optimizatin results the assay A B C Discriminatin actr Discriminatin actr Discriminatin actr AT+CG 4AT+CG 5AT 5AT+CG 5AT+CG 7AT+CG MH stem structure MH-t-WT cncentratin rati 2-MB+5-FNP 2-MB+ 9-FNP 2-MB+2-FNP 7-MB+2-FNP 5-MB+2-FNP Length duplex in sandwich structure Figure S. The ptimizatin the MH and the sandwich design: stem length and sequence MH (A), and the MH-t-WT cncentratin rati (B), length the rmed duplex in the sandwich structure (C). S-8

9 2.2 The Thermdynamic Penalties r Discriminatin Single-nucletide Variants Fr discriminatin actr study, the test system cmpses the capture DNA-MB (C), the signaling DNA-FNP (S), the masking hairpin (MH), the single-nucletide variant (SNV) r the wild-type sequence (WT). The relevant reactins r discriminatin SNV are: where,, G respectively. G 2 G 3 C + SNV + S C-SNV-S () C + SNV + MH C-SNV-MH (2) C + WT + MH C-WT-MH (3) C + WT + S C-SNV-S (4) and are the reactin standard ree energies r reactins ()~(4), G 4 Fr measuring the signal by SNV r WT, initial cncentratins C, S and MH are the same in each reactin, respectively, and initial cncentratins SNV and WT equal. G GC-SNV-S GC GSNV GS G G G G G G G G G G G4 GC-WT-S GC GWT GS 2 C-SNV-MH C SNV MH 3 C-WT-MH C WT MH The values G and G 2 in the wrk are deined as thermdynamic penalties unintended reactins as cmpared t energies intended reactins. Fr Reactins () and (2), G G G G G G G 2 C-SNV-MH C-SNV-S MH S Fr Reactins (3) and (4), similarly, G G G G G G G C-WT-S C-WT-MH S MH G G G G G G G 2 C-WT-S C-SNV-S C-SNV-MH C-WT-MH Fr a given SNV, G cmes rm tw parts, the single-base mismatch by WT bund t S and the single-base mismatch by SNV bund t MH. Using NUPACK stware (nupack.rg) based n S-9

10 DNA energy parameters (SantaLucia, 998), the change standard ree energy in hybridizatin can be estimated (Table S3). S-

11 Table S3 Thermdynamic penalties r discriminatin DNA SNV by NUPACK predictin. Sites a G WT-S G SNV-S (kcal/ml) G SNV-MH G WT-MH (kcal/ml) G (kcal/ml) A T C G IA IT IC IG D A T C G IA IT IC IG D A T C G IA IT IC IG D WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT a : Mismatched sites n signaling sequence. S-

12 2 A susbtitutin T susbtitutin C susbtitutin G susbtitutin A Insertin T Insertin C Insertin G Insertin Deletin G (kcal/ml) Site n signaling sequence Figure S2. The result energy calculatin dierent mutated sites n the signaling sequence calculated by NUPACK. S-2

13 2.3 The lw-abundance results artiicial SNVs synthetic targets. A 4,T>C B C D 4,T>G 4,T Insertin 4,Deletin Cunts Cunts Cunts Cunts.5%.%.4% % % %.5%.%.4% % % % E F G H 6,A Insertin 6,G Insertin 6,C Insertin.5%.%.4% % % %.5%.%.4% 6,T Insertin % % % Cunts Cunts Cunts Cunts I.5%.%.4% 6,Deletin % % % J.5%.%.4% 9, A Insertin % % % K.5%.%.4% 9,Deletin % % %.%.4% % % % Cunts Cunts Cunts.%.4% % % %.5%.%.4% % % %.5%.%.4% % % % Figure S3. Cunts r artiicial SNVs synthetic targets at dierent abundances. S-3

14 2.4 Flurescence micrscpic images r SNV detectin G2A(c.35G>C) MT WT Blank mm mm mm G2V(c.35G>T) mm mm mm G2D(c.35G>A) mm mm mm G3C(c.37G>T) G3D(c.38G>A) mm mm mm G3V(c.38G>T) mm mm mm G2V(c.35G>U) mm mm mm G2D(c.35G>A) mm mm mm Figure S4. The lurescence micrscpic images FNPs r the DF value 6 mst requently ccurring KRAS mutatins and 2 pssible RNA SNVs r Figure. S-4

15 G2A(c.35G>C) % % %. %.5 % mm mm mm mm mm mm G2V(c.35G>T) % % %. % mm mm mm mm mm % % %. %.5 % G2D(c.35G>A) mm mm mm mm mm mm G3C(c.37G>T) % % %. %.5 % G3D(c.38G>A) % %.4 %. %.5 % mm mm mm mm mm mm G3V(c.38G>T) % % %. %.5 % mm mm mm mm mm mm G2V(c.35G>U) % %.4 %. % mm mm mm mm mm G2D(c.35G>A) % % %.4 %. % mm mm mm mm mm mm Figure S5. The lurescence micrscpic images FNPs r the detectin 6 mst requently ccurring KRAS SNVs and 2 pssible RNA SNVs at dierent abundances r Figure 2. S-5

16 % %.4 %. %.5 % % % %.4 %. % % %.4 %. %.5 % Figure S6. The lurescence micrscpic images FNPs r the multiplexed lw-abundance detectin KRAS mutatins (green FNP r KRAS 2D (c.35g>a), blue FNP r KRAS 2V (c.35g>t), red FNP r KRAS G2A (c.35g>c)) at dierent abundances r Figure 3A. % %.4 %.4 %. % % %.4 %. %.5 % % %.4 %. %.5 % Figure S7. The lurescence micrscpic images FNPs r the multiplex detectin KRAS mutatins (blue FNP r KRAS G2V (c.35g>t), green FNP r KRAS 3V (c.38g>t), red FNP r KRAS 3C (c.37g>t)) at dierent abundances r Figure 3B. % % %.4 %. % % mm mm mm mm mm mm % % % Figure S8. The lurescence micrscpic images FNPs r the multiplex detectin tw RNA mutatins (green FNP r KRAS 2D (c.35g>a), red FNP r KRAS 2V (c.35g>u)) at dierent abundances r Figure 3C. S-6

17 % %.5 %. %.5 % mm mm mm mm mm mm Figure S9. The lurescence micrscpic images FNPs r detectin KRAS G2D (c.35g>a) at dierent abundances llwing PCR n synthetic DNA samples r Figure 4B. % %.5 %. %.5 % mm mm mm mm mm mm Figure S. The lurescence micrscpic images FNPs r detectin KRAS G2D (c.35g>a) at dierent abundances llwing PCR n cell genmic DNA samples r Figure 4C. % %.4 %. %.5 % mm mm mm mm mm mm Figure S. The lurescence micrscpic images FNPs r detectin KRAS G2D (c.35g>a) at dierent abundances using urther generalized design by FNP cunting methd r Figure 5. S-7

18 2.5 Cmparisn with appraches using cmpetitive r masking systems reprted since 25. Table S4. Cmparisn between the prpsed methd with appraches using cmpetitive r masking systems reprted since 25. Methds Discriminatin Factr Abundance Sensitivity Enzyme Ampliicatin Reerence.5% r mst lines Hairpin masking with Range: SNVs, FNP cunting Median:545(9 SNVs).5% r cell N N This wrk Simulatin-guided DNA Range: prbe and sink design Median: 89 (44 SNVs) % r Cell line N N Sequestratin-assisted Range: % KRAS G2D mlecular beacn Median: 7 (2 SNVs) (c.35g>a) N N 2 Prtected DNA strand Range: displacement (SD) Median: 26.4 (2 SNVs) NA N N 3 Piezelectric plate.% r KRAS sensr, lcked nucleic NA G2V acid prbe N N 4 Energy driven cascade Range: 45 9 recgnitin Median: 7 (6 SNVs) NA N N 5 g/μl r BRAF Electrchemical PNA NA and KRAS in clamps assay cdnas N N 6 Single-Mlecule Cunting n ttal internal.% r KRAS <3 relectin lurescence c.34g>a N N 7 micrscpy (TIRF) DNA Clutch PNA clamps NA.% ctdna N N 8 LNA-integrated X-shaped DNA prbe Cmpetitin and catalytic ampliicatin Mdular prbes Abasic site mdiied lurescent prbe and lambda exnuclease 37 r A > G.% N Yes 9 23 r A>G and 5 r let7a rm let 7c & let 7e NA N Yes > r 56-nt-lng target rm human genmic NA N Yes DNA.5% r Range: JAK2V67F and Median: 499 (9 SNVs) JAK2V67F Yes Yes 2 SD and selective digestin Range: Median: 95.7 (5 SNVs).2% r KRAS cdn 2 35G>A Yes Yes 3 S-8

19 Methds Discriminatin Factr Abundance Sensitivity Enzyme Ampliicatin Reerence Tehld SD & endnuclease IV with Range:57 abslute discriminatin.5% r BRAF V6E (c.799t > Yes Yes 4 TIRF Median:49(8 SNVs) A) Dynamic sandwich assay and EXPAR 393 r A > G mutatin..5% mutant/wild type Yes Yes 5 Endnuclease IV based cmpetitive DNA prbe assay Range: 5 79 r G:X mismatches.3.5% r KRAS G2A, KRAS G2V and KRAS G2S Yes Yes 6 Lambda exnuclease and a chemically mdiied DNA substrate structure Range: Median: 3 (2 SNVs).5% r JAK2V67F Yes Yes 7 Nucleic acid sel-assembly circuitry and exnuclease III arund r EGFR-L86Q (c.2582 T>A) and NRAS-Q6K (c.8 C>A) % r EGFR-L86Q, NRAS-Q6K Yes Yes 8 sequence-selective and temperature-rbust ampliicatin NA.% in allele requency Yes Yes 9 S-9

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