Supplementary Information for:

Online Reaction based Single-step Capillary Electrophoresis-Systematic Evolution of

Ligands by Exponential Enrichment for ssDNA Aptamers Selection

Chao Zhu1, Linsen Li1, Ge Yang1, Senbiao Fang2,3, Manjiao Liu2,3, Murtaza Ghulam1, Chenxu Hao1,

Yubao Chen2,3, Feng Qu1*

1 Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute

of Technology, 5 South Zhongguancun Street, Beijing 100081, China

2 Beijing Computing Center, Beijing Academy of Science and Technology

3 The Key Laboratory of Beijing Cloud Computing Technology and Application

(*)Email: qufengqu@bit.edu.cn

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The amino acid sequences of H-Thr proteinMAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRANTFLEEVRKGNLERECVEETCS

YEEAFEALESSTATDVFWAKYTACETARTPRDKLAACLEGNCAEGLGTNYRGHVNITRSGIECQLW

RSRYPHKPEINSTTHPGADLQENFCRNPDSSTTGPWCYTTDPTVRRQECSIPVCGQDQVTVAMTPRS

EGSSVNLSPPLEQCVPDRGQQYQGRLAVTTHGLPCLAWASAQAKALSKHQDFNSAVQLVENFCRNP

DGDEEGVWCYVAGKPGDFGYCDLNYCEEAVEEETGDGLDEDSDRAIEGRTATSEYQTFFNPRTFGS

GEADCGLRPLFEKKSLEDKTERELLESYIDGRIVEGSDAEIGMSPWQVMLFRKSPQELLCGASLISDR

WVLTAAHCLLYPPWDKNFTENDLLVRIGKHSRTRYERNIEKISMLEKIYIHPRYNWRENLDRDIALM

KLKKPVAFSDYIHPVCLPDRETAASLLQAGYKGRVTGWGNLKETWTANVGKGQPSVLQVVNLPIV

ERPVCKDSTRIRITDNMFCAGYKPDEGKRGDACEGDSGGPFVMKSPFNNRWYQMGIVSWGEGCDR

DGKYGFYTHVFRLKKWIQKVIDQFGE

The determination formula of bound fraction.Referring to the literature [1, 2], the bound fraction was defined by fitting to the following

formula:

Bound fraction (% )=A−A0

A×100 %

where A is the peak area of the unbound ssDNA library in the absence of target, and A0 is the peak area of the unbound ssDNA library peaks in the presence of target.

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A BH-ThrssDNA library

Figure. S1 Electrophoretogram of ssDNA library (A) and H-Thr (B) by CE-UV.

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Figure S2 Calorimetric titration of H-Thr and Seq-1.The top is the raw titration data showing the heat resulting from each injection of Seq-1 into the H-Thr solution. The bottom shows the integrated heats after correcting for the heat of dilution.

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Figure S3 The specificity analysis of the four sequences to different protein. A: H-Thr; B: lactoferrin; C: bovine thrombin; D: IgG; E: lysozyme.

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To further stress the possibility of aptamers application, we performed the affinity probe CE assay in human serum sample. First, we investigated the influence of serum matrix on the performance of ssDNA. Human serum sample was diluted 50 times by ddH2O as the serum matrix (or control sample). Seq-1 was added in diluted serum as affinity probe, and its electropherogram was compared with that in ddH2O (Fig. S3A). Seq-1 peak in serum matrix decreased significantly, probably due to the nonspecific interaction between serum matrix component and Seq-1. Then, we prepared the solutions containing 500 nM, 250 nM, 100 nM, 50 nM, 25 nM of H-Thr in diluted serum matrix, and added Seq-1 in each of them. Fig. S3B showed the Seq-1 peak height gradually decreased with H-Thr concentration increase, and the peak hight reduction of Seq-1 was refered as its bound fractions. Meanwhile the visible complex peak appeared at about 5.5 min at 250 nM H-Thr. In the examined range of H-Thr (50–500 nM), a good linear correlation was obtained as y = 0.0005x + 0.4414 （ R2=0.9432 ） between H-Thr concentration and Seq-1’s bound fraction, which indicates the selected aptamer possessed the potential in real application.

Figure S4 Application of Seq-1 as an affinity probe in serum sample. A: the electrophoregrams of Seq-1 in ddH2O and in diluted serum matrix. B: the electrophoregrams of Seq-1 added in serum matrix spiked different H-Thr concentration and the linear relationship between H-Thr concentration and Seq-1’s bound fraction.

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The molecular docking simulations and binding sites between Seq-1, Seq-3, Apt-29, and H-ThrSeq-1

Figure S5-1. The molecular docking simulation of Seq-1 and H-Thr.

Table S1-1 Interactions sites of Seq-1 and H-Thr.Interaction Pair ofH-Thr and Seq-1

Distance (Å)Interaction Pair ofH-Thr and Seq-1

Distance (Å)

Y391@N…A36@N7 3.01 N394@ND2…T67@O2’ 2.97Y391@OH…G38@O2’ 2.95 Y434@OH…G66@N1 3.10Y391@OH…G38@O3’ 2.83 Y434@OH…G66@N2 3.21R393@NH1…G38@O5’ 3.24 K302@NZ…G62@O2’ 3.05R393@NH1…G39@OP 3.20 K302@NZ…G61@O4 3.11R393@O…T67@O2’ 2.91 K302@NZ…G61@O3 3.10

R390@ NH1…C68@O2 2.91 K301@NZ…T60@O3 3.05R390@ NH1…C68@O3 3.01 K301@O…T60@O4 3.11R390@ NH2…C68@O3 3.04 K301@O…G61@O4 3.10R393@NE…T67@OP1 2.90 F431@N…A47@N7 2.98N394@ND2…T67@O3’ 2.98 S288@ OG…A47@ O2’ 2.73

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Table S1-2. Interactions sites of Seq-2 and H-Thr

Interaction pair of H-Thr and Seq-2 Distance (Å)

R320@ NH1…T51@ O3 3.01R320@ NH2…C52@ OP 2.95S325@ OH…T56@ O4 2.83N394@ NH2…G65@ N3 3.20R393@ NH1…G66@ O2 2.91R393@ NH1…G69@ N3 2.91Y434@ OH…T63@ N3 3.03Y434@ OH…T74@ O4 3.24

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Seq-3

Figure S5-2. The molecular docking simulation of Seq-3 and H-Thr.Table S1-3 Interactions sites of Seq-3 and H-Thr.

Interaction Pair ofH-Thr and Seq-3

Distance (Å)Interaction Pair ofH-Thr and Seq-3

Distance (Å)

E305@N…G34@O3’ 3.01 K302@NZ…G60@ N2 2.97N394@ND2…T64@OP1 3.01 R393@NH…G65@ OP2 2.97

E305@N…A35@OP1 2.95 K302@NZ…G61@ N3 3.10L298@O…C33@ O2’ 2.83 D433@OD2…G62@O2’ 3.21K302@NZ…C33@ O2 3.24 Y434@OH…G62@O2’ 3.05Y316@OH…C51@OP1 3.20 R320@NH1…T52@O4’ 3.11S387@OG…G8@N2 3.01 D318@OD2…G43@N2 2.97

D326@OD2…C7@O2’ 2.95 R320@NH2…G43@N2 3.10R310@NH2…C51@OP2 2.83 R320@NH2…C51@O2 3.21Y316@OH…C51@O3’ 3.24 R320@NH2…T52@O4’ 3.05Y316@OH…C51@OP1 3.20 R320@NH1…T52@O4’ 3.11S387@OG…G8@N2 3.01 D318@OD2…G43@N2 2.97

R393@NH…G65@ OP1 2.95

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Apt-29

Figure S5-3. The molecular docking simulation of Apt-29 and H-Thr.Table S1-4 Interactions sites of Apt-29 and H-Thr.

Interaction Pair ofH-Thr and Apt-29

Distance (Å)Interaction Pair ofH-Thr and Apt-29

Distance (Å)

Q476@N…G22@N2 3.01 R393@NH2…C15@ O2’ 3.21Q476@O…G20@N2 2.95 R393@NZ…C15@ O2’ 3.01R388@NH1…T19@N3 2.83 Q476@N…G22@ N3 2.83R388@NH2…T18@O4 3.24 Q476@O…G21@ N2 3.02T389@OG1…T18@O4 3.20 R388@NH1…T20@ N3 3.05T389@OG1…T19@O4 2.90 R388@NH2…T19@O4 3.11R390@NH1…G17@N3 2.98 R388@NH1…T19@O4 3.10R390@NH1…G17@O2’ 2.97 T389@OG1…T20@ O4 2.98R393@NH1…A16@ O4’ 3.10 T389@ OG1…T19@O4 2.73

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Figure S6 The ssCE electrophoregrams of DNA library with six proteins and their complex analysis. A: neuron-specific enolase; B: human lactoferrin; C: bovine lactoferrin; D: human liver ferritin; E: programmed death 1; F: programmed death ligand 1.

[1] C.M. Rose, M.J. Hayes, G.R. Stettler, S.F. Hickey, T.M. Axelrod, N.P. Giustini, S.W. Suljak, Capillary electrophoretic development of aptamers for a glycosylated VEGF peptide fragment, Analyst, 135 (2010) 2945-2951.[2] S. Mendonsa, M. Bowser, In vitro selection of high-affinity DNA ligands for human IgE using capillary electrophoresis, Anal. Chem., 76 (2004) 5387-5392.

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