Supplementary Information

Single-probe multistate detection of DNA via aggregation-induced emission on

a graphene oxide platform

Abhishek Tyagi1, Kin Leung Chu1, Irfan Haider Abidi1, Aldrine Abenoja Cagang1, Qicheng

Zhang1, Nelson L. C. Leung2, Engui Zhao2, Ben Zhong Tang2, Zhengtang Luo1, *

1Department of Chemical and Biomolecular Engineering and the Hong Kong Branch of Chinese

National Engineering Research Center for Tissue Restoration & Reconstruction, The Hong Kong

University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong.2Department of Chemistry and Division of Biomedical Engineering, The Hong Kong University

of Science & Technology, Clear Water Bay, Kowloon, Hong Kong.

*Correspondence to: keztluo@ust.hk

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Fig. S1. Gel electrophoresis and AFM analysis. (a) The gel electrophoresis conducted to confirm

DNA hybridization in 2% agarose at 100V for 30 minutes. The marker used was 25 bp to 500 bp

in “Marker” lane; hybridized dsDNA prepared by annealing both comDNA strands in a

thermocycler in lane 1; AIE and hybridized dsDNA in lane 2; AIE, hybridized dsDNA and GO

in lane 3; AIE, ssDNA, GO and comDNA prepared at room temperature in lane 4; AIE, ssDNA

and comDNA prepared at room temperature in lane 5; and AIE-ssDNA-ssDNA prepared at room

temperature, showing a thick band around the 42bp region, in lane 6. (b) The AFM

characterization of a GO monolayer approximately ~1 nm in thickness.

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Fig. S2. A kinetic study of an AIE-ssDNA complex after the addition of GO and comDNA. (a)

The kinetics for the PL quenching of AIE-ssDNA with graphene oxide, with the initial

concentration marked by square dots. (b) The kinetics for comDNA (100 nM) with an AIE-

ssDNA-GO complex, with the initial concentration marked by square dots.

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Fig. S3. A hybrid simulation system for DNA adsorption on a graphene nanosheet (GNS)

surface, with a top-view representation of three alignments of ssDNA and dsDNA above the

GNS surface. (a) and (d) Simulated snapshots of the initial structures of ssDNA and dsDNA; (b)

and (e) their structure after 50 ns, and (c) and (f) after 100 ns. The water and ions are omitted for

clarity. The adsorption of ssDNA on the GNS surface is higher than that of dsDNA.

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Fig. S4. A hybrid system with a DNA-GNS in a water box, with a front-view representation of

three alignments of ssDNA and dsDNA above the GNS surface. Simulated snapshots of (a) and

(d) the initial hybrid structures of ssDNA and dsDNA, (b) and (e) their structures after 50 ns, and

(c) and (f) after 100 ns. The nucleic acid backbone is in red and nucleobases in dark blue. The

water box is depicted in cyan with Na+ and Cl- ions shown in light blue and yellow respectively.

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Fig. S5. A root-mean-square fluctuation (RMSF) calculation for DNA adsorbing on a GNS. The

average per-base RMSF is represented as a function of the base number of the ssDNA (5’ to 3’)

shown by the red lines, while the square dots represent the per-base position along the X-axis.

The RMSF values for the first strand (5’ to 3’) and second strand (5’ to 3’) are shown by the

green and blue lines respectively. The ssDNA experiences larger fluctuations in the bases

compared to dsDNA, indicating a greater contribution to the adsorption across the GNS.

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MMGBSA Binding Energy Calculations

Fig. S6a and Fig. S6b shows the overall binding free energy was derived from nonpolar

contributions such as van der Waals (Evdw) and Gsol_np. It was also observed that the total enthalpy

of individual base pairs is precisely close to the van der Waals contribution, suggesting that the

nucleotide interactions with the GNS are primarily through van der Waals forces. The binding

energy decomposition results reveal that the extra five-membered aromatic rings in the A and G

nucleobases contribute considerably to and account for the higher binding energy of ssDNA

compared to dsDNA (Table S1). The stronger binding between these aromatic species is likely

due to the π-π interactions with GNS.[1]

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Fig. S6. The MM/GBSA decomposition analysis for DNA on a GNS. (a) and (b) The

decomposition of van der Waals, electrostatic, polar/non-polar solvation contributions and the

total contribution of enthalpy for bases of ssDNA and dsDNA are shown by the green, black,

blue, pink and red lines respectively. The decomposition of energy contributes Evdw and ΔGsol_np

and provide relevant information about the significance of van der Waals forces in the adsorption

process.

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MM/GBSA energy contribution to the binding energy of DNA adsorption on a GNS (kcal mol-1).

S No Nucleic ∆Htot T∆S ∆G

1. ssDNA -272.17 -13.92 -258.25

2. dsDNA -125.86 13.40 -139.26

Table S1. MM/GBSA free binding energy calculations. The components of binding free energy

for dsDNA and ssDNA that are adsorbed on GNS (kcal mol-1). The data for ssDNA and dsDNA

used in these calculations were derived from 50-100 ns simulations; the thermodynamics

properties used in the simulations were estimated by quasi-harmonic approximation (QHA).

Fig. S7. The thermodynamic energy contribution of dsDNA. The observations for free binding

energy, enthalpy and entropy are shown by the green, black, and red lines respectively. The

analysis was performed for a duration of 50 ns, with trajectories selected from the first 250 ns of

simulations to analyze thermodynamics properties using the QHA method. The binding free

energy increases over time, demonstrating that the binding affinity increases over the course of

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the experiment as the adsorption of dsDNA on the GNS increases. The total enthalpy

contribution is also likely to be beneficial for the adsorption process.

Fig. S8. Snapshots of dsDNA on a GNS. The snapshots depict different orientations of dsDNA.

(a) After 14 ns, the first opening is initiated between guanine and cysteine. (b) After 42 ns,

another opening occurs and the dsDNA starts leaning towards the GNS. We observed dsDNA

moves closer to the GNS during the simulation, although it retains its helical shape and is

visualized after (c) 150 ns, (d) 200 ns, and (e) 250 ns.

The π-π stacking interactions between DNA and GNS:

We calculated the contribution of the aromatic rings to the interactions between the DNA and

GNS, and proposed a method to calculate π-π interactions by identifying three different

orientations of π-π interactions. The aromatic rings parallel to GNS are considered stable,

vertical and metastable, and the rest are considered unstable as shown in Fig. S9. The main

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contributing factors to the binding free energy are π-π stacking interactions between aromatic

rings in nucleotides and GNS, either face-to-face or T-shaped.[2] The symmetrical structural

orientation of DNA allows a wide distribution of dispersive interfacial contacts due to small

intermolecular distances between ligand and receptor. Past studies have shown [3] that the π-π

stacking distance is larger than the van der Waals radius, and π-π stacking is extensive and

contributes to the interactions among biomolecules and nanomaterials, carbon nanotubes and

graphene.[4].[5]

Fig. S9. A schematic representation of the distribution of π-π interactions. The interactions are

calculated for stable, metastable, and unstable orientations. Aromatic rings are categorized as

stable under 20˚, metastable at 10˚, and unstable at 60˚.

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Table S2. Distribution of pi-pi interaction for stable, metastable, and unstable nucleotides on the

surface of GNS.

a. The π-π interaction table for dsDNA and ssDNA for stable, metastable, and unstable

nucleotides.

Nucleic acid Stable Metastable Unstable

dsDNA 4 10 28

ssDNA 15 1 5

b. The π-π interaction chart of dsDNA for the first and second strands, as compared with

ssDNA.

Bases

dsDNA ssDNA

First strand Second strand

Stable Metastable Unstable Stable Metastable Unstable Stable Metastable Unstable

A 0 3 3 0 1 2 5 0 1

T 0 0 3 0 2 4 2 0 1

G 0 0 0 2 5 5 8 1 3

C 2 1 9 0 0 0 0 0 0

References

[1] J. Liu, Z. Liu, X. Luo, X. Zong, J. Liu, RAFT Controlled Synthesis of Biodegradable Polymer Brushes on Graphene for DNA Binding and Release, Macromolecular Chemistry and Physics 214(20) (2013) 2266-2275.[2] C.D.M. Churchill, S.D. Wetmore, Noncovalent Interactions Involving Histidine: The Effect of Charge on π−π Stacking and T-Shaped Interactions with the DNA Nucleobases, The Journal of Physical Chemistry B 113(49) (2009) 16046-16058.

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[3] K. Shibasaki, A. Fujii, N. Mikami, S. Tsuzuki, Magnitude of the CH/π Interaction in the Gas Phase:  Experimental and Theoretical Determination of the Accurate Interaction Energy in Benzene-methane, The Journal of Physical Chemistry A 110(13) (2006) 4397-4404.[4] X. Zhao, Self-Assembly of DNA Segments on Graphene and Carbon Nanotube Arrays in Aqueous Solution: A Molecular Simulation Study, The Journal of Physical Chemistry C 115(14) (2011) 6181-6189.[5] M. Chehel Amirani, T. Tang, Binding of nucleobases with graphene and carbon nanotube: a review of computational studies, Journal of Biomolecular Structure and Dynamics 33(7) (2014) 1567-1597.

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