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Single-Molecule Analysis of Human Telomere Sequence Interactions with G-quadruplex Ligand Ling Zhang, Kaixiang Zhang, Duo Dong, Yang Liu, Jinghong Li, and Sana Rauf Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00555 • Publication Date (Web): 25 Mar 2016 Downloaded from http://pubs.acs.org on March 26, 2016

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Analytical Chemistry

Single-Molecule Analysis of Human Telomere Sequence Interactions with G-quadruplex Ligand Ling Zhang, Kaixiang Zhang, Sana Rauf, Duo Dong, Yang Liu and Jinghong Li* Department of Chemistry, Beijing Key Laboratory for Analytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China.

*to whom corresponding should be addressed. Phone: 86-10-62795290; Fax: 86-10-62771149 Email: [email protected] 1

ACS Paragon Plus Environment


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Abstract Ligands that selectively promote the formation of G-quadruplexes in human telomeres have great potential for cancer treatment by inhibiting the telomere extension by telomerase. Thus, understanding the interactions of the G-quadruplex ligands with the telomere sequence at single-molecule level is of significant importance. Here, human telomere sequence interactions with a small molecule ligand pyridostatin (PDS) were analyzed via α-hemolysin protein nanopore, and a nanopore thermodynamic analytical method was proposed. The prolonged unraveling time of the telomeric DNA G-quadruplex after PDS binding demonstrated the potent stabilization effect of ligand on G-quadruplex structure. The signature two-level electronic blocks generated by K+-PDS-G-quadruplex complexes suggested a two-state unraveling process, including the dissociation of the inter-quartet cation and the unraveling of the cation-free ligand-bound G-quadruplex. The translocation studies and the analysis of free-energy changes demonstrated a ligand binding mode that PDS molecule and K+ were simultaneously bound to one G-quadruplex structure with the coordinated effect on G-quadruplex stabilization. The single-molecular nanopore platform permits the efficient and accurate determination of ligand affinity constants without the requirement for labeling, amplification or ligand/receptor titration, which provides a general analytical tool for effectively monitoring and quantifying the G-quadruplex/ligand interactions, possessing important implications for the design and screen of anti-cancer drugs.

Keywords Human Telomere sequence, Nanopore, single-molecular analysis, G-quadruplex ligand, Pyridostatin, thermodynamic analysis, label free

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Introduction A telomere is a special region of nucleotide sequences at each end of linear chromosomes in eukaryocytes, which plays a significant role in protection of the genome and regulation of cell division cycle.1-3 Human telomere contains hundreds of 5’-TTA GGG-3’ repeat sequences with a full length of several thousand base pairs.4 Since there are abundant monovalent cations in human cells, these repetitive sequences have the propensity to fold into G-quadruplex (G4) structures, a DNA structure comprising four strands of stacked guanine (G)-tetrads formed by the coplanar arrangement of four guanines, held together by Hoogsteen bonds.5,6 The G4 structures formed in telomeres have been shown to be resistant to the length extension by telomerase, a ribonucleoprotein which is responsible for telomere maintenance by adding tandem repeats to the ends of chromosomes.7-9 Telomerase is one of the most common cancer markers and up-regulated in a vast majority of human tumors. Some small molecules such as pyridostatin (PDS), a highly selective G4-binding small molecule, have been proved to induce DNA-damage response and growth arrest in human cancer cells.10 With an electron-rich aromatic scaffold, PDS is considered to interact with a terminal G-quartet of a G4 structure through a stacking mode.11 Because of the potential for efficient cancer treatment, those small molecules have attracted a huge research interest to discover and design of telomeric G4 targeting ligands for cancer treatment.12-14 However, the exact binding mode of these ligands to telomeric G-quadruplex remains unclear and is still under research. There is therefore a strong need for developing efficient and robust bioassays to monitor and quantity telomeric DNA/ligand interactions. In previous studies, NMR spectroscopy,15,16 X-ray diffraction,17 circular dichroism (CD) spectroscopy18,19 and electrospray ionisation mass spectroscopy (ESI-MS)20 have been utilized to investigate the structural changes of G4 structures upon binding ligands. Other quantitative analytical methods such as thermal melting analysis,21 isothermal titration calorimetry (ITC)22 and surface plasmon resonance (SPR)23 have also been carried out for determining thermodynamic and kinetic 3



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parameters of the interactions. However, these techniques are all ensemble assays which could not obtain single-molecular information, and lack sensitivity. To solve that issue, some single molecule methods such as single-molecule fluorescence resonance energy transfer (FRET) spectroscopy24,25 and optical tweezers26 have also been used for the interaction study and obtained dynamic conformational and mechanical information. However, despite the high sensitivity, the time-consuming and costly fluorescent labeling and chemical modification are still required in these methods. Nanopore technology using a molecular-scale pore structure for single molecule study is very sensitive for detecting the size, charged status, and the conformation of a single molecule.27-30 Specifically, α-hemolysin (α-HL), a protein complex extracted from the bacterium Staphylococcus aureus, has been proven as a powerful platform for single molecule analysis.31-34 It can spontaneously insert into a lipid bilayer membrane and form a ~1.4 nm diameter pore.35 Based on the principle of Coulter Counter,36 when a transmembrane electric potential is applied, an ionic current through the nanopore is created. Once a biomolecule is electrophoretically driven through the pore, the ionic current will be interrupted and this perturbation can be recorded as a characteristic electric signal of the individual biomolecule. On the basis of the sensitive and label-free nanopore technique, various interactions between a biomolecule and its binding target have been investigated, such as the aggregation transition of β-amyloid peptide induced by small molecules,37 the photo-regulated interactions between an RNA aptamer and spiropyran,38 and the forming dynamics of DNA-doxorubicin complex.39 Furthermore, this nanopore system has been proved as a great tool for DNA secondary structure analysis.40,41 Various G4 conformations have been discriminated and analyzed in the α-HL nanocavity.42,43 Herein, we studied the interactions between human telomere sequence and G4 ligand PDS with α-HL nanopore technology at the single-molecule level. To simulate the physiological condition in cells, 100 mM K+ was chosen as a key factor for investigating its effect on telomeric DNA/ligand interaction. Distinctive current signatures for the linear telomeric DNA probe (LDNA), K+-G-quadruplex (K+-G4), 4


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PDS-G-quadruplex (PDS-G4) and K+-PDS-G-quadruplex (K+-PDS-G4) were observed, allowing the nanopore to readily monitor the changes in telomeric DNA structure induced by ligands and cations. Based on the relationship between molecule concentration and translocation event frequency, we further developed a simple nanopore assay for efficiently quantification of the telomeric DNA/ligand binding thermodynamics. In this method, rapid and accurate determination of affinity constants for ligand binding was achieved without labeling, amplification or ligand/receptor titration. By translocation studies and free-energy analysis, PDS and K+ were demonstrated to simultaneously bind to one telomeric G4 with the coordinated stabilization effect.

Experimental Section Reagents

Wild-type α-HL was purchased from Sigma-Aldrich (St. Louis, MO) and

used without purification. 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Tris·HCl, n-decane, and n-hexane were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pyridostatin was purchased from ChemieTek Inc. (Indianapolis, IN, USA). Other regents of analytical grade were obtained from Beijing Chemical Co. (Beijing, China). All solutions were prepared using ultrapure water. The DNA sequences were purchased from Shanghai Sangon Biological Engineering Technology & Services Co. Ltd (Shanghai, China). All DNA sequences were purified through denaturing polyacrylamide gel electrophoresis (PAGE). Sequences of oligonucleotide probes used in this work are listed as follows: Probe sequence: 5’- A25TAG GGT TAG GGT TAG GGT TAG GGT T A25 -3’; Control sequence: 5’- A25 TAC ATA TAC ACT TAG CTC TAC AAT T A25 -3’. Buffer A (0.5 M TMACl, 5 mM Tris, pH 7.4) and buffer B (0.5 M TMACl, 100 mM KCl, 5 mM Tris, pH 7.4) were used as test buffers. For DNA testing, 100 nM DNA sequence was pre-incubated in 1 mL test buffer A or B for 30 min at room temperature before nanopore testing. For DNA/PDS interaction, 100 nM DNA 5



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sequence was firstly pre-incubated in 1 mL test buffer A or B for 30 min at RT. Then, PDS was added in the solution and this mixture was incubated for another 30 min at RT before testing. Single-Channel Recording Single-channel current recordings were performed with an individual α-hemolysin (α-HL) nanopore inserted into a vertical lipid bilayer. The vertical chamber setup was assembled by two compartments, a cuvette with a 150 µm aperture drilled on the side (cis) and a bilayer chamber (trans) (Warner Instruments, Hamden, CT, USA). After pre-paint both sides of the cuvette aperture with 0.5 mg/mL DPhPC/hexane, both chambers were filled with 1 mL test buffer. The lipid bilayer was created by applying 30 mg/mL DPhPC/decane to the pretreated aperture. WT-α-HL protein was added in cis compartment from the stock solution of the protein monomers in storage buffer (10 mM Tris, 50 mM NaCl, pH 8.0, 10% v/v glycerol). After a single protein nanopore was formed in the lipid bilayer, positive potentials of 200 mV were applied across the lipid bilayer with Ag/AgCl electrodes. The cis compartment was defined as the virtual ground. The electrical current was recorded with a patch-clamp amplifier (HEKA EPC10; HEKA Elektronik, Lambrecht/Pfalz, Germany). Recordings were collected using a 3 kHz low-pass Bessel filter at sampling frequency of 20 kHz with a computer equipped with a LIH 1600 A/D converter (HEKA Elektronik). All measurements were carried out at room temperature. Data Analysis Data analysis was performed using MATLAB (R2011b, MathWorks) software and OriginLab 8.0 (OriginLab Corporation, Northampton, MA, USA). The current blockades are described as I/I0, where I0 is the ionic current of open nanopore and I is the blockage current produced by the analyte. Events with current blockades larger than 70% and dwell time longer than 0.05 ms were analyzed as DNA translocations. The mean dwell time for current spikes was obtained from the dwell time histograms by fitting the distributions to single exponential functions by the Levenberg-Marquardt procedure. The mean value of I/I0 was obtained by the fitted Gaussian distributions. All the data are presented as mean±s.d. of three independent experiments. 6


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Results and Discussion Translocation studies of the telomeric G-quadruplex induced by K+

The

principle of detecting the interactions between the human telomere sequence and ligand PDS with nanopore is depicted in Scheme 1. A natural human telomere sequence 5′-(TAG GGT)4 T-3′ linked with two poly(dA)25 single-stranded overhangs on both ends is designed as the telomeric DNA probe. The 25 nt human telomere sequence in the probe has been proved to have the capability to fold into a three-tetrad, two-cation quadruplex in the presence of cations such as K+ and Na+.42,43 The addition of nucleotide tails could greatly assist the G4 structure to enter the vestibule and interact with the constriction of α-HL nanopore, inducing easier threading of the DNA into the β-barrel of α-HL and higher translocation rates. The designed telomeric DNA probe was firstly tested in the buffer without any metal ions or ligands. Specifically, tetramethylammonium chloride (TMACl) was chosen for preparing electrolyte solutions because it didn’t promote the formation of G4 structures. Spike-like ionic current blockades were observed in the current traces, with a short average dwell time of 0.22±0.10 ms and current blockade I/I0 of 0.85±0.03 (Figure 1A, Table 1). These typical single-stranded DNA translocation events indicate that the telomere sequences couldn’t form G4 or any other secondary structures in the absence of metal ions or ligands. Thereafter, considering K+ is one of the most abundant cations in cells under the physiological condition, 100 mM KCl was then brought into the buffer. Previous studies showed that the human telomere sequence can become into different G-quadruplex folding modes (hybrid, basket, and propeller) under different cation and context-dependent conditions. Each folding mode had particular dimension and produced distinctive current signals while interacting with the α-HL ion channel.42,43 It was evidenced that in an aqueous KCl solution, the human telomere sequence will form hybrid-folded structure.44 As shown in Figure 1B and Table 1, events with a longer duration of 0.30±0.08 s were obtained and I/I0 rose a little to 0.89±0.02 in K+. 7



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The drastically prolonged dwell time suggests that the formation of K+-G4 led to a slow unraveling process when interacting with the protein cavity to achieve the final translocation. The larger current blockade was induced by the relatively larger dimension of the hybrid-folded K+-G4 structure located in the pore cavity during unraveling. To further explain the G4 unraveling process, different transmembrane potentials of 150, 180 and 200 mV were applied and the dwell time was shortened from 1.41±0.32 s to 0.30±0.08 s as the voltage increased from 150 mV to 200 mV (Figure S1). The voltage-dependent durations demonstrate the unfolding and translocation of G4. Then, a linear control sequence, with the same overall length and two poly(A)25 overhangs as the designed telomeric DNA probe, in which the 25-base sequence 5’-TACATATACACTTAGCTCTACAATT-3’ substituted between the overhangs was incapable of folding into a G4, was designed and analyzed by nanopore. In the presence of K+, only spike-like signals with short durations of 0.23±0.10 ms and I/I0 of 0.84±0.03 were observed in the nanopore experiments of the linear control DNA molecules (Figure S2A), illuminating the control DNA remained single stranded in K+ and underwent a direct translocation. The difference in event durations between the K+-G4 and the linear control DNA further implied the formation of the telomeric DNA G4 induced by K+.

Translocation studies of the telomeric G-quadruplex induced by PDS

After

the translocation studies of K+ stabilizing telomeric G-quadruplexes, we next analyzed the interactions of the human telomere sequence with PDS in the absence of K+. Several prolonged events were observed in the current traces (Figure 2A), which are ascribed to the stabilization property of PDS to G4 structures. These signals shared the similar shape with those of K+-G4, demonstrating the G4 formation was induced by PDS binding. However, the dwell time 3.72±0.95 s of PDS-G4 was about ten times longer than 0.30±0.08s of K+-G4 under the same transmembrane voltage (Table 1). In other words, PDS-G4 needed more time to overcome the energy barrier of the unraveling process. This result indicates that, compared to K+, PDS possessed greater capability to increase mechanical stability of the telomeric G4 structures. Meanwhile, 8


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the mean current blockade (I/I0) displayed a shift from 0.89±0.02 of K+-G4 to 0.96±0.01 of PDS-G4 (Table 1). The larger current blockage for PDS-G4 could be due to the different conformation of G4 caused by ligand binding.45 The linear control DNA probe was also tested in 1 μM PDS, and only short spike-like events were obtained, which demonstrates the great selectivity of PDS for telomere sequence (Figure S2B). Additionally, PDS molecules didn’t generate any signal during the tests (Figure S3) because PDS molecules were positive charged in buffer and they would be driven away from the nanopore side to the bulk solution in cis chamber under a positive voltage.

Translocation studies of the K+-PDS-bound telomeric G-quadruplex To

further

study the ligand binding in the presence of K+, PDS molecules were added into the cis chamber to interact with the K+-G4. Emerging unique two-level events were observed in the current traces (Figure 3), which are attributed to the newly formed K+-PDS-G4 complexes and suggest a two-state unraveling process of an individual G4 structure. Here, the two current levels could be assigned to I1/I0 and I2/I0. As displayed in Figure 3C and Table 1, Level 1 (L1) had a dwell time of 1.16±0.40 s and an I1/I0 of 0.89±0.02. Interestingly, the current blockade distribution of L1 almost shared the same region in the histograms with that of K+-G4, which has the hybrid-folded structure.44 The similar current blockades implied the similar DNA structures, revealing that even after PDS binding, the G4 in KCl still remained as the hybrid-fold form with unaltered dimension. This conclusion is in agreement with previous studies regarding the G4 structural polymorphism upon binding to ligands.46 After L1, the current dropped to consequential L2 with a much longer duration of 4.30±0.82 s and an I2/I0 of 0.95±0.01, which could be attributed to an intermediate form of a partially unfolded K+-PDS-G4. Both the dwell time and current blockade distributions of L2 were nearly the same as those of PDS-G4, implying that the intermediate form during K+-PDS-G4 unraveling process was PDS-G4 and the K+ ions in G4 had already been released before L2. Therefore, for the K+-PDS-G4 translocation, it firstly entered the vestibule of α-HL resulting in a ~88% current blockade (Level 1). Then the G4 9



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structure remained resident in the vestibule area until the K+ ions in G-quartets were ejected, and sequentially proceeded to Level 2. With the ~96% current blockade and long duration, Level 2 is attributed to an intermediate form of the PDS-G4 in the vestibule. Eventually, the PDS-G4 was unfolded and passed the constriction of α-HL after the dissociation of PDS. The L2 duration time was ~4-fold longer than that of L1, implying G4 structures conquered larger energy barrier to release PDS than K+. Then, the control DNA probe with linear sequence was tested in the presence of K+ and PDS. Only short-time events were observed under the same condition, for a direct translocation process of the linear control DNA (Figure S2C). From the translocation studies, PDS was found to strengthen K+-G4, leading to the prolonged dwell time at the same transmembrane potential. The PDS promoting effect on K+-G4 stabilization can be quantified as an increase in G4 folding energy, which is calculated by using: 𝛥𝐸 = 𝑅𝑇𝑙𝑛(𝜏off K+−PDS−G4 /𝜏off K+−G4 )

(1)

where 𝜏off K+−PDS−G4 and 𝜏off K+−G4 are the mean unraveling time of K+-PDS-G4

and K+-G4, respectively, R is the gas constant with a value of 8.314 J/(K⋅mol) and T is

absolute temperature with a value of 298.15 K. ΔE was obtained as 7.2±0.5 kJ/mol. Besides, the emerging two-level signals demonstrate that the ligand wouldn’t displace all the K+ cations in G-quartets or disrupt the hybrid-folded structure of K+-G4. The K+ and ligand were found to strengthen an individual telomeric G4 simultaneously.

Nanopore thermodynamic analytical method

Next, we carried out a nanopore

method to quantity the thermodynamic information of PDS binding to the telomeric G-quadruplexes in K+, which is critical for understanding the drug function in biomedical research. On the basis of the following binary complex formation mode: R+L ⇌ R-L

(2)

where R (receptor) refers to K+-G4, L (ligand) refers to PDS and R-L refers to the K+-PDS-G4 complex, as the ligand binding to the receptor, the concentrations of the receptor and ligand consumed under equilibrium of reaction are equal to that of the 10


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produced R-L complex, respectively. From the translocation studies, the four distinctive current signals can be used as signatures to identify the LDNA, K+-G4, PDS-G4, and K+-PDS-G4, respectively, while the event frequency f (f=1/τon) can be used to quantify the remaining receptor or the receptor-ligand complex produced. Particularly, the relationship of K+-G4 concentration with its translocation event frequency was investigated via nanopore testing and displayed in Figure S4, it is clear that the translocation frequency f (f=1/τon) for K+-G4 is directly proportional to its concentration, and hence can be used to quantify the remaining DNA receptor after the reaction reaches the equilibrium state. According to the thermodynamic calculation method in the Supporting Information, the affinity constant Ka of PDS binding to K+-G4 can be rapidly obtained with ~15 min nanopore testing requiring only one concentration of ligand (PDS) and receptor (K+-G4). With 100 nM receptor interacting with 1 μM ligand, the binding ratio Φ of K+-G4 and the affinity constant Ka were obtained as 0.74±0.04 and (3.1±0.8)×106 M-1, respectively. This Ka value is similar to the result previously evaluated by a laser tweezers method.26 To further validate the accuracy and reliability of this nanopore thermodynamic assay, different concentrations of PDS from 100 nM to 5 μM were mixed and interacted with 100 nM telomeric DNA in K+ for Ka measurements, respectively. Figure 4A shows the representative current recordings for the tests of telomeric DNA sequences in various PDS concentrations. Clearly, as the PDS concentration increased, the frequency of K+-G4 events decreased while those of two-level K+-PDS-G4 events increased. By the nanopore thermodynamic assay, the increasing binding ratio Φ and the similar affinity constant Ka values were obtained with various ligand concentrations (Figure 4), showing the Ka calculation in the nanopore method is independent of ligand concentration and possesses good reliability and accuracy. Compared to the current affinity constant assays suffering from complicated testing process, this sensitive and label-free nanopore method dramatically simplifies the thermodynamic detection process without varying the concentration of either ligand or receptor. Besides, this single-molecule platform can easily distinguish the K+-PDS-G4 complexes with the substrate K+-G4 structures in a complex media, 11



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displaying great anti-interference capability to other secondary nucleic acid structures. These advantages are especially beneficial for screening purposes in complex biological samples.

Coordinated effect of the ligand and K+ on the formation of telomeric G-quadruplex To investigate the effect of K+ on telomeric DNA/ligand affinity, the affinity constant for PDS binding to the linear telomeric DNA (LDNA) in the absence of K+ was also evaluated by using the proposed nanopore thermodynamic assay (Supporting Information). With 100 nM receptor interacting with 1 μM ligand, the binding ratio Φ of LDNA was obtained as 0.15±0.02. The Ka was found to be (1.9±0.6)×105 M-1, which is smaller than (3.1±0.8)×106 M-1 in K+, indicating the promotion effect of K+ on PDS affinity to the telomeric DNA. The increasing binding ratio Φ of LDNA and the similar affinity constant Ka values of LDNA/PDS interaction were also obtained with the enhanced ligand concentration (Figure 4). Then, to evaluate the equilibrium constant for the G4 formation induced by K+, the two-cation G4 model was used.42,43 With a nanopore testing of the pre-incubated 100 nM telomeric DNA probe in 100 mM K+, the equilibrium constant for the K+-G4 formation was determined to be 720±60 M-2. The affinity of the ligand PDS to the telomeric DNA was further analyzed in a Gibbs free energy perspective. The change in standard free energy (ΔGθ) was calculated using the formula: 𝛥𝐺 𝜃 = −𝑅𝑇𝑙𝑛𝐾 𝜃

(3)

where Kθ is standard equilibrium constant. By the nanopore tests of 100 nM telomeric DNA probe and 1 μM PDS, the values of ΔGθ for the PDS affinity to the telomeric DNA in the presence and absence of K+ were -(37.0±1.3) and -(30.2±0.8) kJ/mol, respectively. This result indicates that the ligand PDS shows better affinity to telomeric DNA in K+, confirming the coordinated effect of the ligand and K+ on the formation of telomeric G-quadruplex, which is in agreement with the results by the nanopore translocation studies. The ΔGθ for G4 formation in K+ was -(16.3±0.4) kJ/mol, which is much smaller than those with ligand. The difference demonstrates 12


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the ligand shows apparently stronger stabilization effect on G4 than K+, indicating the capacity of ligand to maintain the length of human telomere in opposition to the action of telomerase by promoting the folding of telomeric DNA G-quadruplexes.

Conclusion In summary, human telomere sequence interactions with a G-quadruplex ligand pyridostatin (PDS) was analyzed at single-molecule level and a nanopore thermodynamic analytical method was proposed for the affinity constants determination by using α-hemolysin protein. The translocation studies evidence that PDS selectively binds to human telomere sequence and promotes the folding of this G-quadruplex. Using the nanopore method, the ligand binding affinity constants were rapidly assessed. Compared to the conventional bulk assays, this method remarkably simplifies the thermodynamic assay without the requirement for labeling, amplification or ligand/receptor titration and presents a general efficient analytical platform for other biologically relevant ligand–receptor systems. Besides, with the translocation studies and free-energy analysis, the PDS promotion effect on G-quadruplex stabilization is quantified, and a ligand binding mode is proposed that PDS molecule and K+ simultaneously bind to one G-quadruplex structure with the coordinated stabilization effect, which is helpful for the future drug design.

Acknowledgement This work was financially supported by the National Natural Science Foundation of China (No. 21235004, No. 21327806), and Tsinghua University Initiative Scientific Research Program. The authors thank Yuxing Li and Prof. Tianling Ren from Institute of Microelectronics, Tsinghua University, for their help to provide MATLAB programs for data analysis.

Supporting Information Additional information as noted in text. This material is available free of charge 13



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via the Internet at http://pubs.acs.or

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Table 1. Dwell times and current blockades of the translocation events for telomeric DNA analytesa Analyte

Dwell time (s)

I/I0

LDNA

(0.22±0.10)×10-3

0.85±0.03

K+-G4

0.30±0.08

0.89±0.02

PDS-G4

3.72±0.95

0.96±0.01

1.16±0.40 (L1)

0.89±0.02 (L1)

4.30±0.82 (L2)

0.95±0.01 (L2)

K+-PDS-G4 a

The histograms of dwell times and current blockades for LDNA, K+-G4, PDS-G4

and K+-PDS-G4 are obtained from Figure 1-3, respectively. Data of the values are based on three separated experiments.

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Figure Legends

Scheme

1.

Schematic

illustration

of

the

K+-G-quadruplex

(K+-G4),

pyridostatin-G-quadruplex (PDS-G4) and K+-pyridostatin-G-quadruplex (K+-PDS-G4) translocating through α-HL nanopores (From left to right). A PDS-G4 complex generates an event with longer duration than K+-G4. The K+-PDS-G4 generates a two-level blockage with much longer duration than the others.

Figure 1. Nanopore detection of the telomeric DNA probes without (A) and with (B) K+. (a) Representative single-channel current traces for the tests of the telomeric DNA probes. (b) Histograms of dwell times for the tests of the telomeric DNA probes. Histogram for DNA without K+ was fit to an exponential function and the histogram for DNA with K+ was fit to a Gaussian distribution. (c) Histograms of normalized current blockade I/I0 for the tests of the telomeric DNA probes. Each histogram was fit to a Gaussian distribution. All tests were performed with 100 nM DNA probes in the buffer containing 0.5 M TMACl, 5 mM Tris (pH=7.4), with the transmembrane potential of +200 mV. Each experiment was repeated three times.

Figure 2. Nanopore detection of the telomeric DNA probes in the presence of PDS. (A) Representative single-channel current traces for the tests of the telomeric DNA probes in the presence of PDS. Red arrowheads mark the PDS-G4 events in the current traces. (B) Histograms of dwell time for the PDS-G4. (C) Histograms of normalized current blockade I/I0 for the PDS-G4. Each histogram was fit to a Gaussian distribution. All tests were performed with a pre-incubated mixture of 100 nM DNA probes and 1 μM PDS in the buffer containing 0.5 M TMACl, 5 mM Tris (pH=7.4), with the transmembrane potential of +200 mV. Each experiment was repeated three times.

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Figure 3. Nanopore detection of the telomeric DNA probes in the presence of K+ and PDS. (A) Representative single-channel current traces for the tests of the telomeric DNA probes in the presence of K+ and PDS. Blue arrowheads mark the two-level events for the translocation of K+-PDS-G4 through the a-HL nanopore. (B) Expanded view of a typical two-level event indicated in the trace in (A) by the blue box. The two current levels are labelled as Level 1 (L1) and Level 2 (L2). (C) Histograms of dwell times (left) and normalized current blockade I/I0 (right) for Level 1 and Level 2. Each histogram was fit to a Gaussian distribution except that the histogram of Level 1 dwell time was fit to an exponential function. All tests were performed with a pre-incubated mixture of 100 nM DNA probes and 1 μM PDS in the buffer containing 0.5 M TMACl, 100 mM KCl, 5 mM Tris (pH=7.4), with the transmembrane potential of +200 mV. Each experiment was repeated three times.

Figure 4. Measurement of the affinity constant between PDS and the telomeric DNA. Representative single-channel current traces for the tests of 100 nM telomeric DNA sequences with various concentrations of PDS with (A) and without (B) K+. The tests were performed in buffer containing 0.5 M TMACl, 5 mM Tris (pH=7.4) with (A) and without (B) 100 mM KCl, with the transmembrane potential of +200 mV. (C) Fraction of the K+-G4 and LDNA bound with PDS versus concentration of PDS. The plots were fit to the single site binding model. Error bars are standard deviations from three experiments. (D) PDS concentration-independence of the affinity constants Ka for K+-G4/PDS and LDNA/PDS interactions.

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Scheme 1

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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For TOC Only

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Single-Molecule Analysis of Human Telomere ... - ACS Publications

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