Study of pH-Induced Folding and Unfolding Kinetics of the DNA i-Motif

Nov 13, 2012 - Using the stopped-flow circular dichroism (SFCD) technique, we investigate the kinetics of the pH-induced folding and unfolding process...
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Study of pH-Induced Folding and Unfolding Kinetics of the DNA i‑Motif by Stopped-Flow Circular Dichroism Chun Chen,† Ming Li,*,‡ Yongzheng Xing,† Yingmei Li,† Carl-Christian Joedecke,† Juan Jin,† Zhongqiang Yang,† and Dongsheng Liu*,† †

Key Laboratory of Organic Optoelectronics & Molecular Engineering of the Ministry of Education, Department of Chemistry, Tsinghua University, Beijing 100084, China ‡ School of Physics, University of Chinese Academy of Sciences, Beijing 100190, China S Supporting Information *

ABSTRACT: Using the stopped-flow circular dichroism (SFCD) technique, we investigate the kinetics of the pH-induced folding and unfolding process of the DNA i-motif. The results show that the molecule can fold or unfold on a time scale of 100 ms when the solution pH is changed. It is also found that the folding and unfolding rates strongly depend on the solution pH. On the basis of quantitative data, we propose theoretical models to decipher the folding and unfolding kinetics. Our models suggest that the cooperativity of protons is crucial for both the folding and unfolding process. In the unfolding process, the cooperative neutralization of two protons (out of the total six protons in the i-motif molecule) is the only rate-limiting step. In the folding process, there exists a critical step in which three protons bind cooperatively to the DNA strand. These results offer an in-depth understanding of the folding and unfolding kinetics of the DNA i-motif and may give precise guidance for constructing novel nanodevices based on the DNA i-motif.

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INTRODUCTION The i-motif is a four-stranded DNA structure formed by cytosine-rich oligonucleotides under slightly acidic conditions, with intercalated hemiprotonated C•C+ base pairing.1,2 The i-motif widely exists in genomic DNA3 and plays an important role in the biological process, for instance, gene transcriptional regulation.4−8 Other than its biological roles, the i-motif has received increasing attention as an excellent building element, especially in constructing responsive systems because of its unique proton-induced folding and unfolding property.9−15 In 2003, Liu et al. first proposed that a proton-fueled nanomotor can be built from the i-motif solely by employing the conformational change in the i-motif under different pH conditions.16 Since then, a series of molecular devices such as smart surfaces, logic gates, and nanosprings have been developed.17−25 The rate of the folding and unfolding process determines how fast these i-motif motors and responsive materials can respond and work. Therefore, the kinetic study of the i-motif becomes an important research topic, not only in understanding the fundamental phenomena but also as being beneficial to the design of responsive DNA nanodevices. Until now, the kinetics of the i-motif has been studied by several methods, such as surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), and nuclear magnetic resonance (NMR).26−32 However, the method of studying the kinetics of the unmodified i-motif in solution at a relatively high time resolution (i.e., the millisecond scale) has not been reported. Here, we employed the stopped-flow circular dichroism (SFCD) techinique,33 which is dependent on base−base interactions and exquisitely sensitive to nucleic acid secondary structure avoiding any modification,34 to measure the pH-induced folding and unfolding rate of i-motif structures in solution on the millisecond scale. © 2012 American Chemical Society

A model has been proposed to illustrate the folding and unfolding pathways (Figure 1) based on the obtained data.

Figure 1. Schematic illustration of the pH-induced folding and unfolding pathways of the 21-nt-long DNA i-motif.

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EXPERIMENTAL SECTION

Materials. The 21-mer oligonucleotide 5′- CCC TAA CCC TAA CCC TAA CCC- 3′ was purchased from Invitrogen Biotech (Beijing, China) and

Received: September 25, 2012 Revised: November 3, 2012 Published: November 13, 2012 17743

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PAGE purified. All chemicals were reagent grade or better and used as received. Water used in all experiments was ultrapure Milli-Q water (18.2 MΩ·cm). The concentration of DNA was estimated from the optical absorbance at 260 nm by a Cary 100 UV/vis spectrometer (Varian Company). Sample Preparation. To get a DNA sample solution, DNA strands were added to a buffer solution containing 90 mM NaCl and 5 mM Na2HPO4 (pH 4.97 or 8.01) to get a final DNA concentration of 50 μM. Then the mixtures were heated to 95 °C for 5 min and cooled to room temperature over 2 h to form the desired DNA structures. The phosphate buffer solutions were prepared by dissolving Na2HPO4 in water to get a final concentration of 50 mM and were adjusted with H3PO4 to different pH values. In addition, the phosphate buffer solutions were filtered with a 0.22 μm filter unit (Millex-GV, Millipore). Circular Dichroism Spectroscopy. The circular dichroism (CD) spectra were collected on an Applied Photophysics Chirascan spectropolarimeter. The DNA sample solution (50 μM DNA sample in 5 mM, pH 4.97 Na2HPO4 buffer with 90 mM NaCl) was mixed with the phosphate buffer solution (50 mM Na2HPO4 buffer at different pH values) in a volume ratio of 1:4, and the final pH values were 5.00, 5.51, 7.51, and 8.01 respectively. The scanning was carried out at the range of 220−350 nm at room temperature in a quartz cell of 1 mm optical length, and the scanning speed was 60 nm/min. Stopped-Flow Circular Dichroism. The SFCD measurements were carried out on an Applied Photophysics Chirascan spectropolarimeter equipped with a stopped-flow accessory (Figure S1). In an SFCD measurement, the DNA sample solution was rapidly mixed with the phosphate buffer solution in a mixing ratio of 1:4 by being forced through a mixer and optical cell, on emerging from which the flow was suddenly stopped and the reaction was monitored by a CD detector. The change in ellipticity as a function of time under each condition was recorded at least five times. All of the SFCD curves were collected at 288 nm (determined by CD spectra) at room temperature. The optical path length was set to 10 mm with a dead time of 1 ms. The bandwidth was set to 8.0 nm.

Figure 2. Circular dichroism spectra of the oligonucleotides in various pH solutions.

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RESULTS AND DISCUSSION CD Characterization for the Formation of the DNA i-Motif. In this work, we selected a typical i-motif sequence (5′-CCC TAA CCC TAA CCC TAA CCC-3′) containing four cytosine stretches, which is the same as the reported proton-fueled nanomotor.16 To obtain the best resolution in the kinetics studies, the wavelength that exhibits the maximal changes during the folding/ unfolding process was determined with CD spectroscopy before the SFCD measurements. As shown in Figure 2, we obtained a series of CD spectra of the oligonucleotides at various pH values (5.00, 5.51, 6.02, 6.49, 7.03, 7.51, and 8.01). At pH 5.00 and 5.51, the CD spectra showed a strong positive peak near 288 nm and a negative peak near 258 nm with a crossover at around 272 nm indicating that the i-motif structure was formed.35 In contrast, at pH 6.49, 7.03, 7.51, and 8.01 the characteristic i-motif peak at 288 nm disappeared, resulting in a dramatic decrease in the CD signal. Therefore, 288 nm was selected as the fixed wavelength in the following SFCD measurements. Typical SFCD Curves and Fitting Results. The typical folding and unfolding process monitored by SFCD is shown in Figure 3. It is observed that the fastest folding/unfolding process takes more than 100 ms to complete. With a noted SFCD dead time of 1 ms, it can be regarded that DNA keeps its initial configuration during the fast process of solution mixing. Therefore, after mixing, the changes in ellipticity as a function of time could reflect the configuration relaxation under the desired pH value. To analyze the folding or unfolding kinetics, we choose the first-order exponential decay function (eq 1) to fit the raw CD data (the reason for choosing this function will be explained later), as indicated in Figure 3 y(t ) = y0 + Ae

−t / t1

Figure 3. Typical (A) unfolding and (B) folding data (black squares) of the DNA i-motif monitored by SFCD and their fitting curves (red solid line). (A) Unfolding profile: DNA sample solution at an initial pH of 4.97 mixed with pH 8.99 phosphate buffer, resulting in a final pH of 8.22. (B) Folding profile: DNA sample solution at an initial pH of 8.01 mixed with a pH 4.48 phosphate buffer, resulting in a final pH of 4.82. The fitting was accomplished by Origin 8.0.

where y(t) is the raw CD signal recorded at time t and y0, A, and t1 are fitting parameters. t1 is the characteristic folding or unfolding time (1/t1 is actually the folding or unfolding rate), which is the

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main focus of this work. For each CD record, data fitting gives the estimated t1. The experiment and data fitting procedure were repeated at least five times for each pH condition, and all averaged t1 data for different pH values are summarized in Tables 1 (for the unfolding process) and 2 (for the folding process). These tables

lg t1 = − lg k1 − n lg[OH−] = − n × pH − lg k1 + 14n

where lg t1 is the logarithm of t1. To use eq 3 to analyze the data in Table 1, we first transformed t1 values into lg t1 values and then plotted lg t1 against pH (black dots in Figure 4). Finally, the linear function was chosen to fit the lg t1∼pH data. A perfect fitting was found (red solid line in Figure 4). The fitting also gives n ≈ 2 (i.e., B = −1.88), which is close to an integer as the model requires.

Table 1. Characteristic Unfolding Time at Different Final pH Values (pHinitial 4.97) final pHa

averaged t1 (s)

8.22 7.97 7.58 7.04 6.52

0.115 ± 0.009 0.20 ± 0.01 1.49 ± 0.03 14.05 ± 0.09 150 ± 3

(4)

The final pH was obtained by measuring the mixing solution after SFCD measurements.

a

Table 2. Characteristic Folding Time at Different Final pH Values (pHinitial 8.01) final pH

averaged t1 (s)

4.82 5.12 5.36 5.69 5.94 6.10

0.141 ± 0.008 0.21 ± 0.01 0.57 ± 0.02 3.0 ± 0.2 17.9 ± 0.4 99.9 ± 0.7

Figure 4. pH-induced unfolding kinetics of the i-motif at high pH values. The black square represents lg t1 at different pH values, and the red solid line is the linear fitting result.

This gives further support to the validity of our model. These results offer new insight that there must be a rate-limiting step in the unfolding process in which two protons (out of all six protons) are cooperatively neutralized. n ≈ 2 also implies that there may be other steps in the unfolding pathway in which other protons are neutralized. To get more information about the entire pathway, one can propose more complicated kinetic models. In principle, for multistep configuration transitions, the kinetics can be described generally by a set of linear ordinary differential equations. Therefore, the timeevolution function of any conformational species (e.g., I or I*) is definitely a linear combination of several exponential functions (as illustrated in the following subsection, model II). In other words, higher-order exponential decay functions can be used for data fitting. In fact, we have tried second-order and higher-order exponential functions to fit the raw CD data but did not get a statistically significant estimate of all fitting parameters. That means that some steps in the unfolding pathway are hard to resolve by SFCD probably because they are too fast or make negligible CD signal changes. Therefore, model I is sufficient to interpret the experimental data. pH-Dependent Folding Kinetics. The above modeling and fitting procedure can be followed to analyze the folding kinetics. If model I is directly employed, then one can plot lg t1 against pH on the basis of Table 2 and determine whether it agrees with the linear relation predicted by eq 3. Unfortunately, the lg t1−pH relation in the folding case is far from linear (data not shown), which implies that the folding kinetics is much more complicated and cannot be described by simple schemes such as model I. Therefore, we proposed the following two possible schemes, assuming that the folding is nearly irreversible at low pH values

indicate that the i-motif can unfold or fold on a 100 ms time scale at very low or very high pH values. The tables also show that the folding and unfolding rates are strongly dependent on the concentration of H+ or OH−, which provides valuable information for uncovering the folding and unfolding kinetics (e.g., rates and pathway) of the i-motif. pH-Dependent Unfolding Kinetics. To analyze the unfolding kinetics, we assumed that the unfolding process is nearly irreversible at high pH values and proposed the following reaction scheme K

1 ImH+ + nOH− → I*(m − n)H+ + nH 2O

(model I)

where I represents the folded configuration that may contain m protons, I* represents completely or partially unfolded configurations that show a negligible CD signal, and k1 is the intrinsic unfolding rate constant. According to this model, CD signal changes can be observed only when n protons (H+) in the i-motif are neutralized cooperatively by solution OH−. Because there are 6 protons in total in the i-motif molecule,36 n should be an integer less than 6. This model can be rewritten as k′

I → I*, k′ ≡ k1[OH−]n

(2) −

where k′ is the pseudo-first-order rate constant and [OH ] is the concentration of OH− in solution. The kinetic equation of the i-motif concentration [I] is d[I]/dt = −k′[t], which has the simple solution [I](t) = [I]0e−k′t. Therefore, by considering the linear relationship between [I] and the CD signal y, we can choose eq 1 to fit the raw data as previously mentioned. Here 1 1 t1 ≡ = k′ k1[OH−]n (3)

k1

k2

k −1

nH

CmH+ XooY C*mH+ ⎯⎯⎯→ I(m + n)H+ +

is the characteristic unfolding time. Equivalently,

(model IIA)

or 17745

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k2

CmH+ + nH+ XooY C*(m + n)H+ → I(m + n)H+ k −1

(model IIB)

where C represents the unfolded configuration of ssDNA, which may bind m protons, and C* represents the possible intermediate configuration. In either C or C*, the bases are not stacked as in the well-folded configuration, so C and C* show no CD signals. According to these models, CD signal changes can be observed only if other n protons bind cooperatively to the ssDNA. For model IIA, the kinetic equations for [C], [C*], and [I] are ⎛ [C] ⎞ ⎛ [C] ⎞ ⎟ ⎜ ⎟ d⎜ ⎜[C*]⎟ = M ⎜[C*]⎟ , dt ⎜ ⎟ ⎜ ⎟ ⎝ [I] ⎠ ⎝ [I] ⎠

⎛ − k1 0⎞ k −1 ⎜ ⎟ M ≡ ⎜ k1 −(k −1 + k 2*) 0 ⎟ ⎟ ⎜ ⎟ ⎜ 0⎠ k 2* ⎝ 0

k 2* ≡ k 2[H+]n

Figure 5. pH-induced folding kinetics of the i-motif at low pH values. The black square represents lg(t1−t0) at different pH values, and the red solid line is the linear fitting result. Here, the trial value of t0 is taken as 0.13 s.

(5)

A simple calculation shows that the mathematic solutions of the above equations (i.e., the time evolution of [C], [C*], and [I]) can all be written as a linear combination of e−λ+t and e−λ−t. Here 1⎡ λ± = ⎣(k1 + k −1 + k 2*) ± 2

Furthermore, the fitting gives n ≈ 3 (i.e., B = 2.98), which gives support to the validity of model IIA. According to this model, when the solution pH is switched to low values, ssDNA coil C (probably bound to protons) undergoes a reversible and pH-independent transition to an intermediate configuration C* in which hemiprotonated C•C+ pairs are not formed. The C•C+ pairs can be formed and well stacked only once three protons bind to C* cooperatively in a subsequent single step, and then the molecule is irreversibly folded into the i-motif structure. The experimental data can also be well fitted by model IIB. A similar analysis leads to the same conclusion that n = 2.98. However, the transition from C to C* is pH-dependent and reversible in this model, which is more or less unreasonable because proton binding to C can be quite irreversible in low-pH solution. Therefore, model IIA seems to be much more appropriate than model IIB for data fitting and interpretation. A further examination of all two-step reaction schemes shows that model IIA and model IIB are the only two appropriate models for data fitting. Additionally, schemes containing three steps or more did not give statistically significant estimates of all of the fitting parameters. Therefore, we reach the conclusion, based on either model IIA or model IIB, that there exist at least two steps in the folding pathway (one reversible and the other irreversible), in one of which three protons (out of the total of six protons) bind to the DNA molecule cooperatively.

⎤ (k1 + k −1 + k 2*)2 − 4k1k 2* ⎦ (6)

are the two nonzero eigenvalues of the coefficient matrix M in eq 5 (obviously, the third eigenvalue of M is zero). Hence, one can choose a second-order exponential decay function such as ae−λ+t + be−λ−t + c to fit the raw CD data, here a, b, c, are also fitting parameters. Unfortunately, such a fitting did not give a statistically significant estimate of all parameters, and only the first-order exponential decay function gave meaningful fitting results. In other words, the apparent time evolution of the CD signal can be properly described by first-order exponential decay function A + Be−k′t rather than ae−λ+t + be−λ−t + c. This can be possible if one of the two exponentials λ+ and λ− is much larger than the other. A simple calculation shows that if we further assume (k1 + k−1 + k2*)2 ≫ 4k1k2* then the real kinetics can be approximately described by A + Be−k′t. Here, k′ = (k1k2*)/ (k1 + k−1 + k2*) and the characteristic folding time can be defined as t1 ≡

k + k −1 1 1 = + 1 k′ k1 k1k 2[H+]n

(7)

or equivalently k 1 + k −1 − n × lg[H+] k1k 2 k + k −1 1 = n × pH + lg 1 , t0 ≡ k1k 2 k1

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lg(t1 − t0) = lg

CONCLUSIONS In this work, we used a new strategy, the stopped-flow circular dichroism technique, to monitor in situ the configuration changes of the DNA i-motif, allowing a quantitative investigation of the folding and unfolding kinetics of the DNA i-motif. On the basis of the experimental results, we succeeded in building theoretical models to uncover valuable details of the pH-dependent folding and unfolding pathways (Figure 1). Our model suggests that the cooperativity of protons is crucial for either the folding or unfolding process. In the unfolding process, the cooperative neutralization of two protons is the only ratelimiting step. In the folding process, however, there must be at least two steps, and in one of these steps there are three protons cooperatively binding to the DNA molecule. Furthermore, it is also observed that the DNA i-motif can rapidly fold in a solution

(8)

In contrast to eq 3, eq 8 predicts that there exists an asymptotic value (t0) of t1 when the pH value is decreased. This can be understood if one regards t0 as approximately the intramolecular diffusion time of the single DNA strand required to form the intermediate conformation. To use eq 8 to analyze the data in Table 2, one should first transform the original t1 data into lg(t1−t0). This can be done by trying different t0 values. For each trial value t0, we plotted lg(t1−t0) against pH and checked whether the linear relation predicted by eq 8 holds. It is found that when t0 = 0.13 s (k1 = 8.3 s−1) the lg(t1 − t0)∼pH plot (black dots in Figure 5) indeed shows a perfect linear relation (red line in Figure 5). 17746

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of pH ∼5 and unfold in a solution of pH ∼8, both on a time scale of 100 ms, which is comparable to the operating rate of natural protein nanomachines. This indeed guarantees a rapid pH responsiveness of the i-motif molecule if it is used as a practical nanomachine.37−41 All of these results can offer an in-depth understanding of the folding and unfolding kinetics of the DNA imotif and give precise guidance for the construction of novel nanostructures based on the DNA i-motif.

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ASSOCIATED CONTENT

S Supporting Information *

Schematic illustration of instrument setup of stopped-flow circular dichroism. pH values of the DNA sample solutions, phosphate buffer solutions and final solutionsa in each SFCD experiment. Characteristic unfolding time t1 at different final pH values. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(M.L.) E-mail: [email protected]. (D.L.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank National Basic Research Program of China (973 program, no. 2013CB932803), the National Natural Science Foundation of China (nos. 21121004 & 91027046), NSFC-DFG joint project TRR61, and the CAS/SAFEA International Partnership Program for Creative Research Teams for financial support.

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