Effect of Cavity Size of Mesoporous Silica on Short DNA Duplex Stability

Apr 16, 2018 - (error bars) for the experimental data points were obtained from at least three independent measurements. ... On the basis of the two-s...
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Effect of Cavity Size of Mesoporous Silica on Short DNA Duplex Stability Tsubasa Masuda,† Yuuta Shibuya,† Shota Arai,† Sayaka Kobayashi,† Sotaro Suzuki,† Jun Kijima,† Tetsuji Itoh,‡ Yusuke Sato,§ Seiichi Nishizawa,§ and Akira Yamaguchi*,† †

Institute of Quantum Beam Science, Ibaraki University, 2-1-1 Bunkyo, Mito, Ibaraki 310-8512, Japan Research Institute for Chemical Process Technology, National Institute of Advanced Industrial Science and Technology (AIST), Nigatake 4-2-1, Sendai 983-8551, Japan § Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980-8578, Japan ‡

S Supporting Information *

ABSTRACT: We studied the stabilities of short (4- and 3-bp) DNA duplexes within silica mesopores modified with a positively charged trimethyl aminopropyl (TMAP) monolayer (BJH pore diameter 1.6−7.4 nm). The DNA fragments with fluorescent dye were introduced into the pores, and their fluorescence resonance energy transfer (FRET) response was measured to estimate the structuring energies of the short DNA duplexes under cryogenic conditions (temperature 233−323 K). The results confirmed the enthalpic stability gain of the duplex within sizematched pores (1.6 and 2.3 nm). The hybridization equilibrium constants found for the size-matched pores were 2 orders of magnitude larger than those for large pores (≥3.5 nm), and this size-matching effect for the enhanced duplex stability was explained by a tight electrostatic interaction between the duplex and the surface TMAP groups. These results indicate the requirement of the precise regulation of mesopore size to ensure the stabilization of hydrogen-bonded supramolecular assemblies.

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the silica mesopore.9 The enhanced duplex stability was likely explained by the tight confinement of the duplex from the fact that complementary and single-mismatched duplexes exhibited similar structural stabilities. The aim of the present study is to clarify the pore size effect for 4-bp DNA duplex formation. The duplex is a simple DNA secondary structure formed by cooperative hydrogen bonding and base stacking. Knowledge of its stabilization mechanism will contribute to the elucidation of the structural stabilities of biomacromolecules and artificial supramolecular complexes confined inside mesosized cavities. In the present study, short (4- or 3-mer) DNA fragments were confined within amine-functionalized mesoporous silica (AMS) with cylindrical pore channels (Figure 1), and their hybridization equilibria were examined to quantify the degree of the size-matching effect for duplex stabilization. Aiming to use a DNA duplex with a diameter of 2 nm, we synthesized AMSs with different BJH pore diameters (DBJH) ranging from 1.6 to 7.4 nm. We designated AMS as AMSxx, where xx means DBJH. The short DNA fragments with a donor or acceptor dye were introduced into the AMS pores, and their fluorescence resonance energy transfer (FRET) response was measured

INTRODUCTION Mesosized cavities are a promising reaction field for macromolecules and supramolecular complexes since inter- and intramolecular arrangements can be regulated according to the structure and chemical characteristics of the cavity.1−11 Reversed micelles5,6 and mesoporous silica,7−11 which have tunable and uniform mesosized cavities, have been used to study the cavity size effect for the structural regulation of various supramolecular complexes,7,8 oligonucleotides,5,6,9 and proteins.10,11 Some of these studies have proposed that size matching between the cavity size and molecular dimensions can enhance the structural stability of macromolecules and molecular complexes.7−11 However, because of a lack of quantitative studies, structural stabilization inside size-matched cavities remains an obscure phenomenon. There have been no studies to reveal the effect of the cavity size on the structuring energy and/or equilibrium constant of the confined molecules, which are useful parameters for investigating the degree of the size-matching effect. We recently found that confinement inside an aminefunctionalized silica mesopore (pore diameter 2.4 nm) could significantly enhance the stabilities of the 3- and 4-bp DNA duplexes through Watson-Click base pairing.9 The short DNA duplexes composed of AT base pairs are hardly formed in bulk water, whereas effective duplex formation takes places inside © XXXX American Chemical Society

Received: February 7, 2018 Revised: April 16, 2018

A

DOI: 10.1021/acs.langmuir.8b00437 Langmuir XXXX, XXX, XXX−XXX

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a MAC Science/Bruker M21X with Cu Kα radiation. The SAXS patterns of other AMSs were recorded on a Rigaku Smartlab with Cu Kα radiation. A Yanaco model CHN MT-6 elemental analyzer was used to determine the nitrogen weight percentages in the AMSs. The structural parameters of AMSs are listed in Table 1. All AMSs have uniform hexagonal pores with a narrow pore size distribution (Figures S1 and S2). The surface densities of the TMAP group were around 1 group nm−2 for all AMSs. FRET Measurements. The 5′ FAM-modified DNA (d-TTTT and d-TTT) and 3′ TAMRA-modified DNA (a-AAAA, a-TTTT, and aAAA) were custom synthesized and HPLC purified (>97%) by Nihon Gene Research Laboratories Inc. For the adsorption of DNA fragments into AMS pores, each DNA fragment was dissolved in a 10 mM sodium cacodylate buffer solution (pH 7.0) containing 100 mM NaCl and 1 mM EDTA. AMS (1 mg) was added to 400 μL of the 5 μM FAM-modified DNA solution. After being shaken for 5 h at room temperature (25 °C), the TAMRA-modified DNA solution was added to the mixture. The final mixture was then shaken overnight at room temperature (25 °C) before being centrifuged at 14 000 rpm for 20 min. The supernatant was subjected to absorption spectrum measurements to estimate the amount of DNA adsorbed (Table 1).9 Under our experimental conditions, almost all DNA molecules were adsorbed at AMSs, and the ratio of FAM- and TAMRA-modified DNA molecules within AMSs was 0.98 ± 0.03. The numbers of DNA molecules adsorbed within AMSs are listed in Table 1. The AMS with DNA fragments was carefully rinsed with the buffer solution and then suspended in a 70:30 (v/v) mixture of ethanol and aqueous buffer (50 mM NaCl, 10 mM sodium cacodylate buffer, and 1 mM EDTA, pH 7.0). The suspension of AMS with DNA fragments was poured into a quartz cell (1 cm × 1 cm) with a screw cap. The fluorescence spectra were recorded with a spectrofluorophotometer (F-4500, Hitachi) equipped with a cryostat (CoolSpek USP-203, Unisoku). The excitation wavelength was 455 nm for all fluorescence measurements. The fluorescence spectrum of the sample suspension was measured while decreasing the temperature from 323 K at a rate of 2 K min−1. After reaching the target temperature, the sample suspension was stored for 15 min prior to a measurement. This holding time was enough to reach hybridization equilibrium because the spectral shape did not change after 5 min of holding time. The standard deviations (error bars) for the experimental data points were obtained from at least three independent measurements.

Figure 1. Schematic illustration of the 4-bp DNA duplex confined inside a size-matched pore (DBJH = 2.3 nm) and inside large pores (DBJH = 3.5 and 5.7 nm).

under cryogenic conditions (temperature 233−323 K) to estimate the structuring energy of the short DNA duplexes. Guanine is well known to quench the fluorescence of FAM and TAMRA, which are used as donor and acceptor dyes.9 We hence used oligoadenosines and oligothymidines to study the structural stabilities of the DNA duplexes by FRET experiments. The results clearly confirmed the significant enthalpic stability gain of the duplex within size-matched pores (1.6 and 2.3 nm).

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EXPERIMENTAL SECTION

Preparation and Characterization of AMS. We prepared AMSs with different BJH pore diameters (DBJH) ranging from 1.6 to 7.4 nm.12−15 FSM-type mesoporous silica powders were synthesized using alkyltrimethylammonium [CnH2n+1N+(CH3)3] with different alkyl chain lengths (n = 16 and 18), according to the method reported by Inagaki et al.12 The modification of the FSM-type mesoporous silica powders with TMAP groups13 provided AMS1.6 and AMS2.3. SBAtype mesoporous silica powders were synthesized using Pluronic P123 as a template surfactant,14 and the modification of the SBA-type mesoporous silica with TMAP groups provided AMS3.5 and AMS5.7. Pore-expanded mesoporous silica powder was synthesized using 1,3,5trimethylbenzene as a pore expander according to the method reported by Zhao et al.15 The TMAP modification of the poreexpanded mesoporous silica provided AMS7.4. The pore structures of AMSs were characterized by nitrogen adsorption and desorption isotherms, X-ray diffraction (XRD), or small-angle X-ray scattering (SAXS) measurements. The nitrogen contents in AMSs were measured by elemental analysis measurement. The nitrogen adsorption and desorption isotherms were measured on a Micrometrics ASAP 2020 instrument. The specific surface area was calculated using the multiple-point BET method, and the pore size distribution was calculated from the adsorption branch of the isotherm using the BJH method. The XRD pattern of AMS3.5 was recorded on

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RESULTS AND DISCUSSION The AMS with the DNA fragments was dispersed in the 70:30 (v/v) ethanol/water mixture for the fluorescence measurement to suppress the desorption of negatively charged DNA fragments. For all AMS systems, the desorption of the DNA fragments was not observed during the fluorescence measurement. The FRET responses for the AMS systems hence indicate the duplex formation inside the AMS pores. Figure 2A,B shows typical fluorescence spectra obtained for complementary 4-mer DNA pairs (d-TTTT/a-AAAA) within AMS2.3 and AMS3.5. Fluorescence spectra for the 4-mer DNA pairs within other AMSs are shown in Figure S3. In any AMS system, a temperature decrease induces the quenching of donor

Table 1. Structural Parameters of AMSs and Amount of DNA Adsorbed at AMSs AMS1.6 AMS2.3 AMS3.5 AMS5.7 AMS7.4

dBJH/nma

Vpore/cm3 g−1b

DTMAP/molecules nm−2c

1.6 2.3 3.5 5.7 7.4

0.44 0.75 0.16 0.30 0.50

0.8 0.9 0.8 1.1 0.9

ADNA,4/μmol mg−1d

ADNA,3/μmol mg−1e

± ± ± ± ±

3.7 ± 0.2 3.9 ± 0.2 3.9 3.9 ± 0.1 3.8

3.6 3.6 3.0 3.4 3.5

0.1 0.1 0.2 0.1 0.1

a

BJH pore diameter of AMS. bPore volume of AMS. cSurface density of TMAP groups. dNumber of 4-mer DNA molecules adsorbed in AMS. Number of 3-mer DNA molecules adsorbed in AMS. Errors denote the standard deviation of three independent measurements.

e

B

DOI: 10.1021/acs.langmuir.8b00437 Langmuir XXXX, XXX, XXX−XXX

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where Rmax and Rmin are maximal and minimal R(T) values, respectively. On the basis of the two-state model, the DNA hybridization equilibrium is described as follows ⎧ 4 ⎫ ⎧ ⎫ ΔHapp ΔSapp x ⎬ ⎬=− ln⎨ + + ln⎨ 2 RT R ⎩ (1 − x) ⎭ ⎩ Capp ⎭ ⎪

⎪

⎪

(2)

where ΔHapp and ΔSapp are enthalpy and entropy changes upon duplex formation. Capp is the apparent concentration of DNA within AMS. For the 4-mer complementary DNA pairs, as shown in Figure 3, the relationships between R(T) and T took the form of sigmoidal curves that could be analyzed using eqs 1 and 2. Similarly, the sigmoidal curves for the 3-mer DNA pairs within AMS1.6, AMS2.3, and AMS5.7 could also be analyzed (Figure 3B). Since the FRET responses for the 3-mer DNA pairs within AMS3.5 and AMS7.4 were too small (Figure 3B), we did not analyze those FRET responses. The small FRET responses indicate that the 3-bp DNA duplex was not easily formed within AMS3.5 and AMS7.4; that is, the stabilities of 3bp within AMS3.5 and AMS7.4 are much smaller than those within other AMSs. The analysis of the FRET response provides values of ΔHapp/R and (ΔSapp/R + ln[4/Capp]). The apparent hybridization enthalpy, ΔHapp, can thus be directly obtained by data analysis, but an estimation of the total DNA concentration is required to obtain the apparent hybridization entropy, ΔSapp. Herein, the apparent DNA concentration, Capp, was calculated using ADNA/Vpore, where ADNA is the amount of DNA within AMS and Vpore is the pore volume of AMS (Table 1). Figure 4 shows the impact of the pore diameter (DBJH) on ΔHapp and ΔGapp(273) obtained for the complementary 4-mer DNA pair (d-TTTT/a-AAAA). The most noteworthy result, shown in Figure 4, is that the thermodynamic values for AMS1.6 and AMS2.3 are significantly larger than those for other AMSs. The BJH pore diameters of 1.6 and 2.3 nm are close to the width of the 4-bp DNA duplex (2 nm) as schematically shown in Figure 1. Since the BJH pore diameter is slightly (ca. 15%) smaller than the real pore size,17 AMS1.6 would also have enough space for duplex formation. It can therefore be concluded that size matching between the BJH pore diameter and the duplex width significantly enhances the structural stability of the 4-bp DNA duplex. The 4-bp DNA duplex would be oriented along the pore axis (Figure 1). Both −ΔHapp and −ΔGapp for the size-matched pores are about 2-fold larger than for the large pores (DBJH ≥ 3.5 nm), as shown in Figure 4. These relationships indicate that duplex formation inside the pores is an enthalpy-driven reaction, as in bulk aqueous solution, and that the enthalpic stability gain due to size matching is responsible for the enhanced stability of the

Figure 2. Typical fluorescence spectra for complementary 4-mer DNA pairs within (A) AMS2.3 and (B) AMS3.5 and for complementary 3mer DNA pairs within (C) AMS2.3 and (D) AMS3.5.

emission and is accompanied by a rise in acceptor emission. These FRET responses can be ascribed to DNA−DNA hybridization through A-T Watson−Crick hydrogen bonding, as reported in the literature.9 This view is supported by the sequence dependency of FRET responses. Noncomplementary DNA pairs (d-TTTT/a-TTTT) within AMS2.3 exhibited no significant FRET response at decreased temperatures (Figure S4). As shown in Figure 2, the complementary 4-mer DNA pair within AMS2.3 exhibited a larger FRET response with temperature than that within AMS3.5, indicating enhanced duplex stability of the 4bp-DNA duplex within AMS2.3. In the complementary 3-mer DNA pair (d-TTT/a-AAA), a similar pore size effect for the FRET response was recognized (Figure 2C,D). The FRET response of the 3-mer DNA pair within AMS3.5 was much smaller than that within AMS2.3. Fluorescence spectra for the 3-mer DNA pairs within other AMSs are shown in Figure S5. In the present study, the conventional donor-quenching method was used to analyze FRET responses.16 The donorquenching ratio, R(T), is the ratio of fluorescence intensities in the presence (FDA) and absence (FD) of an acceptor. Herein, FD was measured for AMS containing only d-TTTT (Figure S3(D)). The relationship between the hybridized fraction of DNA, x, and R(T) is described by eq 1

R(T ) − R max =x R min − R max

⎪

(1)

Figure 3. Typical temperature dependency of the FRET response obtained for each AMS with different BJH pore diameters. R(T) values found for the 4-mer DNA pair and 3-mer DNA pair are represented by shaded red circles and open black circles, respectively. The solid line shows results by a best fit using eqs 1 and 2. C

DOI: 10.1021/acs.langmuir.8b00437 Langmuir XXXX, XXX, XXX−XXX

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between the DNA bases.19,20 Tight confinement within the size-marched pore suppresses molecular fluctuations of the duplex. The combination of charge neutralization, exclusion of solvated molecules, and suppression of molecular fluctuations is likely to be responsible for the significant enthalpic gain seen in duplex stabilization inside precisely size-matched pores. Using the linearized Poisson−Boltzmann model,21 ΔG for the electrostatic adsorption of the 4-bp duplex on a planar TMAP layer (ΔGsurf) was calculated to be −29 kJ mol−1, and the ΔG value decreases exponentially with increasing distance between the DNA and the TMAP layer (Figure S5). This strong, distance-dependent electrostatic interaction is likely the key factor in duplex stability depending on pore size. This view is suggested by the fact of no significant duplex formation inside size-matched pores without TMAP modification: the complementary 4-mer DNA pair (d-TTTT/a-AAAA) adsorbed onto unmodified mesoporous silica with size-matched pores (DBJH = 2.0 nm) exhibited no significant donor quenching due to duplex formation (Figure S3E). The inner pore surface of unmodified mesoporous silica is negatively charged due to the dissociation of surface silanol groups. We conclude that this electrostatic DNA−TMAP interaction, which is a function of the distance between DNA and the TMAP layer (Figure 1), is the chief determinant of the pore size effect on the 4-bp DNA duplex stability. The thermodynamic values for the 4-bp DNA duplex in a bulk solution cannot be experimentally determined due to the energetic instability.9 We hence calculated −ΔG(273) for the 4bp DNA duplex in bulk water from the nearest-neighbor model.22,23 The calculated values were 3.0 to 4.3 kcal mol−1. Keeping in mind the destabilization effect of ethanol,24−26 −ΔG(273) for the 4-bp DNA duplex in a 70:30 (v/v) ethanol/ water mixture is expected to be more than 2-fold smaller than that in bulk water. Since −ΔGapp(273) values for the 4-bp DNA duplex inside large pores are around 3 kcal mol−1 (Figure 4B), the 4-bp DNA duplex might be slightly stabilized at the surface TMAP layer. On the other hand, it can be considered that the stability of 4-bp DNA duplex inside the size-matched pores is much larger than that in bulk systems. As shown in Figure 3, inflection temperatures of the sigmoidal FRET responses are lower for 3-mer DNA pairs than for 4-mer DNA pairs. Since the number of 3-mer DNA molecules adsorbed at AMS is slightly larger than that of 4-mer DNA molecules, the lower inflection temperatures indicate less stability of the 3-bp DNA duplex. In each AMS system, differences in −ΔGapp(273) between the 4-bp and 3-bp duplexes were estimated to be 3 to 4 kcal mol−1 (Table 2). These difference values are somewhat larger than the those predicted by the nearest-neighbor model (1.9 kcal mol−1).22,23 This disagreement of ΔG(273) as a function of the base number would be due to the high ionic strength in the vicinity

Figure 4. Effect of DBJH on enthalpy (ΔHapp) and Gibbs free energy at 273 K (ΔGapp(273)) with respect to duplex formation. The standard deviations (error bars) for the data points are obtained from three independent measurements.

duplex. The 2-fold difference in ΔGapp means that the hybridization equilibrium constant in the size-matched pore is 2 orders of magnitude larger than that in large pores. This sizematching effect on duplex stabilization is lost if the pore diameter is increased from 2.3 to 3.5 nm (Figure 4). These findings indicate that precise (nanometer-level) pore size regulation of the mesoporous silica host is needed for the stabilization of the duplex structure. The 4-mer DNA fragments are confined inside the AMS pores by electrostatic interaction between negatively charged backbone PO2− groups and the positively charged TMAP layer.9 The surface charge density of the TMAP layer (1 TMAP group nm−2) was calculated to be 0.16 C m−2, which was consistent with that of a DNA molecule (−0.15 C m−2).18 In large pore systems (DBJH ≥ 3.5 nm), the duplex is merely adsorbed onto the surface of the TMAP layer, with a substantial portion of it exposed to the solution phase (Figure 1). Inside size-matched pores, on the other hand, the phosphate groups in each DNA fragment are individually attached to the TMAP layer, as schematically shown in Figure 1, and the charges of the DNA fragments can be completely neutralized by the TMAP layer. When a duplex is formed inside a size-matched pore, the polar protic solvent molecules (H2O and ethanol) around the duplex are effectively excluded by the duplex formation, which results in the enhancement of the hydrogen bonding forces

Table 2. Comparison of Thermodynamic Values for 4-bp and 3-bp DNA Duplexes 4-bp duplex −ΔHapp/kcal mol−1 AMS1.6 AMS2.3 AMS3.5 AMS5.7 AMS7.4 a

28 33 11 19 14

± ± ± ± ±

3 4 2 2 1

3-bp duplex −ΔGapp/kcal mol−1

−ΔHapp/kcal mol−1

−ΔGapp/kcal mol−1

± ± ± ± ±

25 ± 3 15 ± 1

2.3 ± 0.9 2.4 ± 0.2

6.0 6.6 2.9 3.5 3.5

0.5 1.3 0.3 0.1 0.2

a

24 ± 1 a

a

0.7 ± 0.4 a

Weak FRET response due to less duplex stability. D

DOI: 10.1021/acs.langmuir.8b00437 Langmuir XXXX, XXX, XXX−XXX

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equilibrium constant in the size-matched pore is 2 orders of magnitude larger than those in large pores. The size matching between the pore size and the dimensions of the DNA duplex could significantly enhance the duplex stability, but the sizematching effect for the enhanced duplex stability was sensitive to the nanometer-level pore size. These results strongly recommend the necessity of precise pore size regulation of rigid inorganic mesopores for duplex stabilization. This recommendation will be applied to the stabilization of hydrogen-bonded supramolecular assemblies such as the DNA duplex.

of the DNA duplex. The nearest-neighbor parameters were established for (dA-dT) DNA duplexes in bulk water (1 M NaCl). In contrast, the apparent ionic strength in the vicinity of the duplex was calculated to be about 3 M by assuming that the quaternary amine groups and their counteranions contribute to the ionic strength.9 The base number dependency of the Gibbs free energy change for the duplex formation becomes larger with increasing ionic strength.23 We therefore consider that the lower stability of the 3-bp DNA duplex is ascribed to the decrease in one A-T base pair for the duplex formation. For 3-mer DNA pairs within AMSs, larger −ΔGapp(273) values were estimated for the size-matched pores of AMS1.6 and AMS2.3 (Table 2); −ΔGapp(273) values for AMS1.6 and AMS2.3 are about 3-fold larger than those for AMS5.7. This result indicates that the 3-bp DNA duplex is also stabilized inside the size-matched pores as in the 4-bp DNA duplex. Inside the size-matched pores, the short DNA duplexes would be oriented along the pore axis by the tight electrostatic interaction with the positively charged TMAP layer (Figure 1). Among large pore systems of AMS3.5, AMS5.7, and AMS7.4, −ΔHapp values obtained for AMS5.7 seem to be larger than those for others (Table 2). This larger −ΔHapp may imply the possibility of a specific microenvironment in the vicinity of the short DNA duplexes. Another possibility is the specific binding conformation of the short DNA duplex to the surface TMAP groups. The short DNA duplexes are surrounded by the surface TMAP groups and solvent molecules. Since the density of surface TMAP groups for AMS5.7 is almost the same as those for other AMSs (Table 1), pores of all AMSs would provide the same microenvironment for duplex formation. We hence consider the specific binding conformation for the possibility of large −ΔHapp for AMS5.7. When the width of the short DNA duplex is matched to the pore diameter, the duplex aligns along the pore axis as schematically shown in Figure 1. When the pore diameter is large enough, on the other hand, a bent DNA duplex may adsorb along the pore circumference. The rolling up of a long DNA duplex is often found for DNA− protein and DNA−bacteriophage conjugates, and the curvature radius for the tightly bent DNA has been reported to be more than 3 nm.27 In artificial system, a curvature radius of 3.3 nm is found for a bent DNA duplex around a cylindrically shaped dendronized polymer.28 With these curvature radii, curvature radii of the short DNA duplexes may be matched to those of the pores of AMS5.7, resulting in an efficient electrostatic interaction between the duplex and the surface TMAP groups. This prediction of the structure and orientation of the short DNA duplexes will be verified by molecular dynamic simulations based on the thermodynamic values obtained in this study.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.8b00437. SAXS and XRD data and additional results on fluorescence experiments (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Seiichi Nishizawa: 0000-0001-6303-2999 Akira Yamaguchi: 0000-0003-3029-3775 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by JSPS KAKENHI grant numbers 16H04160, 17K19022, and 26620112. ABBREVIATIONS AMS, amine-functionalized mesoporous silica; TMAP, trimethyl aminopropyl; FAM, fluorescein; TAMRA, tetramethylrhodamine

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REFERENCES

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CONCLUSIONS In the present study, we estimated thermodynamic values for the formation of short DNA duplexes (4- and 3-bp DNA duplexes) inside amine-functionalized silica mesopores with different pore sizes. For both DNA duplexes, it was revealed that the confinement of the duplexes inside size-matched pores resulted in enhanced duplex stability. The enhanced duplex stability for the size-matched pores could be explained by the electrostatic interaction between the surface TMAP layer and the DNA duplex. The structuring energy (−ΔGapp) of duplex formation inside size-matched pores (1.6 and 2.3 nm) was 2fold greater than that inside large pores (DBJH ≥ 3.5 nm). The 2-fold difference in ΔGapp means that the hybridization E

DOI: 10.1021/acs.langmuir.8b00437 Langmuir XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.langmuir.8b00437 Langmuir XXXX, XXX, XXX−XXX