Article pubs.acs.org/Macromolecules
Influence of Tetra(ethylene glycol) (EG4) Substitution at the Loop Region on the Intramolecular DNA i-Motif Yuhe Yang,† Yawei Sun,‡ Yang Yang,‡ Yongzheng Xing,†,‡ Tao Zhang,§ Zeming Wang,∥ 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 ‡ National Center for Nanoscience and Technology, Beijing 100190, China § Department of Chemistry, Renmin University of China, Beijing 100872, China ∥ Department of Chemistry, University of Science and Technology of China, Hefei 230026, China ABSTRACT: In this research, we employed tetra(ethylene glycol) (EG4) to substitute bases at the loop region of the intramolecular DNA i-motif formed by (CCCTAA)3CCC, and systematically studied the influence of such nonbase components on the stability and conformation of the formed structures by circular dichroism (CD), UV−vis spectroscopy and gel electrophoresis. We found that with all loop bases substituted, the i-motif structure can still form. The stability of the i-motif generally got weaker with the increase of the substitution number. Substitution at different positions might lead to different topologies. The findings above demonstrate that bases at the loop region play an important role on the stability and topology of the intramolecular DNA i-motif.
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INTRODUCTION i-motif is a four-stranded DNA structure formed by C-rich oligodeoxynucleotides (ODNs), a part of well-known human telomere.1−8 i-motif, associating with G-quadruplex,9 plays an important role in aging, cancer10,11 and gene expression regulation.12,13 Therefore, many efforts have been made to understand its detailed structure and intercalation topology. In 1993, Gehring and Leroy revealed that hemiprotonated base C forms a Hoogsteen base pair (C·C+), which leads to a parallelstranded duplex. Such two duplexes in an antiparallel orientation associate into a quadruplex under slightly acidic condition.1 The following studies found that i-motif can also be formed inter- or intramolecularly other than formed by four DNA strands.14 Owing to the inherent property and pH responsiveness, DNA i-motif have been employed in many applications. For example, DNA i-motif was used as basic building blocks to form 1-D extended nanowires based on non-Watson−Crick base pairing,15 not only DNA, but also DNA2−RNA2 i-motif, and RNA2−PNA2 i-motif have also been formed with the potential of a building block for structural RNA nanotechnology.16,17 More recently, a new type of DNA hydrogel was constructed from three-armed DNA nanostructures through the formation of intermolecular i-motif structures, furthermore, it can be reversibly switched between sol and gel state upon pH changes.18 With the help of DNA i-motif, it is possible to construct DNA nanomachines,19,20 and the key role of driving such nanomachines is the pH responsiveness of i-motif structure.21 Other noncontact stimuli (e.g., electricity, light) were also utilized to trigger the pH responsiveness,22,23 and © 2012 American Chemical Society
allow DNA nanostructures to be integrated into devices, thus performing more complex tasks. It has shown that the i-motif structure is influenced by many factors, including pH, ion strength, temperature, molecular crowding, sequence length and composition, etc.8,24−26 However, the influence of loop sequences on the intramolecular i-motif is underexplored.27 Herein, we introduce a nonbase component, tetraethylene glycol (EG4), at the loop region and study its influence on the DNA i-motif.
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EXPERIMENTAL SECTION
Materials. All chemicals were purchased from Aldrich. The organic solvents were dried according to published methods. THF was distilled from sodium under N2 immediately prior to use. Water used in all experiments was Millipore Milli-Q deionized (15.6 MΩ cm). As shown in Figure 1, the tetra(ethylene glycol) (EG4) was introduced into the loop region of the ODNs via the phosphoramidite process according to the literature.28 Synthesis of 12-O-Dimethoxytrityltetraethylene Glycol (1). A solution of dimethoxytrityl chloride (DMT-Cl, 7.45 g, 22 mmol), tetra(ethylene glycol) (3.88 g, 20 mmol) in pyridine (2 mL), and CH2C12 (150 mL) was mixed and stirred at 0 °C overnight. The organic phase was extracted with 5% of aqueous NaHCO3 (2 × 25 mL) then with water. The organic phase was dried over Na2SO4, filtered then evaporated. The compound was purified by column chromatography (CH2Cl2/methanol as eluant, 50:1, v/v). Synthesis of 12-O-Dimethoxytrityltetraethylene Glycol-1[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (2). The Received: February 1, 2012 Revised: March 1, 2012 Published: March 13, 2012 2643
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Figure 1. Synthetic route of 12-O-dimethoxytrityltetraethylene glycol (1) and 12-O-dimethoxytrityltetraethylene glycol-1-[(2-cyanoethyl)-(N,Ndiisopropyl)]-phosphoramidite (2).
Scheme 1. (A) Schematic Illustration of EG4 Modified ODNs;a (B) Illustration of the Molecular Structures of EG4 Modified ODN
a
Each orange ball stands for one EG4 unit. at 4 °C overnight in 100 MES buffer (pH 5.0). After the sample was loaded on a native 15% polyacrylamide gel, the gel electrophoresis was run at 4 °C. All gels were stained by Stains All and then analyzed on a Beijing Sage Creation phosphorimager instrument. Circular Dichroism (CD) Spectra. The ODN samples were dissolved in 100 mM MES buffer (pH 5.0) to get a concentration of 2 μM, heated to 95 °C for 5 min, and gently cooled to room temperature. The CD spectra were collected on an Applied Photophysics Chirascan Spectropolarimeter, the determination was carried out at the range of 220−320 nm at room temperature in a 1.0 cm length cell. The reported spectra correspond to the average of at least three scans. The scan of the buffer alone was used as a control and subtracted from the average scan for each sample. CD melting curves were measured at 289 nm, and the temperature was scanned at a heating rate of 1 °C/min. The melting point (Tm) was obtained from the peak of the corresponding differential melting curve.
compound (1) obtained above (2.2 g, 4.4 mmol) was kept under vacuum overnight then dissolved in CH2C12 (10 mL) containing N,Ndiisopropylethylamine (DIPEA, 1.1 g, 8.8 mmol). 2-Cyanoethyl diisopropyichlorophosphite (PAM-Cl, 1.3 g, 5.3 mmol) was added under nitrogen atmosphere with stirring. After 30 min, the mixture was poured into 50 mL of ethyl acetate and extracted with 10% of aqueous NaHCO3 (2 × 80 mL) then water. The organic phase was dried over Na2SO4, filtered then evaporated. The compound was purified by column chromatography (CH2Cl2/THF/Et3N as eluant, 50:49:1, v/v/ v). Synthesis of ODNs Modified with EG4. The syntheses were conducted automatically on an Applied Biosystems 394 DNA Synthesizer, then purified by denatured polyacrylamide gel electrophoresis (20% polyacrylamide, 1 × TBE (Tris−borate−EDTA), 7 M urea) and desalted by C18 columns purchased from Glen Research. The molecular weights of the purified ODNs were determined by BIFLEX III MALDI−TOF mass spectroscopy. Modified ODNs strand concentration was determined by Varian Cary 100 UV−vis spectrometer at 260 nm, in a pH 8.0 buffer. The single strand extinction coefficient was calculated from mononucleotide and dinucleotide data of a nearest-neighbor approximation.29 Gel Electrophoresis Analysis. The purity of EG4 modified ODNs were checked by 15% denatured PAGE at a concentration of 40 μM in pH 8.0 TBE buffer at room temperature. To verify whether the EG4 modified ODNs can form secondary structures, the samples were slowly annealed from 95 °C to room temperature and then incubated
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RESULTS AND DISCUSSION Design of EG4 Modified ODNs. In this paper, we studied the influence of loop region on the formation of i-motif structure, in particular, when the base at loop region was replaced by nonbase unit, tetra(ethylene glycol) (EG4), which locally destroys the base stacking,14 thus loosens the interaction between bases, meanwhile, the charge density keeps unchanged as the phosphate part still remains after the modification. 2644
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We also carried out denaturing gel electrophoresis to confirm the purity of EG4 modified ODNs. As shown in Figure 2A, all modified ODNs had a clean single band, indicating the synthesis was efficient and the product was pure, this
Scheme 1 shows the design of EG4 modified ODNs, including (i) unmodified ODNs (as control); (ii) modified ODNs with one or three EG4 at one, two or three loops. The structure includes 3 loops as indicated in the left top image: the loop near the 5′ end, in the middle, and near the 3′ end were named as loop 1, loop 2, and loop 3, respectively. We adopted the following convention for all the ODNs: the letter S indicates single substitution, which refers to base A at the middle of one loop substituted with EG4 and the letter T indicates triple substitution where all three bases in one loop were substituted. L1, L2, and L3 represent the loop positions of the EG4 unit. For example, T-L1L3 represents three EG4 units substituted by three bases at loop 1 and 3, and no replacement at loop 2. Synthesis and Characterization of ODNs. In order to introduce EG4 into the loop region of the ODNs, the conventional phosphoramidite process was conducted.28 In a typical experiment, as shown in Figure 1, EG4 was first modified with DMT to get compound (1) and then reacted with PAMCl to obtain compound (2). Normally, automatic synthesizer uses acetonitrile (ACN) as solvent, in case of compound (2), which has good solubility in ACN, therefore, it is possible to treat compound (2) as a common base to synthesize modified ODNs directly. The position of EG4 at loop region could be programmed by automatic synthesizer. The yield of the modified ODNs was over 90%, and this synthetic method provides a general guidance on inserting functional groups into DNA. In order to characterize EG4 modified ODNs, the MALDITOF mass spectroscopy was conducted, see Table 1. It is shown that the observed molecular weight has a good agreement with the calculated value and the error was below 0.4%, indicating EG4 modified ODNs were successfully synthesized.
Figure 2. (A) Denaturing gel electrophoresis of EG4 modified ODNs at pH 8.0. (B) Native gel electrophoresis of EG4 modified ODNs at pH 5.0. The red number in the middle represents the substitution number of EG4 unit.
observation, when combined with the result from mass spectroscopy in Table 1, giving further evidence that the EG4 modified ODNs with high purity were indeed obtained. Formation of i-Motif Sturcture. We sought to determine if the modification of EG4 influences the formation of intramolecular i-motif, and CD spectra and native gel electrophoresis at pH 5.0 were employed. As shown in Figure 3, the black line represents the unmodified sequence, with a positive peak at 289 nm, a negative peak at 258 nm and a crossover at 273 nm, which are characteristic peaks of C·C+ pairs,30 indicating intramolecular i-
Table 1. Summary of Molecular Weights of EG4 Modified ODNs name control S-L1 S-L2 S-L3 S-L1L2L3 T-L1 T-L2 T-L3 T-L1L2 T-L2L3 T-L1L3 T-L1L2L3 a
sequence (5′-3′)a CCC TAA CCC TAA CCC TAA CCC CCC T*A CCC TAA CCC TAA CCC CCC TAA CCC T*A CCC TAA CCC CCC TAA CCC TAA CCC T*A CCC CCC T*A CCC T*A CCC T*A CCC CCC *** CCC TAA CCC TAA CCC CCC TAA CCC *** CCC TAA CCC CCC TAA CCC TAA CCC *** CCC CCC *** CCC *** CCC TAA CCC CCC TAA CCC *** CCC *** CCC CCC *** CCC TAA CCC *** CCC CCC *** CCC *** CCC *** CCC
calcd [g/mol]
found [g/mol]
error (%)
6200
6207
0.11
6152
6148
0.07
6152
6150
0.03
6152
6151
0.02
6056
6036
0.33
6038
6044
0.10
6038
6039
0.02
6038
6042
0.07
5876
5882
0.10
5876
5880
0.07
5876
5884
0.14
5714
5720
0.11
Figure 3. CD spectra of EG4 modified ODNs at pH 5.0.
motif structure was formed at pH 5.0.31 It is observed that after bases at different loop regions were substituted with different numbers of EG4, the positive peaks still appeared around 289 nm, at a range of 288−291 nm, and the negative peaks and crossovers also showed a similar behavior; therefore, it is concluded that the i-motif structure still formed with EG4 modified ODNs.
An asterisk stands for one EG4 unit. 2645
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Table 2. Summary of Tm of EG4 Modified ODNs
Stability Influenced by EG4 Substitution. The stability of the intramolecular i-motif structures formed by 12 sequences derived from (CCCTAA)3CCC by substitution of the TAA loops with EG4 spacer is investigated. We sought to examine the melting point (Tm) of ODNs by CD, and evaluate the influence of different substitution numbers and positions of EG4 on the stability of modified ODNs. Five typical sequences with substitution numbers of 0, 1, 3, 6, and 9 were chosen, as shown on the top of Figure 4, based on the following rules: unmodified ODN was used as control; S-L1 contained 1 EG4 unit at the middle of loop 1; T-L1 contained 3
name
number of EG4
Tm (±0.5 °C)
control S-L1 S-L2 S-L3 S-L1L2L3 T-L1 T-L2 T-L3 T-L1L2 T-L2L3 T-L1L3 T-L1L2L3
0 1 1 1 3 3 3 3 6 6 6 9
64.9 61.7 64.9 62.2 62.5 58.2 58.8 56.9 49.3 50.7 52.2 45.4
Next, we looked into the triple substitution at loop regions. Triple substitution at one loop was shown in the third column of Scheme 1, corresponding to T-L1, T-L2, and T-L3, their Tm decreased to 58.2, 58.8, and 56.9 °C, respectively. Such reduction was not only attributed to the disruption of π−π stacking between loop bases as that occurred in single substitution, but also the stacking between A·T base pair and its adjacent C·C+, which are considered to make i-motif highly stable.14 The reduction of Tm became more pronounced as the number of triple substitution increased to two, shown in the last column of Scheme 1, corresponding to T-L1L2, T-L2L3, and T-L1L3, and their Tm further decreased to 49.3, 50.7, and 52.2 °C, respectively, which has a good agreement with our explanation above. Note that the EG4 substitution at loop 2 showed less decrease on Tm comparing to that at other two loops. This effect has the same trend as that in the single substitution, suggesting that either single or triple substitution at loop 2 has less influence on Tm than that at loop 1 and 3. Finally as all bases at all loop regions were substituted, shown on the bottom left of Scheme 1, T-L1L2L3, the Tm decreased to 45.4 °C, almost 20 °C lower than the control, this result when combined with Tm data above, suggests that in case of triple substitution, the more EG4 units in the loop region, the lower the Tm and thus the lower the stability of the system. We make a final observation regarding to the influence of substitution position on topology. We studied the native gel electrophoresis at pH 5.0, at which condition, it is well-known that intramolecular i-motif structure forms.8 In Figure 2B, lanes 2, 3, and 4 were ODNs with single substitution, it appeared that substitution at both loop 1 and loop 3 (S-L1 and S-L3) resulted in a band position proximal to that of unmodified one (lane 1), however, the ODN modified at loop 2 (S-L2) exhibited a faster migration. Similar results were observed with the triple substitution. Both T-L1 (lane 6) and T-L3 (lane 8) had similar migration rate, but slower than T-L2 (lane 7). Since ODNs with a same number of EG4 possess the same molecular weight and charge, the different migration rates might be caused by different topologies. It is known that loops 1 and 3 correspond to wide grooves, and loop 2 corresponds to a narrow groove, the faster migration might be attributed to the more compact structure of ODNs induced by the modification of EG4 at the narrow groove. This provides interesting information for other DNA sequence modification designs.
Figure 4. CD melting curves of EG4 modified ODNs at pH 5.0.
EG4 units at loop 1; T-L1L2 contained 6 EG4 units, one triple substitution at loop 1 and another at loop 2; T-L1L2L3 contained 9 EG4 units, showing that all bases at all loop regions were replaced. The CD melting curve in Figure 4 showed a clear trend that with the increase of substitution number from 0, 1, 3, and 6 to 9, the Tm continuously decreased from 64.9 to 61.7, 58.2, 49.3, and 45.4 °C, respectively, indicating that substitution of EG4 at the loop region disturbed the stability of DNA i-motif. This might be attributed to the break of, such as π−π stacking between loop bases in case of single substitution, the stacking between base A and C·C+ and the base stacking between A·T base pair and C·C+,14 the latter two in particular for the case of triple substitution. In order to further investigate the influence of both substitution numbers and positions on the stability of modified ODNs, we characterized all Tm by CD and the results are listed in Table 2. We examined single substitution as shown in the second column of Scheme 1, S-L1, S-L2, and S-L3, and their corresponding Tm were 61.7, 64.9, and 62.2 °C, respectively, showing a relatively small difference from the control sequence (Tm = 64.9 °C). Note that the influence of single substitution at loop 2 on Tm is smaller than that at loop 1 and loop 3, this will be further discussed in triple substitution section. Moreover, when three discrete bases at the middle of all loops were substituted, S-L1L2L3, shown on the left of the second row in Scheme 1, its Tm was 62.5 °C, which is only 2.4 °C lower than the control, suggesting that single substitution has little influence on the stability of the system. The preservation of the stability indicates that single substitution of EG4 is a feasible option of adding functional groups in DNA i-motif.
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CONCLUSION In this work, we studied the influence of the bases at the loop region on the intramolecular i-motif structure of human 2646
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telomeric DNA. We discovered that though i-motif structure can still form, the stability and topology of EG4 modified ODNs varied with substitution numbers and positions. In general, single substitution at all loops preserved the stability of i-motif, while the triple substitution of three bases at one loop resulted in a significant decrease of stability, and the more EG4 substitutions, the less stable of the system. The substitution at the narrow groove results in a faster migration in agarose gel comparing to those at other two wide grooves, suggesting modification at different positions has different extents of influence on the topology. These results would benefit to the in-depth understanding of the folding topology of i-motif and their future applications in drug discovery and diagnosis.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: (D.L.)
[email protected]; (Z.Y.) zyang@ tsinghua.edu.cn. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank the National Basic Research Program of China (973 program, No. 2011CB935701), the National Natural Science Foundation of China (No. 21121004, 91027046, and 20974030), and the NSFC-DFG joint project TRR61 for financial support.
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