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. 2021 Oct 14;6(42):28263–28269. doi: 10.1021/acsomega.1c04346

A pH and Mg2+-Responsive Molecular Switch Based on a Stable DNA Minidumbbell Bearing 5′ and 3′-Overhangs

Jiacheng Zhang †, Yang Wang †, Liqi Wan ‡, Yuan Liu §,*, Jie Yi †, Sik Lok Lam ‡, Pei Guo †,*
PMCID: PMC8552455  PMID: 34723023

Abstract

graphic file with name ao1c04346_0007.jpg

Minidumbbell (MDB) is a non-B DNA structure of which the thermodynamic stability is sensitive to a chemical environment such as pH, serving as a potential structural motif in constructing DNA-based molecular switches. This work aims to design thermodynamically stable MDB structures bearing 5′ and 3′-overhanging deoxyribonucleotides in order to examine the possibility of MDB to be functionalized. Via making use of 5-methylcytosine and adjusting the pH of solution to be acidic, MDBs bearing 1-nucleotide (nt) or 2-nt overhanging residues at the 5′ and 3′-ends have been obtained. Based on one of the new MDB sequences, we have designed a molecular switch that could respond to dual inputs of pH and Mg2+. The MDB strand and its partner strand formed a duplex (the “ON” state) upon inputting pH 7 and Mg2+, whereas the duplex dissociated to restore the MDB structure (the “OFF” state) upon inputting pH 5 and EDTA. The demonstration on the ability of MDB to sustain 5′ and 3′-overhanging residues and the construction of a pH and Mg2+-responsive molecular switch will extend the application of MDB structures in dynamic DNA nanotechnology.

1. Introduction

A DNA molecule can exist in various conformations apart from the classical B-form structure. Non-B DNAs, such as the triplex,1 G-quadruplex,2 and i-motif,3 have been found to associate with different biological functions and genetic instabilities.4 In recent years, non-B DNAs have attracted extensive attentions owing to their potential applications in DNA nanotechnology. In particular, molecular switches based on non-B DNAs can respond to a variety of inputs such as pH, metal ions, and light,5−7 offering an advantage over those governed solely by Watson–Crick base pairs.6 The non-B DNA, which is sensitive to chemical change, can serve as a template to guide the assembly of metal nanoparticles8,9 and control drug release10,11 and can also be used as a probe in biosensing when reporter molecules are conjugated with the non-B DNA.12,13 These properties, supplemented with their biocompatibility, pose the non-B DNA as a promising structural candidate in bio-related research.

Minidumbbell (MDB) is a non-B DNA structure formed by eight to ten-nucleotide sequences.14,15 It is composed of two tetraloops or pentaloops with extensive interactions between intraloop and interloop residues. For the MDBs containing two type II tetraloops, the first (L1/L1′) and fourth (L4/L4′) loop residues form a loop-closing base pair, the second loop residue (L2/L2′) sits in the minor groove, and the third loop residue (L3/L3′) stacks on the nearby loop-closing base pair in each loop (Figure 1).14 The MDB structures have been found to form in expandable CCTG and ATTCT repeats of which expansion mutations associate with myotonic dystrophy type 2 and spinocerebellar ataxia type 10 neurodegenerative diseases, respectively, suggesting a potential role of MDB in mediating genetic instabilities of these repeats.14,15 It has been proposed that MDBs could escape from mismatch repair via a competing mechanism.16 Small ligands that can target the MDBs may offer a strategy for disease therapy.4 Apart from its potential biological roles, the MDB has been utilized to construct chemical stimuli-responsive molecular switches.17,18 For instance, upon repeatedly adjusting the pH from 7 to 5 and vice versa, formation and dissociation of a DNA duplex could be controlled in a reversible manner at room temperature.17 Due to the small molecular size, MDB-based molecular switches were easier to be manipulated compared to those using triplexes19 and quadruplexes6 formed by longer sequences or those relying on strand displacement.20

Figure 1.

Figure 1

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Averaged NMR solution structure of the MDB containing two CCTG tetraloops (PDB ID: 5GWL).

Further functionalization of MDB, such as conjugation with gold nanoparticle, lipophilic moiety, and fluorophore/quencher, will greatly extend the application of MDB. However, the ability of MDB to sustain functional modification depends on whether the thermodynamic stability of MDB is high enough, as any chemical addition at the termini of an MDB is expected to weaken the 5′ and 3′-terminal stacking that is essential to the MDB formation. So far, all reported MDBs formed by natural sequences have mild thermodynamic stabilities, i.e., their melting temperature (Tm) values ranged from 17 to 46 °C,14,15,17,21,22 and it is unclear whether MDBs are stable enough to be functionalized. This work aims to investigate if any MDBs can sustain functional modifications. We added 5′ and 3′-overhanging residues at the termini of the MDB to mimic the presence of functional modifications. Their structures and thermodynamic stability were examined using solution nuclear magnetic resonance (NMR) and ultraviolet (UV) spectroscopy, respectively. It was found that MDBs formed by natural sequences could not sustain overhanging residues. Upon using 5-methylcytosine (mC) and adjusting the pH of solution to be acidic, stable MDBs bearing 5′ and 3′-overhanging residues were obtained. On the basis of a newly designed sequence, we have constructed a DNA molecular switch that could switch to the “ON” state (a duplex) upon inputting pH 7 and Mg2+ and switch back to the “OFF” state (two separated strands) upon inputting pH 5 and EDTA. Results of this work pave the way for functionalization of MDBs and extend their applications in dynamic DNA nanotechnology.

2. Results and Discussion

All DNA sequences used in this study are shown in Table 1. We first investigated if the MDB formed by 5′-CCTGCCTG-3′ at pH 5 could sustain 5′ and 3′-overhangs, as this sequence formed the most stable MDB among all reported natural sequences,17 and it was named as (CCTG)2 here. Based on this model sequence, we designed two sequences by adding (i) a 5′-T and a 3′-T and (ii) a 5′-A and a 3′-T to (CCTG)2, which were named as T(CCTG)2T and A(CCTG)2T, respectively. Further, we modified the core MDB-forming sequence in T(CCTG)2T and A(CCTG)2T by substituting its minor groove cytosine residues (C2 and C6) with 5-methylcytosine residues (mC2 and mC6). The two newly obtained sequences were named as T(CmCTG)2T and A(CmCTG)2T, respectively. To investigate if the methylated MDBs could sustain extra overhanging residues, a sequence named as TT(CmCTG)2TT was designed. For constructing a DNA molecular switch based on T(CmCTG)2T, two sequences were used, including 5′-CAGGCAGG-3′ and 5′-TAGGCAGG-3′, which were named as S1-wc and S2-tg, respectively.

Table 1. DNA Sequences Used in this Study.

name sequence
(CCTG)2 5′-CCTGCCTG-3′
T(CCTG)2T 5′-TCCTGCCTGT-3′
A(CCTG)2T 5′-ACCTGCCTGT-3′
T(CmCTG)2T 5′-TCmCTGCmCTGT-3′
A(CmCTG)2T 5′-ACmCTGCmCTGT-3′
TT(CmCTG)2TT 5′-TTCmCTGCmCTGTT-3′
S1-wc 5′-CAGGCAGG-3′
S2-tg 5′-TAGGCAGG-3′
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2.1. MDB Was Unable to Form in T(CCTG)2T and A(CCTG)2T

We first study the solution structures of T(CCTG)2T and A(CCTG)2T to examine if the MDB could sustain 5′ and 3′-overhanging residues, which were a mimic of adding functional modifications at the two termini of MDBs. In the reported (CCTG)2 MDB at pH 5,17 (i) C1 and C2 were fully protonated at their N3 sites, (ii) C1+-G4 and C5-G8 formed the Hoogsteen and Watson–Crick loop-closing base pairs, respectively, (iii) C2+ and C6 formed a reverse wobble mispair via three hydrogen bonds, and (iv) T3 and T7 stacked on C1+-G4 and C5-G8 base pairs, respectively (Figure 2A). These structural features lead to corresponding NMR features, including (i) the downfield shifted C1+ and C2+ amino H41/H42 proton signals at ∼9.0 to 10.5 ppm due to protonation at their N3 sites, (ii) the C1+ H3 signal at ∼17.0 ppm in the C1+-G4 Hoogsteen base pair and the G8 H1 signal at ∼13.0 ppm in the C5-G8 Watson–Crick base pair, (iii) C6 H41/H42 at ∼8.5/6.9 ppm together with C2+ H41/H42 at ∼9.8/9.3 ppm in the C2+·C6 reverse wobble mispair, and (iv) upfield shifted T3 and T7 H7 signals at ∼1.4 ppm due to the shielding effect of their nearby loop-closing base pairs.17 Based on the reference NMR spectrum of the (CCTG)2 MDB, we first acquired 1D 1H NMR spectra for T(CCTG)2T and A(CCTG)2T.

Figure 2.

Figure 2

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(A) Schematic of the (CCTG)2 MDB. (B, C) 1D 1H NMR spectra of T(CCTG)2T and A(CCTG)2T at pH 7, 6, and 5 in comparison with the reference spectrum of (CCTG)2 MDB at pH 5. All spectra were acquired at 0 °C. The H1/H3 and H41/H42 signal intensities were amplified by fourfolds relative to the methyl H7 signals.

Figure 2B,C shows the 1D 1H NMR spectra of T(CCTG)2T and A(CCTG)2T at pH 7, 6, and 5 in comparison with the reference spectrum of (CCTG)2 MDB at pH 5. Unfortunately, upon lowering the pH from 7 to 5, we did not observe any characteristic NMR signals belonging to the MDB in T(CCTG)2T and A(CCTG)2T, such as the C1+ H3 signal at ∼17 ppm, G8 H1 signal at ∼13 ppm, C1+ and C2+ H41/H42 signals at 9.0–10.5 ppm, and upfield shifted T3 and T7 H7 signals at ∼1.4 ppm, revealing that the MDB was not formed in these two sequences.

The original (CCTG)2 MDB has a Tm value of ∼46 °C, yet an addition of 5′ and 3′-overhanging residues lead to the failure in forming an MDB even at 0 °C. The result suggested that 5′ and 3′-overhanging residues strongly destabilized the MDB structure. This is different from the effects of overhanging residues on DNA duplexes or G-quadruplexes, wherein addition of one or two overhanging residues could stabilize the structures via gaining base–base stackings between the overhanging residues and terminal base pairs23 and G-tetrads.24,25 However, in the case of MDB structures, the overhanging residues might weaken the base–base stacking between the 3′-terminal G8 and 5′-terminal C1+, which is crucial to the MDB formation. Therefore, designing thermodynamically more stable MDBs that can compensate the destabilization caused by overhanging residues appears to be the prerequisite for functionalization of MDBs.

2.2. T(CmCTG)2T and A(CmCTG)2T Formed Stable MDB Structures

Previously, we have reported that hydrophobic interactions between the methyl groups of minor groove residues and the 2′-methylene groups of loop-closing base pair residues provided substantial stabilization to the MDB structure, e.g., an increase in Tm by ∼12 °C was achieved by introducing such hydrophobic interactions.22 5-Methylcytosine, which serves as an epigenetic biomarker in gene regulations,26 has been reported to enhance thermodynamic stabilities of various types of DNA structures such as duplexes,27 triplexes,28 i-motifs,29 and G-quadruplexes.30 Herein, we substituted the minor groove residues (C2 and C6) in T(CCTG)2T and A(CCTG)2T with 5-methylcytosine residues and obtained T(CmCTG)2T and A(CmCTG)2T.

It was found that upon lowering the pH to 5, both T(CmCTG)2T and A(CmCTG)2T showed characteristic NMR signals of MDB, including (i) the C1+ H3 at 16.9 ppm and G8 H1 at 12.8 ppm, (ii) downfield shifted C1+ H41/H42 (10.1/9.1 ppm) and mC2+ H41/H42 (10.4/8.9 ppm) signals, and (iii) upfield shifted T3 and T7 H7 signals at 1.4 ppm (Figure 3), all of which agreed with those in the reference (CCTG)2 MDB. These suggested that T(CmCTG)2T and A(CmCTG)2T folded into MDB structures bearing 5′ and 3′-overhanging residues at pH 5. Indeed, such characteristic signals were also observed at pH 6 and their peak intensities were similar to those at pH 5, suggesting that C1 and mC2 were almost fully protonated and the MDBs were formed stably at pH 6.

Figure 3.

Figure 3

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1D 1H NMR spectra of (A) T(CmCTG)2T and (B) A(CmCTG)2T at pH 7, 6, and 5 in comparison with the reference spectrum of the (CCTG)2 MDB. All spectra were acquired at 0 °C. The H1/H3 and H41/H42 signal intensities were amplified by fourfolds relative to H7 signals.

As determined by UV melting experiments, the Tm values of these two MDBs were ∼51 and 52 °C, respectively (Table 2), suggesting that they would predominantly remain folded at room temperature or even physiological temperature. Noticeably, the Tm value of T(CmCTG)2T or A(CmCTG)2T was even higher than that of the (CCTG)2 MDB (∼46 °C),17 revealing the great effectiveness of cytosine methylation in stabilizing the MDB structure.

Table 2. Thermodynamic Parameters of the T(CmCTG)2T, A(CmCTG)2T, and TT(CmCTG)2TT MDBsa.

MDB Tm (°C) ΔH° (kcal·mol–1) ΔS° (cal·K–1·mol–1) ΔG°298K (kcal·mol–1)
T(CmCTG)2T 51 ± 2 –24 ± 1 –73 ± 3 –1.9 ± 0.1
A(CmCTG)2T 52 ± 2 –20 ± 1 –62 ± 4 –1.66 ± 0.03
TT(CmCTG)2TT 45 ± 2 –12 ± 2 –38 ± 7 –0.76 ± 0.08
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a

The average and uncertainty values were determined by three replicative UV melting experiments.

2.3. TT(CmCTG)2TT Formed a Stable MDB Structure

Upon successful construction of MDBs carrying a 1-nucleotide (nt) overhang at both 5′ and 3′-ends, we wondered if the MDB could sustain longer 5′ and 3′-overhangs. Accordingly, we designed TT(CmCTG)2TT that carries a 2-nt overhang at both 5′ and 3′-ends. At pH 5, TT(CmCTG)2TT displayed NMR signatures that are suggestive of the MDB formation (Figure 4), including (i) the C1+ H3 at 16.9 ppm and G8 H1 at 12.8 ppm, (ii) the downfield shifted C1+ H41/H42 (10.1/9.1 ppm) and mC2+ H41/H42 (10.9/9.5 ppm) signals, and (iii) the upfield shifted T3 and T7 H7 signals at 1.4 ppm.

Figure 4.

Figure 4

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1D 1H NMR spectra of TT(CmCTG)2TT in comparison with the reference spectrum of (CCTG)2 MDB. The spectra were acquired at 0 °C. The intensity scale of the H1/H3 and H41/H42 regions was increased by fourfolds relative to that of the methyl H7 region.

The Tm of the TT(CmCTG)2TT MDB was determined to be ∼45 °C (Table 2), which was ∼6 °C lower than that of the T(CmCTG)2T or A(CmCTG)2T MDB. The ΔG°298K of TT(CmCTG)2TT was less negative than that of T(CmCTG)2T or A(CmCTG)2T MDB, and this was attributed predominantly to a less favorable ΔH° and partially compensated by the favorable ΔS°. As suggested by the less negative ΔH°, the longer overhangs in TT(CmCTG)2TT might lead to a steric hindrance at the terminal junction of the MDB so that the base–base stackings were weakened.

2.4. Rational Design of a DNA Molecular Switch Based on the T(CmCTG)2T MDB

DNA-based molecular switches responding to external stimuli, e.g., pH, metal ion, and light, show potential applications in bio-related research such as biosensing and controlled drug release.6,19,20 As T(CmCTG)2T, A(CmCTG)2T, and TT(CmCTG)2TT formed stable MDBs sustaining overhanging residues (Figures 3,4), we wondered if they could be used to construct a DNA molecular switch. To test this, T(CmCTG)2T was selected for a proof-of-concept study. We first constructed a system that was composed of the T(CmCTG)2T strand and the S1-wc strand. It was anticipated that T(CmCTG)2T and S1-wc could form a duplex containing eight Watson–Crick base pairs in the presence of Mg2+ at pH 7 (the “ON” state), while the duplex would dissociate to restore the MDB formation upon inputting EDTA and pH 5 (the “OFF” state), as the MDB would become more stable than the duplex at pH 5 especially when the Mg2+ was chelated by EDTA.

CD experiments were performed to monitor the structural conversion. The CD spectrum of T(CmCTG)2T MDB at pH 5 displayed a positive band at ∼290 nm, which served as a reference spectrum (Figure S1). The CD spectrum of the T(CmCTG)2T and S1-wc mixture in 10 mM NaPi (pH 7) and Mg2+ showed a major positive absorption band at ∼265 nm, which was consistent with the CD spectral feature of DNA duplexes31 and thus suggested the hybridization between T(CmCTG)2T and S1-wc (Figure S1). The CD spectrum of the T(CmCTG)2T and S1-wc mixture in 10 mM NaPi (pH 5), Mg2+, and excessive EDTA displayed a positive band at ∼290 nm, which was in line with the CD absorbance of the T(CmCTG)2T MDB, but unfortunately the positive band at ∼260 nm was still clearly observed (Figure S1). This suggests that the duplex did not fully dissociate upon adjusting the pH to 5 and adding EDTA, probably because the difference in the thermodynamic stability between the T(CmCTG)2T MDB and the complementary duplex at pH 5 was not large enough.

Based on the above trial, we rationally designed another DNA molecular switch that was composed of T(CmCTG)2T and S2-tg. The duplex, if formed, would contain a T·G mismatch and seven Watson–Crick base pairs. It has been reported that the Tm of the DNA duplex was lowered by 6–7 °C upon substituting one C-G Watson–Crick base pair with a T·G mismatch.32,33 Therefore, we expected that the thermostability of the duplex formed by T(CmCTG)2T and S2-tg would be lower than that of the duplex formed by T(CmCTG)2T and S1-wc. The CD spectrum of the T(CmCTG)2T and S2-tg mixture in 10 mM NaPi (pH 7) and Mg2+ showed a positive band at ∼265 nm (Figure 5A, blue line), suggesting a vast majority of duplex formation. The CD spectrum of the T(CmCTG)2T and S2-tg mixture in 10 mM NaPi (pH 5), Mg2+, and excessive EDTA showed a major positive band at ∼290 nm, and the positive band at ∼265 was almost vanished (Figure 5A, red line). This reveals that the usage of a T·G mismatch to lower the thermodynamic stability of the duplex facilitated structural conversion to the MDB at pH 5.

Figure 5.

Figure 5

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(A) CD spectra of the T(CmCTG)2T MDB in 10 mM NaPi (pH 5), Mg2+, and EDTA (black line), the T(CmCTG)2T and S2-tg mixture in 10 mM NaPi (pH 7) and Mg2+ (blue line), and the T(CmCTG)2T and S2-tg mixture in 10 mM NaPi (pH 5), Mg2+, and EDTA (red line). (B) Truth table (top) and scheme (bottom) of the molecular switch using T(CmCTG)2T and S2-tg.

In short, we have successfully constructed an MDB-based molecular switch that is composed of T(CmCTG)2T and S2-tg (Figure 5B). In the presence of pH 7 and Mg2+ dual inputs, the system was in an “ON” state, i.e., T(CmCTG)2T and S2-tg formed a duplex. Upon inputting pH 5 and excessive EDTA dual inputs, the system was in an “OFF” state, i.e., T(CmCTG)2T and S2-tg adopted an MDB and a single strand, respectively. The MDB-based molecular switch designed in this study sensitively responds to pH and Mg2+/EDTA in a programmable manner, which shows structural conversions at room temperature with an easy manipulation. More importantly, the 5′ and 3′-overhanging residues in the newly designed stable MDBs may be replaced by reporter molecules such as fluorophores to extend the application of MDBs in biosensing.

3. Conclusions

This study, for the first time, demonstrates that the MDB structure could sustain 5′ and 3′-overhanging residues, suggesting the possibility of functionalization of MDBs. The newly designed MDBs remained stable at room temperature or even physiological temperature. A molecular switch based on one of the new MDBs was constructed. Dual inputs of pH 7 and Mg2+ could maintain the system in an “ON” state, whereas the dual inputs of pH 5 and EDTA could switch the system to an “OFF” state. The result of this work paves the way for extending the application of an MDB structure in dynamic DNA nanotechnology such as biosensing.

4. Materials and Methods

4.1. DNA Sample Preparation

DNA samples were synthesized using an Applied Biosystems Model 394 DNA/RNA Synthesizer. Phosphoramidites used for DNA synthesis were purchased from ChemGenes Corporation (U.S.A). The synthesized DNA products were purified by denaturing polyacrylamide gel electrophoresis and diethylaminoethyl sephacel anion exchange column chromatography and finally desalted using Amicon Ultra-4 centrifugal filter devices. The UV absorbance at 260 nm was measured for DNA quantitation.

4.2. NMR Experiments

NMR samples were prepared by dissolving ∼0.1 μmol purified DNA in 500 μL of 90% H2O/10% D2O (v/v) solutions containing 0.02 mM 2,2-dimethylsilapentane-5-sulfonic acid (DSS) and 10 mM sodium chloride (NaCl). The pH of NMR samples was adjusted by adding hydrochloric acid (HCl) or sodium hydroxide (NaOH). NMR experiments were conducted on a Bruker AVANCE-500 and/or AVANCE-700 spectrometers. In one-dimensional (1D) 1H experiments and two-dimensional (2D) nuclear Overhauser effect spectroscopy (NOESY) experiments, the excitation sculpting pulse sequence34 was employed to suppress the water signal. The 1D 1H spectra were processed by applying an exponential window function with a line broadening of 2 Hz. For 2D NOESY experiments, datasets of 4096 × 512 were acquired and then zero-filled to give 4096 × 4096 spectra with a cosine window function applied to both dimensions. All NMR spectra were acquired at 0 °C unless otherwise specified.

4.3. 1H Resonance Assignments

Sequential resonance assignments of DNA sequences were made from the NOESY H6/H8-H1′ fingerprint region using standard methods.35 The amino proton H41/H42 resonances were assigned based on intra-nucleotide H5-H41/H42 NOEs for cytosine and intra-nucleotide H7-H41/H42 NOEs for 5-methylcytosine. 1H resonance assignments of T(CmCTG)2T, A(CmCTG)2T, and TT(CmCTG)2TT are shown in Figures S2–S7.

4.4. UV Melting Experiments

UV melting experiments were carried out to determine the thermodynamic stabilities of T(CmCTG)2T, A(CmCTG)2T, and TT(CmCTG)2TT. Samples for UV melting experiments were prepared to contain 5 μM DNA in 1000 μL buffer solutions containing 10 mM sodium phosphate (NaPi) at pH 5. A cuvette with a path length of 10 mm was used. The absorbance at 260 nm was monitored as a function of temperature using a Hewlett–Packard 8453 diode-array UV–visible spectrophotometer, and the temperature was measured by a BetaTHERM thermistor temperature sensor inserted into the cuvette. The absorbance was measured from 25 to 80 °C for T(CmCTG)2T, from 20 to 80 °C for A(CmCTG)2T, and from 15 to 80 °C for TT(CmCTG)2TT, with a step size of 1 °C and a holding time of 1 min. Thermodynamic parameters, including the Tm, change in enthalpy (ΔH°), change in entropy (ΔS°), and change in Gibbs free energy (ΔG°298K), were obtained by fitting the melting profiles with a two-state transition model.36 Three replicative UV melting measurements were conducted for each DNA sequence.

4.5. Circular Dichroism (CD) Experiments

To monitor the molecular switching process in our designed DNA molecular switch, CD experiments were performed using a Chirascan V100 CD spectrometer with a bandwidth of 1 nm at room temperature. Five CD samples were prepared, including (i) 15 μM T(CmCTG)2T in 10 mM NaPi (pH 5), MgCl2, and EDTA, (ii) 15 μM T(CmCTG)2T and S1-wc in 10 mM NaPi (pH 7) and MgCl2, (iii) 15 μM T(CmCTG)2T and S1-wc in 10 mM NaPi (pH 5), MgCl2, and EDTA, (iv) 15 μM T(CmCTG)2T and S2-tg in 10 mM NaPi (pH 7) and MgCl2, and (v) 15 μM T(CmCTG)2T and S2-tg in 10 mM NaPi (pH 5), MgCl2, and EDTA. The CD sample was placed in a cuvette of 0.5 mm path length, and the CD spectrum was collected from 200 to 350 nm with a step size of 1 nm. For each sample, three replicates of scans were acquired, and an average value was taken. The CD spectra were background corrected using the corresponding buffer solution.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Project ID: 22004038), the Natural Science Foundation of Guangdong Province, China (Project ID: 2021A1515010102), and the Basic and Applied Basic Research Foundation of Guangzhou, Guangdong, China (Project ID: 202102020066).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c04346.

  • 1H NMR assignments of T(CmCTG)2T, A(CmCTG)2T, and TT(CmCTG)2TT, and additional CD spectra (PDF)

Author Contributions

# J.Z. and Y.W. contributed equally to this work.

The authors declare no competing financial interest.

Supplementary Material

ao1c04346_si_001.pdf (877.2KB, pdf)

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