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A Stable and Label-free Fluorescent Probe based on G-triplex DNA and Thioflavin T Hui Zhou, Qian-Jin Han, Zhi-Fang Wu, Hong-Mei Zhong, Jun-Bin Peng, Xun Li, and Xiao-Lin Fan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04666 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on January 30, 2018

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Analytical Chemistry

A Stable and Label-free Fluorescent Probe based on G-triplex DNA and Thioflavin T Hui Zhou*, Qian-Jin Han, Zhi-Fang Wu, Hong-Mei Zhong, Jun-Bin Peng, Xun Li* and Xiao-Lin Fan College of Chemistry and Chemical Engineering, Gannan Normal University, Ganzhou, 341000, China. *To whom correspondence should be addressed. Tel: +86-797-8393536; Fax: +86-797-8393536; E-mail: [email protected] (H. Zhou); [email protected] (X. Li)

ABSTRACT: G-triplexes have recently been identified as a new kind of DNA structures. They perhaps possess specific biological and chemical functions similar as identified G-quadruplex, but can be formed by shorter G-rich sequences with only three G-tracts. However, till now, limited G-triplexes sequences have been reported, which might be due to the fact that their stability is one of the biggest concerns during their functional studies and application researches. Herein, we found a G-rich sequence (5’TGGGTAGGGCGGG-3’) which can form a stable G-triplex (Tm ~ 60 °C) at room temperature. The stable G-triplex can combine with thioflavin T and function as an efficient fluorescence light-up probe. Comparing with traditional G-quadruplex based probe, this triplex based probe was easy to be controlled and excited. Finally, the probe was successfully applied into constructing a labelfree molecular beacon for miRNA detection. Taking advantage of these abilities of the G-triplex based fluorescent probe, the challenges faced during designing G-rich sequences based fluorescent biosensors can be efficiently solved. These findings provide important information for the future application of G-triplex.

G-quadruplex is a well-known non-canonical DNA structure. When it is noncovalently combined with small molecule ligands to form label-free probes, interesting functions such as catalysis and light-up fluorescense are exhibited.1-5 Due to their biological significance and inherent advantages, G-quadruplex based labelfree probes have been wildly used in biological progress monitoring and biomolecules analyzing.3-10 Wherein, the concomitant conformational change of G-quadruplex occurs, usually becomes the starting point for their application.1, 11, 12 However, controlling and triggering G-quadruplex structures often encounter with some difficulties. For example, in G-quadruplex based label-free molecular beacons (MB),12-15 in order to control the formation of Gquadruplex, a long stem sequence containing many C bases should be inserted to pair with at least two G-tracts and one loop of G-quadruplex, whereas the long stem is always too strong to be opened, and the G-quadruplex is difficult to be triggered on by the target DNA or RNA.12 Therefore, the design restraint is still a fundamental challenge during applying G-quadruplex based fluorescent probe in biosensor. Recently, G-triplex has been identified as a new DNA structure.16, 17 Putative G-triplex structure was prevalently suggested as one of the most plausible intermediates in folding process of Gquadruplex structure.18-20 Recent research demonstrated that Gtriplex structure can be individually formed by G-rich sequences with only three G-tracts,16, 20, 21 and this structure also displayed catalyst functions similar as G-quadruplexes in vitro.22-24 The unique structures perhaps with specific biological functions in vivo remains elusive till date. Limited functional studies and application researches are available on the G-triplexes. Inspired by its similarity to G-quadruplexes, we were interested in exploring their potential roles as a novel label-free fluorescent probe. However, the stability of G-triplexes may be the biggest concern during the application. As far as we know, most G-triplex researches

Scheme 1. Illustration of G-triplex based fluorescent probe (A) and Gtriplex based molecular beacon (MB) for miRNA analysis (B).

are focused on a 11-mer sequence (5’-GGTTGGTGTGG-3’) truncated from thrombin binding aptamer (TBA) quadruplex.16, 21, 22, 24 The melting temperature (Tm) of this observed G-triplex was about 33.5 °C in a buffer solution containing 70 mM K+, suggesting that the structure is instable at physiological temperatures.16 Therefore, exploiting new G-rich sequences which can form stable G-triplex is desired. In this study, we respond to the above challenges by developing a G-rich sequence which could form a stable G-triplex with Tm of 60 °C. Moreover, when they combined with thioflavin T (ThT, a new small molecule ligand which can specifically bind to human telomeric G-quadruplex2) and formed a label-free fluorescent probe, not only the Tm but also the fluorescence intensity of the probe increased dramatically. Based on these findings, this fluorescent probe was applied to construct a label-free MB. Compared with the traditional MB based on G-quadruplex, the MB based on G-triplex requires very less G-C base pairs in the stem to form hairpin structure, which makes the design more flexible.

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EXPERIMENTAL SECTION Materials and reagents All oligonucleotides listed in Table S1 and Table S2 were synthesized and purified by Takara Biotechnology Co. Ltd. (Dalian, China) and Sangon Biotech Co., Ltd. (Shanghai, China). Thioflavin T (ThT) was purchased from Sigma Chemical Co., (St. Louis) and freshly prepared before each experiment. Exonuclease I was purchased from New England Biolabs (Ipswich, MA). Tris, potassium chloride and other reagents were of analytical grade and provided by China National Medicines Co. Ltd. (Beijing, China). Milli-Q water (resistance >18 MΩ/cm) was used throughout for solution preparation. In miRNA detection experiment, all the water, tips and tubes were treated with 1‰ diethypyrocarbon-ate (DEPC, Sangon Biotechnology Co. Ltd. Shanghai, China). Fluorescence measurements G-rich oligomers were diluted in water, and heated to 95 °C for 5 min, then cooled rapidly to room temperature. After this treatment, the oligomers were used in the subsequent experiment. The reaction sample (150 µL) was prepared by mixing 100 nM G-rich oligomer and 6 µM ThT in 25 mM Tris buffer (pH = 7.4, 50 mM KCl) and incubating at 4 °C or room temperature for 2 h. Fluorescence measurements were performed on a LS-55 Fluorescence Spectrometer (Perkin Elmer, USA) under the following instrument parameters: excitation and emission slit width 7.0 nm, range 440 ~ 600 nm, excitation at 435 nm and emission at 492 nm for G3 oligomer, excitation at 442 nm and emission at 487 nm for G4 oligomer. For dynamic study, a volume of 100 µL reaction mixture containing ThT and Tris buffer was prepared. Another volume of 50 µL mixture containing oligomers with different structures (G31 ssDNA, G31 triplex and dsDNA) was also prepared. After recorded the fluorescent signal of ThT mixture for 5 min, 50 µL oligomers mixture was subsequently added for further fluorescent recording. The signal at 492 nm was collected. Circular dichroism (CD) measurements and melting curves CD spectra was performed on a Chirascan CD Spectrometer (Applied Photophysics Ltd, England, UK) at room temperature. Three scans were accumulated and averaged under the following conditions: range from 200 nm to 500 nm, speed of 200 nm/min, response time of 0.5 s, bandwidth of 1.0 nm and quartz cuvette of 0.1 cm path length. All samples were prepared by adding of 10 µM G-rich oligomers in Tris buffer (25 mM, pH 7.4, 50 mM of KCl) and incubating at room temperature for several hours. Before the CD measurement, ThT was injected into the sample and incubated at room temperature for 15 min. The final concentration of ThT was about 100 µM. Melting curves study was carried out on a MOS-500 CD spectrometer (Bio-Logic, France) equipped with a peltier temperature controller. Normalized CD melting curves of G31 triplex and G31 triplex/ThT compound recorded at 265 nm under the following conditions: range from 10 oC to 90 oC, scan rate of 1 oC/min, fentes bandwidth of 5 nm, and quartz cuvette of 1 cm path length. The final concentration of G31 oligomer and ThT were 4 µM and 25 µM, respectively. Exonuclease I hydrolysis assay G31 oligomer was pretreated in different ways. One was denatured at 95 °C and incubated in Tris buffer (25 mM, pH 7.4, 50 mM KCl) at room temperature to form G-triplex. Another was denatured at 95 °C and cooled in ice water to keep ssDNA structure. Both of them were injected into digestion solution containing exonuclease I. The enzymatic degradation was proceeded for 30 min at 37 °C. Prior to ﬂuorescence measurements, 130 µL of Tris buffer (25 mM, pH 7.4, 50 mM KCl) containing ThT was injected into the degradation solution (20 µL) and incubated at room tem-

perature for another 2 h. The final concentration of G31 oligomer and ThT were 100 nM and 6 µM, respectively. MB assay MB was diluted in Tris buffer (25 mM, pH = 7.4, 50 mM KCl, 2.5 mM MgCl2) and heated to 95 °C for 5 min, then cooled to room temperature for 2 h. The reaction sample (150 µL) was prepared by mixing 100 nM MB, different concentrations of target miRNAs or DNAs, 8 U Recombinant RNase Inhibitor, and 6 µL ThT in Tris buffer (25 mM, pH = 7.4, 50 mM KCl, 2.5 mM MgCl2) and incubating at room temperature for 2 h. The final mixture was measured on LS-55 Fluorescence Spectrometer. For G3 MB, the excitation and emission wavelengths were 435 nm and 492 nm, respectively. For G4 MB, the excitation and emission wavelengths were 442 nm and 487 nm, respectively.

RESULTS AND DISCUSSION Fluorescence spectrum of G-triplex based fluorescent probe G-rich sequence (5’-GGTTGGTGTGG-3’) is the most reported G-triplex sequence. However, this G-triplex, possessing two Gtriad layers, is not stable at physiological temperatures. Recent study showed that G-triplexes with three G-triads should be more stable.21 Therefore, we prepared fourteen G-rich sequences (Table S1), most of which possess three G-tracts, and tested their fluorescence and CD spectrum. Finally, we selected a G3 oligomer G31 (5’-TGGGTAGGGCGGG-3’) by truncating the 3’-most Gtract of CatG4 (5’-TGGGTAGGGCG GGTTGGG-3’). CatG4 is a well-known G4 oligomer, which is widely used as G-quadruplex based DNAzyme.15, 25, 26 This G31 oligomer contains only three groups of GGG. The results of gel electrophoresis and melting curves show that the G31 performs a new intramolecular structure rather than G-quadruplex (Figure S1 and S2).22, 23 We further incubated G31 and CatG4 oligomer in K+ solution to form specific secondary structure (probable G-triplex) and G-quadruplex, respectively. The fluorescence was determined after adding ThT and incubating for hours. As shown in Figure 1A, the ThT dye displayed its characteristic absorption spectrum with a maximum at 415 nm and very weak emission profile. Addition of 100 nM CatG4 into the ThT solution shifted the absorption and emission bands of ThT to 442 and 487 nm, which was consistent with the

Figure 1. (A) Excitation and emission spectra of ThT in the presence of G31 and CatG4 DNA oligomers. (B) Emission intensity of ThT with various DNAs. Samples were prepared at 100 nM DNA oligomers in 50 mM Tris-HCl buffer (pH 7.4) containing 50 mM KCl and 6 µM ThT. (C) CD spectra of the G31 oligomer (15 µM) in the absence and presence of ThT (100 µM). The experiment was performed on a MOS-500 CD spectrometer (Bio-Logic, France) in a Tris-HCl buffer containing 50 mM KCl.


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Figure 2. (A) Melting curve of G31 triplex and G31 triplex/ThT compound. (B) Fluorescence of G31 triplex/ThT before and after adding of complementary sequences G31HB. (C) Fluorescence intensity of G31 ssDNA and G31 triplex after exposure to deferent concentration of exonuclease I. The error bars represent the standard deviation of three independent measurements.

previously reported G-quadruplex/ThT probes.2, 27 Strikingly, for G31, the absorption and emission bands were located at 435 and 492 nm, which was distinguished with that of CatG4. Moreover, the emission intensity of ThT was also remarkably increased. The enhancement was found to be as high as ~ 80-fold. The enhancement was slightly lower than 22Ag (one of the most frequently investigated human telomeric G-quadruplex sequences which has been reported as a significant ThT fluorescence enhancer.2, 28-30), and much higher than that of single-stranded G31HB (ssDNA), duplex G31/G31HB (dsDNA) and G-quadruplexed DNA (CatG4, Oxy28) (Figure. 1B). While numerous “light-up” fluorescent dyes for G4 oligomer have been described in literature,31-34 cyclometallated Ir (III) is the sole dye reported for G3 oligomer.35 However, the fluorescence enhancement of cyclometallated Ir (III) dye induced by 5 µM G31 is ~ 10-fold, which is much lower than the enhancement of ThT. Above results indicated a strong interaction of ThT with G31, and the interaction of G31 may be different from that of CatG4 with a G-quadruplex structure. Next, the result of the dynamics study demonstrate that the specific secondary structure of G31 (probable G-triplex) is important for the G31 based fluorescent probes. The structure is required for ThT binding, and the binding occurs in a few seconds (shown in Figure S3). Additionally, the G31 based fluorescent probe exhibits satisfactory photo stability during photo-bleach testing (shown in Figure S4). Binding mechanism of G-triplex based fluorescent probe To comprehend the most probable structure of G31 and the binding mode of ThT to G31, CD and induced CD (ICD) spectra from G31/ThT compounds were analyzed. Since the G-triplet and G-quartet share similar stacking and loop geometry, CD signals reflecting the strand orientation in the G-quadruplex can be also applied to the G-triplex.21 As shown in Figure 1C, the CD spectrum of G31 had a strong positive peak at approximately 265 nm and a negative peak at 240 nm. Both of them remained invariant after the addition of ThT. They can be distinguished by the characteristic of ssDNA and dsDNA which have a positive peak at approximately 280 nm,35 while matches the typical parallel stand arrangement, suggesting a parallel G-triplex would formed for G31 in the presence and absence of ThT.16, 25 Additionally, a negative ICD band was displayed at 425 nm with the solution containing both G31 and ThT, whereas no band at 425 nm was observed with the solution containing G31 or ThT alone. The negative ICD band would denotes an intercalation mode of binding.2 In addition, G31 revealed a similar CD spectrum profile with CatG4 (Figure S5). It should be noted that the CD signal intensity of the G31 G-triplex was much lower than that of the CatG4 Gquadruplex, suggesting the probability of G-triplex formation is less than that of G-quadruplex (Figure S5). However, the fluorescence intensity of ThT induced by the G31 G-triplex was significantly higher than that of the CatG4 G-quadruplex, indicating an

efficient bond between G31 G-triplex and ThT. Taken together, these results led us to presume that ThT probably take an intercalation mode to bind with parallel G31 G-triplex and form an effective G-triplex based fluorescent probe. Stability of G-triplex based fluorescent probe Stability of the G-triplex structure is critical for such G-triplex based fluorescent probe. To the best of our knowledge, most reported G-triplex structures exhibit low Tm values, suggesting that they maybe instable at room temperature.16, 21 For example, the Tm of G3 oligomer TBA11 (5’-GGTTGGTGTGG-3’) and Hum15 (5’-GGGTTAGGGTTAGGG-3’) are about 26.4 °C and 43.2 °C in a diluted buffer containing 100 mM K+ ion.21 Though the values can be improved to 34.3 °C and 52.0 °C in molecular crowding buffer, additional PEG 200 and high dosage of Ca2+ were desired.21 Therefore, in order to study the stability of Gtriplex based fluorescent probe, a series of experiments were conducted: 1. the melting temperature (Tm) of G-triplex based fluorescent probe was analyzed by monitoring the CD absorbance at 265 nm as a function of temperature (Figure 2A). The Tm of G-triplex formed by G31 was about 60 °C which was significantly higher than reported G-triplex sequences.16, 21 Additionally, when it noncovalently binds with ThT to form G- triplex-based fluorescence probe, the Tm value increased to 83 °C, suggesting that the Gtriplex based fluorescence probe is stable enough at physiological temperature; 2. we pretreated G31 oligomer in two different ways to form ssDNA and G-triplex, respectively, and then added G31HB sequences to hybridize. For G31/G31HB duplex, the length was about 13 base-pairs, the Tm was about 56.9 °C according to the

Figure 3. Fluorescence intensity of ThT with various G-rich oligomers. The concentrations of G-rich oligomers and ThT were 100 nM and 6 µM, respectively. Samples were prepared in a Tris-HCl buffer solution (25 mM, pH 7.4) containing 50 mM KCl. Error bars were standard deviation of three repetitive experiments.



Figure 4. Fluorescence intensity of 100 nm G3 probe0 (A), G4 probe1 (B) and G4 probe0 (C) in different complementary stretching probes (300 nm). Their sequences were displayed in inset. Error bars were estimated from at least three independent measurements.

evaluation of mfold software (http://unafold.rna.albany.ed u/?q =mfold). Therefore, the duplex should be easy to be formed. If the G31 G-triplex opened and formed a G31/G31HB duplex, no ThT signals would be generated because of ThT is difficult to combine with dsDNA. However, there was no obvious change in the ThT signal for G-triplex after adding G31HB sequences (Figure 2B), indicating that G31 G-triplex is more stable than expected; 3. an enzymatic degradation was also conducted to examine the stability of G31 G-triplex structure. It is well known that exonuclease I, a single-strand specific exonuclease, degrades nucleotides from the 3' end to 5' end of linear ssDNA.37-40 The exonuclease activity can be inhibited by G-quadruplex or other structure formations.41 In this study, G31 oligomers were pretreated using two different methods to form G31 ssDNA, G31 G-triplex, and then injected into exonuclease I solution for digesting. As shown in Figure 2C, for G31 ssDNA, a remarkable degradation was observed in 0.1 U exonuclease I, and the degradation rate was ~ 87.1% in 10 U exonuclease I. In contrast, the G31 G-triplex showed remarkable degradation in 1 U exonuclease I, and the degradation rate ~ 24.5% in 10 U exonuclease I, suggesting that the G31 G-triplex inhibited the hydrolysis significantly. This result confirms the formation of a stable G-triplex structure to a considerable extent.

resulting from self-assembly of the G-rich fragment of the designed probes can be regulated and controlled by varying the length of complementary strands. For G3probe0, the fluorescence signal decreased dramatically when only one “c” base was extended to complement with the sequence of G- triplex (G3HB1). A plateau was rapidly achieved when the first G-tract was complemented (G3HB3). In contrast, for G4probe1, the plateau was lagged till both the first and the second G-tract were complemented (G4HB6). The result indicated that our G-triplex is more convenient to be controlled. Surprisingly, we observed a sudden increase for G4probe1 when the first G-tract was complemented (G3HB3, G4HB4). Similar result was achieved for G4probe0 with another G-quadruplex fragment CateG4 (Figure 4C). CD spectrum also verified this result (Figure S7). The increase may be relative with the formation of G-triplex for the residual three Gtracts after complementation. According to the above results and the recent reports, G-triplexes displayed similar functions as Gquadruplexes when they complexed with ThT or hemin.22-24 Therefore, this could be the reason why long complementary sequence containing many C bases should be preffered in designing G-quadruplex based biosensor.12-15

Comparison of different G-rich strands We compared ThT fluorescence intensity in the presence of variety of G-rich sequences consisting of truncated froms of G4 oligomers: G31 (CatG4), G38 (c-MYC), G39 (EAD1), G310 (22Ag), G312 (Oxy28), G312 (Hum24), G313 (21CTA) and G314 (TBA).28-30 As shown in Figure 3, several important informations were obtained: 1) the TA loop may be important for G31; 2) G-triplex sequences with short loop maybe beneficial to form G-triplex structure; 3) the fluorescence intensity induced by Gtriplet sequences with a parallel structure is significantly higher than that with parallel/antiparallel mixture and antiparallel structure (Figure S5). Additionally, the effect of ThT dosage, ion species and concentration on emission of G-triplex based fluorescent probe are shown in Figure S6. The probe displayed high fluorescence signal in Tris buffer solution containing 50 mM K+ and 6 µM ThT. Flexibility of G-triplex based fluorescent probe To demonstrate the utility of G-triplex based fluorescent probe, we explored the fluorescence characteristics of our G-rich probes (G3probe0, G4probe0 and G4probe1) by hybridizing with various complementary strands (G3HB and G4HB). As shown in Figure 4, G-rich sequences possess a G-rich fragment and a random tail. The G-rich fragment of G3probe0, G4probe0 and G4probe1 are G31, CatG4 and 22Ag, respectively. The random tail with 20 bases was designed to ensure the hybridization of stretching sequences. The unique G-triplex or G-quadruplex conformation—

Figure 5. (A) Fluorescence response of G-triplex and G-quadruplex based MBs with different stem lengths for target miRNA-141. The concentrations of MB and miRNA-141 were 100 nm and 300 nm, respectively. (B) Linear relationship between the fluorescence intensity and the concentration of miRNA-141. The concentrations of G3M2, G4M4 and ThT were 100 nM, 100 nM and 6 µM, respectively. Samples were prepared in a Tris-HCl buffer solution (25 mM, pH 7.4) containing 50 mM KCl.


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Analytical Chemistry Label-free MB based on G-triplex fluorescent probe To further demonstrate the utility of this stable G-triplex, we applied G-triplex in constructing label-free MB. As shown in Scheme 1B, our G-triplex based MB consists of two regions: a G31 stem sequence and a target recognition loop sequence. In the absence of target, part of G31 sequence was hybridized to form a duplex stem. MB was maintained as a hairpin structure thereby inhibiting the signal generation. In the presence of target, the loop region recognizes the target, and the stem opens to release the G31 sequence. Then, the released G31 sequence self-assembles into G-triplex and activates ThT fluorescence. Considering that the design of the stem is a challenge in constructing G-rich sequences based MB,12-15 we investigated the influence of stem length on the fluorescence response of MB for excess target. As shown in Figure 5A, for G-triplex based MB, the best fluorescence response value appeared in G3M2, which possessed a short stem with 6 base pairs (it is about 5-7 base pairs for conventional MB) and only two “c” bases to complement with the first G-tract of G-triplex. However, for G-quadruplex based MB (G4M1-5, G22M1-5), the value appeared in G4M4 and G22M4, which possessed a long stem with more than 8 base pairs, and at least 6 “c” bases to complement with both the first and the second G-tract of G-quadruplex. In other words, all the stem bases should be restricted to pair with sequences of G-quadruplex. Additionally, long stem not only requires longer target and longer time for opening by the analyte,12 but are also difficult to applied in some amplification systems, e. g. DSNSA.13, 42, 43 As shown in Figure S8, the target should possess 28 bases to open the 22Ag Gquadruplex based MB G22M4, wherein part of them should be restricted as “GGG” and compared with the stem. Therefore, design restraint is a challenge during applying G-quadruplex in label-free MB. In our study, the design of the stem became more flexible when we used G-triplex to construct label-free MB. G3M2 exhibited a good linear relationship between the fluorescence intensity and the miRNA-141 concentration in the range from 1 to 200 nM (Figure 5B). The calibration equation was F= 114.1480 + 2.9458 C, with a correlation coefficient of R2= 0.9922. The detection limit of miRNA-141, based on 3σ rule, was about 8.0 nM. The value was slightly lower than that obtained by using G4M4 (17.2 nM), and slightly higher than that obtained by using traditional dual labeled MB (2.4 nM, Figure S9). The difference was less than one order of magnitude, which may be relative with that all of them were based on the same hybridization mechanism without any signal amplification. If signal amplification has been introduced, the sensitivity of G-triplex based MB could be improved more than one order of magnitude (Figure S10). Additionally, G3M2 also displayed good selectivity shown in Figure S11. A standard addition experiment in healthy human urine and blood serum was further carried out (Table S3 and S4). The recovery ranges from 95.8% to 106.2%, demonstrating a good applicability for the G-triplex fluorescent probe and MB in complex background. Strand displacement amplification system based on Gtriplex fluorescent probe We further designed a strand displacement amplification (SDA) strategy to demonstrate the utility of this stable G-triplex fluorescent probe. As shown in Figure 6, the template involves three regions: a recognition region for target; a nicking site for Nt·AlwI nickase upon the duplex formation; and a signal region for synthesizing of G-rich probe. The SDA reaction was initiated through the hybridization of the template with target (as a trigger).44 The target further extended along the template in the presence of polymerase/dNTPs, resulting in a stable duplex with recognition sites for the Nt·AlwI nickase. Then, the Nt·AlwI nicked the duplex, resulting in a new active site for DNA polymerization and the concomitant displacement of nicked strand. Finally, numerous G-

Figure 6. Illustration of G-rich probe based strand displacement amplification (SDA) strategy for miRNA analysis. The sample was prepared by mixing 50 nM G3 or G4 template, 25 nM target, 2 U Klenow Fregment (3’-5’ exo-), 4 U Nt. AlwI, 8 U Recombinant RNase Inhibitor and 0.2 mM dNTPs in 20 µL 1×NEB buffer 2 at 37 °C for 1 h, and then heated to 95 °C for 5 min. Prior to ﬂuorescence measurements, 100 µL of Tris buffer (25 mM, pH 7.4, 50 mM KCl) containing ThT was injected into the sample (20 µL) and incubated for another 2 h.

rich sequences were generated, displaced and dissociated. Those G-rich sequences self-assembled into G-triplex or G-quadruplex and bound with ThT to generate fluorescent signal. Herein, we designed two SDA systems by using G31 G-triplex and 22Ag Gquadruplex as products, respectively. 22Ag was selected as a control because it is one of the most frequently investigated human telomeric G-quadruplex sequence which has been reported as an effective ThT fluorescence enhancer.2, 28-30 The fluorescence enhancement induced by the same dosage of target in G31 SDA system was much higher than that in 22Ag SDA system. Additionally, the G3 SDA system seems to be more sensitive than G4 SDA system (Figure S12). Instead, the signal of G31 G-triplex was lower than that of 22Ag G-quadruplex when we prepared them along in Figure 1C. That may be relative with the short sequences length of G31. G31 sequence possesses only 13 bases. It is much shorter than 22Ag and other G-quadruplex sequences. Therefore, G31 sequence should be easier to be produced in DNA polymerization.

CONCLUSIONS In summary, we found a G-rich sequence which could form a stable G-triplex with Tm 60 °C. The G-triplex could combine with ThT to form a “light-up” fluorescent probe. The probe was easy to be controlled by hybridizing with complementary probes. Based on the findings, this novel fluorescent probe was applied to construct a label-free MB. Compared with the traditional MB based on G-quadruplex, the MB based on G-triplex does not require many G-C base pairs in the stem to form hairpin structure, which makes the design more flexible. Though the unique structure of G-triplex perhaps possess specific biological functions in vivo and chemical functions in vitro, the studies on G-triplex is still elusive and limited. Our report may add on to the functional study or the application of G-triplex.

ASSOCIATED CONTENT Supporting Information The Supporting Information (including TableS1-S4, Figure S1S12 and corresponding discussion) is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author



* Dr. Hui Zhou and Prof. Xun Li, College of Chemistry and Chemical Engineering, Gannan Normal University, Ganzhou 341000, China. Tel: +86-797-8393536; Fax: (+86) 797-8393536; E-mail: [email protected]; [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We are grateful to the National Natural Science Foundation of China (Grants No. 21405023) and Natural Science Foundation of Jiangxi Province (Grant No. 20161BAB203097 and 20142BAB213008) for their financial support of this work.

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