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Classical Triplex Molecular Beacons for MicroRNA-21 and Vascular Endothelial Growth Factor Detection Shasha Lu, Shuang Wang, Jiahui Zhao, Jian Sun, and Xiurong Yang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00996 • Publication Date (Web): 17 Oct 2018 Downloaded from http://pubs.acs.org on October 20, 2018

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Classical Triplex Molecular Beacons for MicroRNA-21 and Vascular Endothelial Growth Factor Detection Shasha Lu,†, ‡ Shuang Wang,†, ‡ Jiahui Zhao,†, § Jian Sun,*, † and Xiurong Yang*, †, ‡ †State

Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China ‡University

of Science and Technology of China, Hefei, Anhui 230026, China

§University

of Chinese Academy of Sciences, Beijing 100049, China

KEYWORDS: Triplex molecular beacons, MicroRNA-21, Hoogsteen base-pairing, pH responsiveness, Fluorescence resonance energy transfer

ABSTRACT: Triplex molecular beacons (tMBs) possess great potential in the biological sensing because of the pH responsiveness and controllability of binding strength. Here, we systematically investigate and rationally design a classical tMB for convenient detection of microRNA-21, a well-known biomarker of cardio-cerebrovascular diseases. In the tMB, we employ the complementary sequence of miR-21 as the loop and the sequences of protonated cytosine-guanine-cytosine (C-G•C+) and thymine-adeninethymine (T-A•T) as the triplex stem, in which both the Watson-Crick and Hoogsteen base-pairing control the binding strength in cooperation. It is demonstrated for the first time that the presence of miR-21 would only break the Hoogsteen base-pairing in the stem and hybridize with the tMB to form the rigid heterozygous hybrid duplex structure. These would hinder the fluorescence resonance energy transfer (FRET) between the fluorophore (FAM) and quencher (BHQ1) labeled at the ends of the oligonucleotide, and the fluorescence recovery degree of FAM can be used as the standard to quantitate the miR-21. More significantly, the excellent adjustability and sensitivity of our tMBs have been confirmed by constructing the corresponding duplex molecular beacon (dMB) for comparison. The fluorophore FAM in the tMB could be replaced by the fluorescent DNA/silver nanoclusters, which exhibits the universal applicability of energy donor and receptor selection for tMB. Furthermore, our proposed tMB could also be developed as an aptasensor for the detection of vascular endothelial growth factor (VEGF) by only introducing the complementary sequence of its aptamer into the tMB. This work is of great significance for the systematic study of tMBs for the detection of biomarkers such as nucleic acids and proteins.

Molecular beacons (MBs) are typical and well-known oligonucleotide fluorescent probes. Classical molecular beacons are composed of stem, loop, fluorophore and quencher. The existence of targets will open the loop and separate the fluorophore and quencher of MBs, thus recover the fluorescence and the signal can be turned “on”.1,2 Since the MBs were reported by Tyagi and Kramer in 1996,1 they has been widely used in biosensing, bioimaging, trerapy and other fields owing to their characteristics of simple synthesis, easy operation, good selectivity and high sensitivity.3 Many sensors for assaying proteins, small molecules and nucleic acids have been developed by designing the aptamer,4,5 DNase,6,7 special recognition sites8,9 or complementary single-stranded DNA10 into the “loop” structure of MBs. As the “lock” of MBs, the stem structure of MBs should be reasonably regulated by targets or environmental stimulation to actualize the “off-on” of fluorescence signal and the stem is usually consist of two complementary sequences which is bound by Watson-Crick hydrogen bonds.11,12 However, some studies have shown that the stem length of MBs can achieve the best stability with 5-7 bps,13,14 which greatly limits the ability to regulate the strength of the stems, and it is difficult to meet the requirements of various targets. In order to control the strength of stem better, various new strategies have been developed, including the introduction of special DNA structures,15-17 metal ions and

small molecules,18-21 external stimuli,22,23 nucleotide analogues,24,25 etc. Among them, the triplex structures of DNA have been already utilized and exhibited their potential in the stem design of MBs as a special DNA structure. The classical triplex structures include protonated cytosineguanine-cytosine (C-G•C+) and thymine-adenine-thymine (TA•T), in which both the Watson-Crick and Hoogsteen basepairing control the binding strength in cooperation.26 More importantly, these triplex structures are pH responsive and provide more favorable control conditions for their binding strength.27 Therefore, the triplex structures have been used in various DNA-related fields,28 including triplex molecular beacons (tMBs),29-32 DNA nanomachine,17,33 DNA 27 34 nanoswitches, DNA tiles and DNA hydrogels.35,36 In particular, Seitz’s group have constructed a modular tMB for DNA detection by the introduction of T-A•T structure in 2007, in which the “A” is replaced by peptide nucleic acid (PNA) bases, provided important guidance for future research of tMBs.30 And then, Yang’s group have extended the tMBs to the construction of aptamer sensor by introducing a duallabeled oligonucleotide serving as a signal transduction probe (STP),29 as well as the construction of living cell imaging probe by using the nucleic acid-gold nanoparticles conjugate.31 More recently, with the assistance of

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ferrocene⊂cucurbit[7]uril (Fc⊂CB-[7])-modified DNA strand, Wu’s group have applied the tMBs into the nanopore sensing and provided a modular tool for the detection of specific macromolecules,37 greatly expanding the application field of tMBs. However, all of these tMBs applications make the design more complicated to some extent, while ignoring the exploration of the original and convenient application potential of the classical tMBs. MicroRNAs (miRNAs) have attracted more and more attention due to their crucial role in gene expression.38 Many kinds of miRNAs have been used as novel biomarkers of multiple diseases, such as acute myocardial infarction (AMI) , Alzheimer's disease (AD) et. al.39 In past years, various methods have been used to detect the miRNAs.40-42 However, the development of more sensitive and faster nucleic acid probes using different DNA structures still exhibit great potential. Scheme 1. Schematic illustration of the analysis of miRNA using the classical tMB.

In light of this, we have used the classical tMB to construct the detection platform of miRNAs in here. As shown in Scheme 1, the tMB consists of a probe of triplex molecular beacon (ptMB) modified with the fluorophore/quencher pair (FAM/BHQ1) and a stem-forming oligonucleotide (sfO).28,30,37 The complementary sequence of miRNA is introduced into the loop of the ptMB, and the stem is made up of the typical triplex structures (C-G•C+ and T-A•T). Upon the addition of target miRNA, the Hoogsteen pairing of the triplex stem will open, hindering the FRET, accompanied by fluorescence “turn on”. The degree of fluorescence recovery is proportional to the increased concentration of the miRNA. Furthermore, the classical tMB possesses the universal applicability for the detection of different miRNAs by only substituting the complementary sequences of miRNAs in ptMBs. EXPERIMENTAL SECTION Chemicals and apparatus. All of the DNA/RNA was synthesized by Sangon Biotechnology Co. Ltd. (Shanghai, China) and their sequences were listed in Table S1. The underlined parts were the loops of tMBs or dMBs. Silver nitrate (AgNO3) and sodium borohydride (NaBH4) were purchased from Sigma-Aldrich (St. Louis, USA). The vascular endothelial growth factor (VEGF) was purchased from PeproTech, Inc. (USA). All of other chemicals were of reagent grade and used directly. Clinical human serum samples were kindly supplied by the First Hospital of Jilin University (Changchun, China). The ultrapure water (18.25 MΩ.cm, Millipore, USA) was used throughout all the experiments. Fluorescence measurements were carried out using an F4600 fluorescence spectrophotometer (Hitachi). Circular dichroism (CD) spectral measurements were performed on a

Jasco J-820 Circular Dichroism Spectra polarimeter (Tokyo, Japan). Photographs were taken by a commercial digital camera. Optimization of tMB Structure and Experiment Condition. For the optimization of triplex structure length, ptMBs (0.25 μM) and sfOs (0.25 μM) with different lengths were mixed and annealed from 95°C to room temperature in two hours respectively to form the tMBs with different lengths triplex structures. The CD spectra of these tMBs were carried out. And then, commensurable miR-21 (0.25 μM) or isometric PBS buffer (pH = 7.4) were added to tMBs and the fluorescence spectra of them were measured. Similarly, the dMBs with different duplex lengths were annealed firstly and then added miR-21 or PBS buffer to carry out their fluorescence spectra, the concentrations of dMBs and miR-21 were both 0.25 μM. Detection of miR-21 by tMB or tMB-AgNCs. The mixture of commensurable ptMB and sfO was annealed from 95°C to room temperature in two hours to form the tMB. And then, different concentrations of miR-21 were added to the above formed tMB and incubated for two hours at room temperature. After that, the fluorescence intensities were measured. For the tMB-AgNCs, the mixture of commensurable ptMBAgNCs (0.5 μM) and sfO (0.5 μM) was annealed from 95°C to room temperature in two hours. And then, the six-fold freshly prepared AgNO3 (3 μM) was added to the mixture and incubated for one hour in the dark. Following by the addition of six-fold freshly prepared NaBH4 (3 μM). The solution was incubated for another six hours in the dark to form the tMBAgNCs. The following steps were consistent with the detection of miR-21 by tMB. Detection of VEGF by tMB. The mixture of 0.25 μM ptMB-VEGF, sfO and Aptamer was annealed from 95°C to room temperature in two hours to form a duplex hybridization. And then, different concentrations of VEGF were added to the above formed hybridized duplex and incubated for two hours at room temperature to restore the triplex structure. After that, the fluorescence intensities were measured. RESULT AND DISCUSSIONS Construction and Characterization of tMB. Typically, the tMB was constructed by the balanced mix and anneal of ptMB and sfO (Scheme 1), in which the ptMB consist of a complementary sequence of miRNA (cyan part) and two triplex forming sequences (black parts). We have used miR-21 as the model target and poly adenine (A10) as sfO to construct a tMB firstly. As shown in Figure 1A, the ptMB modified with FAM exhibited medium fluorescence intensity (λEm =520 nm) and its fluorescence almost completely quenched after adding sfO. And then, the addition of miR-21 could strongly recover the fluorescence. On the basis of the distance-dependent principle of FRET, the fluorescence of FAM was partially quenched by BHQ1 through FRET when the ptMB was in a free crimp state because of the relatively close distance. With the addition of sfO and the formation of triplex stem, the FAM was very close to the BHQ1 and the fluorescence was almost fully quenched. And then, after the addition of miR-21, the fluorescence was recovered because that the miR-21 would open the triplex stem and form rigid duplex structure with the tMB, which made the FAM keep a certain distance from the BHQ1 and impeded the FRET. The circular dichroism (CD) spectra could be used to explore the secondary structure

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Figure 1. Fluorescence emission spectra (A) and CD spectra (B) of the process of tMB formation and target addition. (C) Characterization of the process of tMB formation and target addition by Native-PAGE: (1) ptMB; (2) ptMB + sfO; (3-6) ptMB + sfO + miR-21; (7) 25 bp Marker. The ratios of tMB: miR-21 in lane 3-6 was 10:1, 5:1, 2:1 and 1:1 respectively. (D) Schematic illustration of the analysis of miRNA using the classical tMB’. (E) Fluorescence emission spectra of the process of tMB’ formation and target addition. consist of ptMB’ and Cy5-modified sfO (Cy5-sfO) was changes during the process of tMB formation and target designed as Figure 1D. The fluorescence spectra were shown addition. As shown in Figuer 1B, the positive peak at 275 nm in Figure 1E. The addition of Cy5-sfO almost completely and negative peak at 245 nm was referred to the single quenched the fluorescence of FAM (λEm =520 nm), and the stranded ptMB.43 The appearance of the strong negative peak fluorescence recover after the addition of miR-21, which was at 212 nm and strong positive peak at 279 nm in presence of corresponding to the formation and dissociation of triplex stem. sfO suggested the formation of the triplex structure, which As for Cy5 (λEm =660 nm), the fluorescence was quenched by proved that the tMB was constructed successfully.44 And then, the BHQ1 and didn’t recover after the addition of miR-21, the addition of miR-21 bring the positive peak at 279 nm blue which was attributed to that the addition of miR-21 only opens shifted to 270 nm, as well as the appearance of another the Hoogsteen pairing and not destroys the Watson-Crick positive peak at 220 nm and two negative peaks at 245 nm and pairing between the ptMB’ and Cy5-sfO. Fortunately, this was 210 nm, which were the characteristic peaks of DNA duplex helpful to improve the sensitivity of our FRET-based detection or DNA:RNA hybrids duplex.45 These results proved the by effectively prolonging the length of the rigid duplex hybridization of tMB with miR-21. Furthermore, the structure after adding miR-21. This was also the advantage of formation process of tMB and miR-21 addition was tMBs over duplex molecule beacons (dMBs), and we would characterized by native polyacrylamide gel electrophoresis discuss in detail later. All of the above results proved that we (Native-PAGE). As shown in Figure 1C, the tMB (lane 2) have successfully constructed a tMB which could be applied moved to a lower position in the Native-PAGE. With the to the detection of miRNAs. gradual addition of miR-21, the bands moved to a higher position and the higher band become brighter with the Optimization of tMB Structure and Experiment increase of the miR-21 (lane 3-6), which confirmed that the Condition. The length and sequence of triplex structure were tMB was hybridized with miR-21 and formed the hybrids with the two key factors that determine the bonding strength of a larger molecular mass. It is noteworthy to mention that the triplex and the detection ability of tMB. Here, the length of tMB (lane 2) could move faster than the ptMB (lane 1), which triplex structure was studied at pH 7.4 firstly and a series of may be due to the random coil structure of single strand ptMB tMBs with different triplex lengths (6 bp-16 bp) were designed. hindered its movement in space. Moreover, no bands of sfO The CD spectra of them were shown in Figure 2A. Previous were found after the addition of miR-21 in the Native-PAGE studies have proved that the positive peak at 220 nm was the experiment, which suggested that the sfO still hybridize with characteristic peak of duplex.45 As we can see, the peak was the ptMB to form the heterozygous structure of DNA duplex gradually decreased when the triplex length was changed from and DNA:RNA hybrids duplex. This phenomenon was very 6 bp to 10 bp and gradually increased when the triplex length interesting and different from the reported tMBs.30,31 Most of was changed from 10 bp to 16 bp, which indicated that there them claim that the addition of the target will cause the falling were explore the effect of triplex length in tMB on the of sfO from the tMB, which would shorten the length of the detection of miR-21, we measured the fluorescence intensity rigid duplex. of tMBs with almost no duplex in tMB at 10 bp. We believe In order to further confirm that sfO and ptMB could still that the tMB reached the most stable state at 10 bp, which may maintain hybridisation after target miRNA addition, the tMB'

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Figure 2. (A) CD spectra of the tMBs with different stem lengths. Fluorescence emission spectra of the tMBs with different triplex lengths before (B) and after (C) the addition of miR-21. Fluorescence emission spectra of the dMBs with different duplex lengths before (D) and after (E) the addition of miR-21. (F) The fluorescence recovery degrees of the tMBs and dMBs with different triplex or duplex lengths. prepared the fluorescence kinetic properties of tMB (10 bp) because the Hoogsteen base-pairing was less stable than the and dMBs (6 bp/10 bp) after the addition of miR-21 (Figure Watson-Crick base-pairing, and if the triplex was too short or S2). The fluorescence intensity of tMB (red line) increased too long, part of the Hoogsteen base-pairing in triplex will be faster than that of dMBs and the fluorescence intensity of tMB broken to form duplex and reduce the stability of tMB. In with 10 bp triplex could reach the platform within 1200s. order to further explore the effect of triplex length in tMB on What’s more, the effect of salt concentration on kinetic the detection of miR-21, we measured the fluorescence properties of tMB (10 bp) and dMB (6 bp) were studied. We intensity of tMBs with different triplex lengths before and found that both of tMB and dMB could maintain the after the addition of miR-21. Before the addition of miR-21, dynamics trend when the salt concentration up to 500 mM the fluorescence intensity was weakest at 10 bp, which was after the addition of miR-21 (Figure S3). Thus we chose 10 bp agreed with the results of CD spectra (Figure 2B). Once the as the research length in the following studies. miR-21 was added, the fluorescence intensities of the tMBs enhanced to different extents (Figure 2C). We have noticed that the tMB of 10 bp showed the strongest fluorescence intensity after the addition of miR-21, which could be attributed to two aspects: (1) The shortlength of duplex couldn’t hinder the FRET effectively, so only part of the fluorescence were recovered;46 (2) when the duplex was two long, the rigidity of duplex would be put to a serious test.47 As the contrast, the corresponding dMBs with different duplex lengths were designed to study the advantages of our tMB. The mechanism diagram was shown in Figure S1. The dMBs were the probe modified with the fluorophore/quencher pair (FAM/BHQ1) itself and the complementary sequence of miRNA is introduced in the loop. The fluorescence intensities of dMBs with different duplex lengths before and after the addition of miR-21 were measured (Figure 2D and 2E). The fluorescence intensity of dMBs only showed the excellent enhancements at the duplex length were 6 bp and 8 bp, because the long duplex (≥ 10 bp) couldn’t be opened by miRFigure 3. (A) The schematic diagram of tMB and the triplex 21. The degrees of fluorescence recovery ((F-F0)/F0) of the sequences with different proportions of T-A•T: 50%, 60%, tMBs and dMBs were shown in Figure 2F, in which F0 and F 80%, 90% and 100%. (B) Histogram of the fluorescence were the fluorescence intensities of tMB or dMB before and recovery degrees of the tMBs with different proportions of Tafter the addition of miR-21. The tMBs exhibited the greatest A•T. (C) The change trends of the fluorescence intensity of fluorescence recovery degree at 10 bp while the fluorescence above tMBs with pH values after the addition of miR-21. (D) recovery degree of dMB decreased as the duplex length The change trends of the fluorescence recovery degrees of increased, which proved that the tMB possess better above tMBs with pH values. adjustability by the sequence. Based on these results, we

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ACS Sensors Next, we designed a series of tMBs by replacing different proportions of T-A•T with C-G•C+ to evaluate the influence of sequence of triplex structure on tMB. The sequences of triplex with different proportions of T-A•T were shown in Figure 3A, and the maximum fluorescence recovery degree was shown at 90% (Figure 3B). Owing to the pH responsiveness of T-A•T or C-G•C+,27 we also studied the fluorescence intensities and fluorescence recovery degrees of tMBs with different triplex sequences at a series of pH values (4.4-10.4). On the one hand, the fluorescence intensity of all tMBs had two plateaus at both ends, and increased sharply in the range of pH 6-8 (Figure S4 and 3C). We believe that the plateau at pH 4.4-6 indicated the stable triplex of tMB while the plateau at pH 8-10.4 indicated that the triplex of tMB were completely opened, which were attributed to the bound of Hoogsteen base-pairing in C-G•C+ at acid environment and the broken of Hoogsteen base-pairing in T-A•T at alkaline environment.36 On the other hand, the fluorescence recovery degrees of all tMBs were first increased and reached the peaks at pH 6.4, and then decreased gradually with the increase of pH values (Figure 3D). The tMBs were very stable as pH < 6.4, which induced the failure of opening tMBs by the addition of miR-21. So the fluorescence recovery degrees were very low and gradually increased with the pH values (Figure S4A-C). However, when the pH values were greater than 6.4, a part of triplex would be broke before the addition of miR-21, which also induced the low fluorescence recovery degrees and graduallydecreased with the pH values (Figure S4E-I). Fortunately, we found that at the pH = 6.4, these tMBs could maintain stable triplex structures before the miR-21 was added and could be successfully opened with the addition of miR-21 (Figure

Figure 4. (A) Fluorescence emission spectra of the tMB with 90% T-A•T in triplex after the addition of different concentrations of miR-21 (0, 0.5, 2.5, 5, 15, 25, 40, 50, 75, 90, 100, 125, 150, 175, 200, 225 and 250 nM from bottom to top, pH = 6.4). (B) The linear relationship between the fluorescence recovery degree and the concentration of miR-21 ranging from 0.5 to 250 nM. Fluorescence emission spectra (C) and fluorescence recovery degrees (D) of the tMB after the mismatched RNAs and different control miRNAs were added in the absence or presence of miR-21.

S4D). It should also be noticed that both the fluorescence intensity and fluorescence recovery degree reached the maximum at the proportions of T-A•T was 90%, which was consistent with our above studies. MiR-21 Detection Assay. The optimized tMB with 10 bp triplex (90% of T-A•T) was used to detect the miR-21 at pH = 6.4. Fluorescence emission spectra of the tMB after the addition of different concentrations of miR-21 were shown in Figure 4A. The fluorescence intensity of tMB was very low before the addition of miR-21 and gradually enhanced with the increase of the concentrations of miR-21, which indicated that the fluorescence of FAM could be quenched effectively due to its proximity to BHQ1, and the miR-21 could open the triplex stem and hybrid with the tMB to separate the FAM and BHQ1, meanwhile hinder the FRET between them. The fluorescence recovery degrees enhanced with the increase of miR-21 concentration and obtained a great liner range from 0.5 to 250 nM and the limit of detection (LOD) could reach to 0.18 nM based on 3S/N. The selectivity and specificity of the tMB were also being studied. One to three base pairs mismatched RNAs (mis-1, mis-2, mis-3) were designed and added to the tMB of miR-21 in the absence or presence of miR-21 as controls. Only miR-21 could induce the enhancement of the fluorescence (Figure 4C), and the corresponding fluorescence recovery degrees were close to 0 in the absence of miR-21 (black columns of Figure 4D) while the tMB occupied high fluorescence recovery degrees in the presence of miR-21 (red columns of Figure 4D). What’s more, a series of cardiocerebrovascular diseases related miRNAs, including miR-499, miR-1, miR-208b, miR-208a, miR-133a, were also added to the tMB of miR-21 in the absence or presence of miR-21 as controls. As shown in Figure 4C and D, these miRNAs also didn’t induce the enhancement of the fluorescence and didn’t interfere with miR-21 detection. These results demonstrated the great selectivity and specificity of tMB. DNA-templated silver nanoclusters (DNA/AgNCs) have attracted great attention because of their simple synthesis, photostability, low toxicity. Therefore, we tried to use the DNA/AgNCs instead of fluorescent molecules (FAM) as energy donors to construct the tMB-AgNCs. The scheme of the analysis of miR-21 using the tMB-AgNCs was shown in Figure 5A. A cytosine-rich single stranded DNA was introduced to the ptMB (ptMB-AgNCs) and used to synthesize fluorescent DNA/AgNCs according to previous literatures.39,48 The ptMB-AgNCs showed a medium fluorescence and the addition of sfO and miR-21 would quench and recover the fluorescence respectively (Figure 5B), which were completely consistent with the results of tMB (Figure 1A). The liner range of tMB-AgNCs was 1 to 500 nM and the LOD was 0.61 nM based on 3S/N (Figure 5C), which was close to that of tMB. These results demonstrated the flexibility of tMB energy donors or receptors selection. VEGF Detection Assay. In fact, such tMB could also be used in the analysis of certain protein with help of the aptamer. Here, a vascular endothelial growth factor (VEGF) detection tMB (tMB-VEGF) was designed by introducing its aptamer (Figure 5D). Different from the tMB, the initial state was the duplex structure which formed by the hybridization of tMBVEGF with aptamer, with the strong fluorescence at the same time. The addition of VEGF would separate the aptamer from

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Figure 5. (A) Schematic illustration of the analysis of miR-21 using the tMB-AgNCs. (B) Fluorescence emission spectra of the process of tMB-AgNCs formation and target addition. (C) Fluorescence emission spectra of the tMB-AgNCs with 90% T-A•T in triplex after the addition of different concentrates of miR-21 (0, 0.1, 1, 10, 20, 50, 80, 100, 150, 200, 300, 400, and 500 nM from bottom to top, pH = 6.4). The insert was the linear relationship between the fluorescence recovery degree and the concentration of miR-21 ranging from 1 to 500 nM. (D) Schematic illustration of the analysis of VEGF using the classical tMB. (E) Fluorescence emission spectra of the tMB with 90% T-A•T in triplex after the addition of different concentrations of VEGF (0, 0.05, 0.5, 1.5, 2, 2.5, 3, 3.5, 4.5, 5, 5.5, 6, 7, 9 and 10 ng/mL from top to bottom, pH = 6.4). (F) The relationship between the fluorescence recovery degrees and the concentration of VEGF. The insert was the linear relationship between the fluorescence recovery degree and the concentration of VEGF ranging from 0.05 to 6.00 ng/mL. the tMB-VEGF and restore the triplex structure of tMB-VEGF, and the fluorescence would be quenched. It should be noticed that a non-base paired end was left between the aptamer and the complementary sequence of the tMB-VEGF, which would facilitate the combination of VEGF and its aptamer, so that the aptamer could be separated from the tMB-VEGF easily. The mixture of same amount of tMB-VEGF and aptamers were annealed to form the hybridization of tMB-VEGF and aptamer firstly. And then after adding different concentrations of VEGF, the fluorescence intensity and fluorescence recovery degree were both decreased with the concentration of VEGF in the range of 0.05-10 ng/Ml (Figure 5E and 5F). However, the liner relationship between the fluorescence recovery degree and the concentration of VEGF was obtained only in the range of 0.05-6 ng/mL, which may be related to the non 1:1 relationship between VEGF and its aptamer. Determination of miR-21 and VEGF in Human Serum Sample. Previous studies have shown that the miR-21 or VEGF in the systemic circulation (plasma/serum) of normal people were main tained at a very low level and these biomarkers of some cardio-cerebrovascular diseases may be flew out to the plasma/serum due to tissue necrosis or apoptosis rupture, so the detection of miR-21 or VEGF in the serum was of great significance. Three serum samples (2.5%) of normal human were used to investigate the performance of our tMB. The certain amounts of miR-21 (Table S2) or VEGF (Table S3) were added to these serum samples and all of the recoveries were around 100%, which proved the potential of our tMB in application in real samples.

In conclusion, we have constructed a classical tMB consisted of ptMB and sfO and applied it to the detection of miR-21 and VEGF. The ptMB included the complementary sequences of miR-21 as the loop part, and the poly thymidine (T10) in its two ends to form the triplex stem of tMB with the sfO (A10). The addition of miR-21 would broke the Hoogsteen basepairing in triplex stem of tMB and hybrid with the loop of tMB, followed by the increasing distance induced fluorescence recovery by the hindrance of FRET. We optimized the length and sequences of triplex structure, as well as the pH to obtain the maximum degree of the fluorescence recovery. The tMB was used to quantify the miR-21, and the linerrange of 0.5-250 nM and the LOD of 0.18 nM were obtained. What’s more, we also replaced the FAM in tMB with the DNA/AgNCs and obtained ideal result. Significantly, we also successfully expanded the tMB to the detection of VEGF by the introduction of its aptamer. We believe that the tMB would be a universal strategy for the detection of miRNAs and biomarkers with suitable aptamer, which hold great potential for the early clinical diagnostics of diseases.

CONCLUSIONS

AUTHOR INFORMATION

ASSOCIATED CONTENT Supporting Information Schematic illustration of the analysis of miRNA using the classical dMB. Fluorescence kinetic test of the tMB and dMBs. Fluorescence emission spectra of the tMBs with different triplex sequences. Sequences of the DNA/RNA used in this article and the results of miR-21 and VEGF concentration in the analysis of human serum samples. This material is available free of charge via the Internet at http://pubs.acs.org.

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ACS Sensors Corresponding Author *Prof. Xiurong Yang, State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. E-mail: [email protected]. Tel.: +86 431 85262056; Fax: +86 431 85689278. *Dr. Jian Sun, State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. E-mail: [email protected]. Phone: +86 431 85262063. Fax: +86 431 85689278.

Author Contributions The manuscript was written through contributions of all authors.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Key Research and Development Program of China (2016YFA0201301), the National Natural Science Foundation of China (Grant Nos. 21435005, 21627808, 21605139), and Key Research Program of Frontier Sciences, CAS (QYZDY-SSW-SLH019).

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