Pinpoint the Positions of Single Nucleotide Polymorphisms by a

Jan 26, 2017 - Institute of Nuclear and New Energy Technology, Collaborative Innovation Center of Advanced Nuclear Energy Technology, Beijing Key Lab ...
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Pinpoint the Positions of Single Nucleotide Polymorphisms by a NanoCluster Dimer Jie Liu, Yuexiang Lu, Lu Feng, Song Wang, Shixi Zhang, Xuewei Zhu, Linfeng Sheng, Sichun Zhang, and Xinrong Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04981 • Publication Date (Web): 26 Jan 2017 Downloaded from http://pubs.acs.org on January 28, 2017

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Analytical Chemistry

Pinpoint the Positions of Single Nucleotide Polymorphisms by a NanoCluster Dimer Jie Liu,a Yuexiang Lu,b Lu Feng,a Song Wang,a Shixi Zhang,a Xuewei Zhu,a Linfeng Sheng,c Sichun Zhang,*,a and Xinrong Zhanga a. Department of Chemistry, Tsinghua University, Beijing 100084, P.R. China b. Institute of Nuclear and New Energy Technology, Collaborative Innovation Center of Advanced Nuclear Energy Technology, Beijing Key Lab of Radioactive Waste Treatment, Tsinghua University, Beijing 100084, P. R. China c. State Key Laboratory of Analytical Chemistry for Life Science, Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, 210023, P.R. China ABSTRACT: Single Nucleotide Polymorphisms (SNPs) are the most fundamental internal causes for many genetic diseases. However, the location information of SNPs in a specific DNA sequence is not well acquired through current SNPs detection methods, except for accurate DNA sequencing. Here we report a fluorescence enhancement phenomenon in the process of two silver nanoclusters (AgNCs) approaching closely to form a Nanocluster Dimer (NCD). The fluorescence intensity is sensitive to the distance between two AgNCs, therefore the NCD lights into different fluorescence intensities upon binding SNPs targets with mismatched bases at different positions. Interestingly, the fluorescence intensities of the NCD decrease linearly when the position of single mismatched base moves gradually from the middle point to the end of the target DNA. The NCD is a single probe acting as a universal platform to pinpoint various SNP positions. With this single probe, we can not only identify the existence of SNPs, but also pinpoint the location of a specific single mismatched base in the adjacent positions. This strategy is feasible to detect specific gene point mutations in clinical samples.

Single-nucleotide variations are closely associated with some human genetic diseases. Among the variations, the single nucleotide polymorphisms (SNPs) are the most abundant component1 and the detection of them is very important for early diagnosis of cancer2. Current methods of SNPs detection include accurate DNA sequencing3 and DNA biosensors4,5. In most situations, it is unnecessary to know all the bases in a specific DNA sequence, so the accurate DNA sequencing methods are overqualified. In recent years, much attention has been paid to DNA biosensors, which are useful for specific gene point mutations such as K-ras mutations6, Sickle cell anemia7,8 and Hb Constant Spring9 etc. Traditionally, DNA biosensors consist of fluorophores and quenchers10-12. Many nanomaterials have been studied as platform to quench the emission of fluorophore, such as gold nanoparticles13-15, carbon nanotubes16, graphene oxide17,18, silicon nanowire19,20, and MoS221,22 etc. The fluorophore-labeled single-stranded DNA (ssDNA) is quenched by the quenchers. And after hybridization, the double-stranded DNA (dsDNA) departs from the quenchers, regenerating the fluorescence. Because of the differences in binding free energy, the changed fluorescence intensities can be observed in the presence of single-base mismatched target DNA. The location of single mismatched base has been investigated with a graphene oxide nanosheets-based fluorometric DNA biosensor23. However two DNA sequences with single mismatched base in adjacent positions cannot be discriminated. Recently, Werner and coworkers24 reported a method based on the discrimination of both fluorescence intensity and color

to detect SNPs using chameleon NanoCluster Beacon (cNCB) sensor. The color of the AgNCs can change substantially depending upon its position relative to an enhancer sequence. When a single mismatched base existed in the target DNA, the position of AgNC relative to the G-rich sequence changed, therefore the color of the AgNC changed. Furthermore, Yeh and coworkers developed a similar sliver cluster probe, termed methyladenine-specific NanoCluster Beacon (maNCB) for N6-Methyladenine (m6A) detection at the single-base resolution25. Based on the fact that the binding affinity of adenine and guanine is stronger than that of m6A and guanine, identification of m6A at the single base level and quantification of adenine methylation in heterogeneous samples were achieved. In their study, however, a particular NCBs probe could only be used to recognize a defined SNP position of the target DNA, due to the specificity of NCB sequences. The multiple probes with different sequences should be designed to identify various SNP positions of DNA targets. This is obviously inconvenient in analytical practices. It is useful to design a single probe acted as a universal platform to pinpoint various SNP positions, but it sounds a very difficult task. Here we developed a new type of sliver cluster probes for the purpose above, based upon the discovery of a nanocluster fluorescence enhancement phenomenon affected by various SNP positions of DNA targets. We termed this new type of sliver cluster-based DNA probe as Nanocluster Dimer (NCD). In our proposed method, two darkish DNA-template sliver nanoclusters were used as the fluorescence reporters. Two hybridization sequences linked to the two nanocluster probes can hybridize with the target DNA sequence. Two AgNCs

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were squeezed with each other in the middle of the hybridized sandwich structure, inducing the enhancement of fluorescence. When the single mismatched base existed in the target DNA, the fluorescence intensity decreased compared to that of the perfectly hybridized sandwich structure. The fluorescence intensity of the NCD decreased linearly when the position of single mismatched base moved gradually from the middle point to the end of the target. By this method, we can pinpoint target DNA sequences with SNPs in the adjacent positions. EXPERIMENTAL SECTION Chemical and Materials All the chemicals are at least of analytical grade. Silver nitrate (AgNO3) and sodium borohydride (NaBH4) were purchased from Alfa Aesar (Shanghai, China). PB buffer I (10 mM, pH=7.4) and PB buffer II (20 mM, pH=7.0) were purchased from Beijing Dingguo changsheng Biotechnology Co,.Ltd. DPBS buffer was purchased from CORNING (Shanghai, China). Centrifugal filtration tube (MWCO = 3 kDa) was purchased from Millipore. All solutions were prepared with ultrapure water (18.2 MΩ·cm) from a Millipore Milli-Q system. All the oligonucleotides were synthesized and purified by Sangon Inc. (Beijing, China).All the Sequences of oligonucleotides used in this work were listed in Table S1. Instrumentation and Chracteriaztion All the fluorescence measurements were performed with multimode plate reader (VARIOSKAN FLASH, Thermo Fisher Scientific Inc.) using a black 96-well microplate (COSTAR, CORNING). Transmission electron microscope (TEM) measurements were performed on Jeol JEM-2100F instrument (JEOL Ltd, Japan). All the DNA solutions were quantified by UV/vis absorption at 260 nm, from JASCO V-550 UV–vis spectrophotometers (Tokyo, Japan). The excited-state emission lifetimes were obtained on the FLSP920 (Edinburgh Instrument). Eppendorf 5418-R centrifuge was used for the human serum ultrafiltration. Synthesis of DNA-template Silver nanoclusters The method for the synthesis of DNA-template AgNCs was followed the previous reported literature26,27 with some modifications. In brief, nucleation probes (NC probes 15 uL 100 uM) dissolved in PB buffer I was mixed with PB buffer II (73 uL). Freshly prepared AgNO3 aqueous solution (6 uL 1.5 mM) was added into the NC probes solution obtained above with vigorous shaking for 30 s. Then, the mixed solution was kept in an ice bath and in the dark. After 30 mins, freshly prepared NaBH4 ice aqueous solution (6 uL 1.5 mM) was added into the solution obtained above, followed by vigorous shaking for 30 s. Before use, the solutions were kept in the dark at 4 ℃ for 6 hours. The fluorescence enhancement of NCD For the forming of NCD-4, NC-probe-0 and NC-probe-4 AgNCs solution were mixed with equivalent, and then perfectly-matched (PM) target DNA dissolved in DPBS was quickly added into the mixed NC-probes solution (NC-probe-0:NC-probe-4: target DNA = 1:1:1). The mixed solutions were kept in 37 ℃ water bath for 30 mins before the following fluorescence measurements. When the NCD-4 was used to investigate mismatched positions, the PM target DNA was replaced by the target DNA with single mismatched bases at different positions. Kinetic experiments For investigate the relation between the reaction rate and mismatched positions, as shown in Figure 6c-d, kinetic experiments were measured at 25 ℃ and other

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experimental conditions were same as hybridization experiments of NCD-4. The fluorescence intensity was measured at 613 nm (excited at 555 nm) every minute after adding the target DNA (PM, P1, P3, P5, P7, P9, P11, P13, P14, P15, P16 and P17). The total measure time was 150 mins. For getting the shorter reaction time, kinetic experiments were measured at 37 ℃. As shown in Figure S9, we can find that the fluorescence intensity did not reach the maximum until about 30 mins, so all the fluorescence intensities were measured 30 mins after adding the target DNA. Tay-Sachs disease (TSD) mutations detection in diluted human serum For the Tay-Sachs disease mutations detection in diluted human serum, the NC-probe-0-tsd and NC-probe-4tsd were used for the synthesis of DNA-template AgNCs. The hybridization experiments conditions were same as that of NCD-4 except that perfectly-matched target DNA sequence was replaced by Normal-C-target or TSD carrier-G-target as shown in Table S1. Before adding human serum into the mixed AgNCs probes solution, the human serum was centrifuged by a centrifugal filtration tube (MWCO = 3 kDa, Millipore) at 10,000 rpm for 90mins.

Figure 1. (a) Schematic illustration of different hybridization structures of NCD-1 (NC probe-0 and NC probe-1), NCD-2 (NC probe-0 and NC probe-2), NCD-3 (NC probe-0 and NC probe-3) and NCD-4 (NC probe-0 and NC probe-4); (b) Fluorescence spectra of NCD-1, NCD-2, NCD-3 and NCD-4 after hybridization; (c) Fluorescence intensities of NCD-1, NCD-2, NCD-3 and NCD-4 after hybridization at maximum emission.

RESULTS AND DISSCUSION In order to explore how the distance between two AgNCs affected the fluorescence emission of hybridized NCD, we designed four different hybridization structures of NCDs and two AgNCs distributed at different locations of the hybridized dsDNA (Figure 1a). Each NCD consisted of two DNAtemplate silver probes. One component DNA sequence of all the four NCDs was the same (NC probe-0) and the other one was different (All these NC-probes sequences of oligonucleotides were listed in Table S1). Among the four NCDs, the NCD-4 had the most obvious fluorescence enhancement. NCD-3 and NCD-4 had the same sandwich hybridization structures with small differences in NC-nucleation sequences (Figure 1a). The fluorescence emission spectra of all the four NCDs after hybridization were shown in Figure 1b. More detailed fluorescence properties of these four NCDs before and after hybridization are shown in Figure S1-S4.The

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fluorescence intensity of NCD-4 was 32-fold stronger compared to that of NCD-1 (Figure 1c).

Figure 2. (a) Schematic illustration of different NCD-4 hybridization structures for seven target DNA sequences (one perfectly matched target DNA sequence and six target DNA sequences with different length of T nucleotides spacers inserted in the middle of perfectly matched target DNA); (b) Fluorescence emission spectra and (c) intensities of NCD-4 with perfectly matched target DNA sequences, six target DNA sequences with different length of T nucleotides spacers and PB buffer without target DNA.

To confirm that the fluorescence enhancement of NCD was decided by the distance between the two AgNCs, we inserted T nucleotides as spacers in the middle of target sequence to change the length of the NCD-4 (illustrated in Figure 2a). As shown in Figure 2b-c, with the number of T nucleotides spacers increasing, the fluorescence intensity of hybridized NanoCluster Dimer decreased gradually. When the number of T nucleotides spacers reached more than 20, there existed little fluorescence enhancement between these two silver nanoclusters. The close proximity arrangement of the AgNCs (NCD-4) before and after hybridization was characterized by high resolution transmission electron microscope (HRTEM, Figure S5). It indicated that the distance between two AgNCs impacted greatly on the fluorescence intensities of them. When two AgNCs approached to each other, interaction between them got stronger, inducing the greater fluorescence enhancement. The excited-state emission lifetimes of NCD-4 before and after hybridization were measured. Before hybridization, the emission lifetime of two AgNCs (NC probe-0 and NC probe-4) is 1.82 ns and 3.48 ns, fitting with a monoexponential function. After hybridization, the emission lifetime of NCD-4 is 0.74 ns and 2.58 ns, fitting with a dualexponential function (Figure S6).The lifetime of two AgNCs (NC probe-0 and NC probe-4) got shorter after they got close. The possible interaction effect between two AgNCs is likely to be the plasmonics effect.

Figure 3. (a) Schematic illustration of three target DNA sequences with single mismatched bases (The SNPs sites and the nucleotides are shown in red and boldface) located at 1st, 5th and 9th positions of target (form the middle point of target); (b) Schematic illustration of different NCD-4 hybridization structures for N1-P1, N1-P5 and N1-P9; (c) Fluorescence intensities of NCD-4 for N1P1, N1-P5 and N1-P9; (d) Schematic illustration of four target DNA sequences with single-base substitution, double-base substitutions, triple-base substitutions and tetrad-base substitutions located at P5 position; (e) Schematic illustration of different NCD-4 hybridization structures for N1-P5, N2-P5, N3-P5 and N4-P5; (f) Fluorescence intensities of NCD-4 for N1-P5, N2-P5, N3-P5 and N4-P5. (Maximum emission is 613 nm, excited at 555 nm. All the fluorescence intensities were measured at least three times).

We also explored how the base substitutions affected the fluorescence intensity of NCDs. We took NCD-4 as an example for all following experiments. For single-base substitution, we designed three DNA target sequences (Figure 3a), with the single mismatched bases at different positions. The single mismatched bases were located at 1st, 5th and 9th positions of target (form the middle point of target), which were identified as N1-P1, N1-P5 and N1-P9, respectively. After hybridization, the fluorescence intensity decreased with the increasing distance between the position of mismatched base and the middle point (N1-P1>N1-P5>N1-P9), as shown in Figure 3c. The results indicated that the distance between two AgNCs increased, therefore the interaction effect between them weakened (Figure 3b). It was found that the fluorescence intensity decreased as the number of mismatched bases increased. We took P5 position as an example, as shown in Figure 3d. At P5 position, when the number of mismatched bases increased from one to four, the distance between two AgNCs increased gradually as shown in Figure 3e, the fluorescence intensity decreased along the rule (N1-P5>N2-P5>N3-P5>N4-P5 in Figure 3f).

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Furthermore, to examine the effects of the mismatched position on fluorescence intensity of NCD-4, we changed the single-base substitution into double-base substitutions, triple-base substitutions and tetrad-base substitutions at different positions. After hybridization, the fluorescence intensity also decreased, similar to that of the single-base substitution (N2P1>N2-P5>N2-P9, N3-P1>N3-P5>N3-P9, N4-P1>N4P5>N4-P9), as shown in Figure S7. For the investigation of the effects of the number of mismatched bases on fluorescence intensity of NCD-4, we changed the position of mismatched bases from P5 position into P1 or P9 positions, with mismatched bases increasing from one to four, the change of fluorescence intensity is similar to that of the P5 position (N1P1>N2-P1>N3-P1>N4-P1, N1-P9>N2-P9>N3-P9>N4-P9), as shown in Figure S8.

Figure 5. (a) Schematic illustration of 17 target DNA sequences with single mismatched bases (The SNPs sites and the nucleotides are shown in red and boldface.) moving from middle point to 5’end (P1-P17); (b) Schematic illustration of different NCD-4 hybridization structures for P1-P13; (c) Schematic illustration of different NCD-4 hybridization structures for P14-P17; (d) Fluorescence emission spectra of the NCD-4 with single mismatched bases at different positions (P1-P13); (e) Linear relationship between the fluorescence intensity and the position of single mismatched base (“0” position means perfect match); Figure 4. (a) Schematic illustration of target DNA sequence perfectly matched and target DNA sequences with different single mismatched bases types (T, C, and G) at P4 position (The SNPs sites and the nucleotides are shown in red and boldface); (b) Schematic illustration of different NCD-4 hybridization structures for four target DNA sequences; (c) Fluorescence emission spectra and (d) intensities of NCD-4 with perfectly matched base (A) and different mismatched bases types (T, C, and G) at P4 position.

In order to evaluate the influence of mismatched base types, we took N1-P4 (Figure 4a-b) as an example, where the T−A bond was changed to T−T (A→T), T−C (A→C), and T−G (A→G), respectively. As shown in Figure 4c-d, the fluorescence of N1-P4 with different mismatched base types showed similar intensities and the fluorescence intensities of N1-P4 with different mismatched base types decreased obviously compared to that of N1-P4 perfectly matched, indicating that mismatched base type changes could be ignored in pinpointing the location of a specific single mismatched base.

All the results above has shown that the fluorescence enhancement of NCD could be used for detecting the DNA mutations and the fluorescence intensity are affected by both the number and position of mutations. For the SNP detection, there is only single base mutation, so to examine the existence of SNP and pinpoint the mutation positon are the main tasks. To discriminate target DNA sequences with single-base substitutions in the adjacent positions, we designed 17 target DNA sequences with single mismatched bases moving from middle point to 5’-end (P1, P2…P16, P17), as shown in Figure 5a. We used the two NC-probe sequences of NCD-4 to detect 17 target DNA sequences above. As shown in Figure 5d-e, the fluorescence intensities of P1, P2…P12, P13 linearly decreased as the distance from middle point increased, and the linear equation is y = -12.54 x + 221.28 (R2=0.98, 0≤x≤13). Therefore, we could precisely pinpoint the location of a specific single mismatched base in target DNA. In addition, the fluorescence intensities of P13, P14, P15, P16, and P17 increased as the distance from the middle point increased, as shown in Figure 6a.

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Figure 6. (a) Fluorescence emission spectra of the NCD-4 with single mismatched bases at different positions (P13-P17); (b) Gibbs binding free energy (∆G, in red) and the fluorescence intensity (in black) with the single mismatched bases at different positions; (c) Kinetic experiments of NCD-4 with single mismatched bases at different positions (P1, P3, P5…P13); (d) Kinetic experiments of NCD-4 with single mismatched bases at different positions (P13-P17). To explain the mechanism why fluorescence intensities linearly decreased at the range from P1 to P13, the different hybridization reaction rates were studied by examining the target DNA sequences with SNPs at different positions, since we considered the reaction rates would be one of the most important factors. As shown in Figure 6c, from P1 to P13, the hybridization reaction rates of DNA sequences with SNPs in the positions of every other base decreased gradually. The changing trends of hybridization reaction rates and fluorescence intensities with single mismatched base from P1 to P13 are in good consistency. It indicated that the different positions of single mismatched bases resulted in the different hybridization reaction rates and the different hybridization reaction rates made the significant difference on the fluorescence intensities of the single mismatched base at different positions. In addition, the increasing hybridization reaction rates from P13 to P17 is a good explanation for the fluorescence intensities increasing when the single mismatched base moves from P13 to P17 (Figure 6d). Furthermore, we compared the Gibbs binding free energy (∆G, for the NC-Probe-0 and DNA target) with the fluorescence intensities of the single mismatched bases at different positions (Figure 6b). Interestingly, we found that the two changing trends had a significant negative correlation with the correlation coefficient r=-0.617 (P