Signal amplification method for miR-205 assay through combining graphene oxide with duplex-specific nuclease

Since microRNA-205 (miR-205) is a predictive biomarker for anti-radiation of nasopharyngeal carcinoma (NPC), quantitative detection of miR-205 is important for developing personalized strategies for the treatment of NPC. In this investigation, based on the graphene oxide sensor and duplex specific nuclease (DSN) for fluorescence signal amplification, a highly sensitive detection method for miR-205 was designed. A target-recycling mechanism is employed, where a single miR-205 target triggers the cleavage of many DNA signal probes. The method shows the ability to analyze miR-205 in solution, and it can detect miR-205 at concentrations as low as 132 pmol L−1 with a linear range of 5–40 nmol L−1. Furthermore, the method is specific in that it distinguishes between a target miRNA and a sequence with single base, double base and three base mismatches, as well as other miRNAs. Considering simplicity and excellent sensitivity/specificity, it is promising for applications in radioresistance studies as well as the early clinical diagnosis of NPC.


Introduction
Nasopharyngeal carcinoma (NPC) is an endemic disease prevalent in Southeast Asia. 1 Radiation therapy has become the standard treatment for locally advanced NPC due to the radiosensitivity of the top and side walls of the nasopharyngeal cavity. 2,3 However, the failure of radiotherapy for NPC will lead to treatment delays. 4,5 It can be seen that the problem of radiation resistance needs to be solved urgently.
MicroRNAs (miRNAs) are a class of endogenous non-coding single-stranded RNA found in eukaryotic cells, usually 19 to 25 nucleotides in length, about 30% of human genes are regulated by miRNAs, and the expression levels of miRNAs are closely related to the occurrence of various diseases in humans. 6,7 Studies have shown that the abnormal expression of miRNAs is closely related to the emergence of a variety of cancers, and can be used as an effective biomarker for the diagnosis, progression and therapeutic response of cancer. [8][9][10][11][12] miR-205, also known as hsa-miR-205, is micro-RNA which includes 22 bases. In the gene sequence based on the protection of Takifugu rubripes and mice, miR-205 was rst predicted by computer and subsequently conrmed in zebra sh and humans. Recent studies have shown that miR-205 is signicantly higher in NPC radiationresistant cells than normal NPC cells, 13,14 indicating that miR-205 is associated with radioactivity in NPC cells. miR-205 may be used as a biomarker to predict radioresistance in NPC. This nding may contribute to the personalized treatment of NPC, thus the method of detecting miR-205 with high sensitivity is of great signicance.
Currently, there are many widely used methods developed for the detection of miRNAs, including northern blotting, 15 quantitative real-time polymerase chain reaction (qRT-PCR), 16 microarray analysis 17 and so on. The northern blot method has the disadvantages of large sample volume, cumbersome operation, long time consumption, multiple interference factors, and radioactive contamination. Due to the short length of miRNAs, the design of amplication primers is very difficult, and most of the traditional PCR techniques are indirectly judged by examining their precursors. Therefore, qRT-PCR does not reect the true level of miRNAs. Microarray technology enables simultaneous detection of multi-component miRNAs. However, the fabrication and detection steps of the chip are cumbersome and costly, and cross-hybridization is prone to occur. 18 Graphene oxide (GO) is a two-dimensional nanomaterial. Due to its good dispersibility and hydrophilicity, high cell absorption rate, rapid uorescence quenching ability and abundant modiable sites, it has been widely used in biomedical elds such as bio-propagation and biosensing. [19][20][21] GO is a two-dimensional monolayer carbon material that strongly adsorbs single-stranded oligonucleotide molecules by p-p stacking effect and efficiently quenches the uorescent signal of uorescent dyes or uorescent nanoparticles labeled on oligonucleotides. 22 When the target miRNA hybridizes with the dyelabeled DNA probe, a heteroduplex is formed that detaches from the GO surface and restores the uorescent signal. 23,24 According to the previous studies, 25 Mn 2+ will be formation with the generation of GO.
Duplex-specic nuclease (DSN), a nuclease isolated from the hepatopancreas of the crab, Kamchatka, which selectively recognizes and cleaves a perfectly matched DNA duplex or the DNA in the RNA/DNA hybrid double strand has no effect on single-stranded DNA, RNA or double-stranded RNA, and can distinguish between target RNA and single-base mismatched RNA. [26][27][28] Therefore, DSN paved a promising way for ultrasensitive and accurate quantitation of miR-205.
Herein, a highly sensitive detection method which in the help of signal amplication has been designed by combining the uorescence quenching effect of a uorescently labeled single-stranded DNA probe with GO, and DSN. Compared with common graphene oxide sensors, this method has higher sensitivity and higher selectivity, and it has broad application prospects in the analysis of biomarkers for predicting radiation tolerance in NPC.

Materials and reagents
Sodium chloride (NaCl), magnesium chloride hexahydrate (MgCl 2 $6H 2 O) and Tris were purchased from China National Pharmaceutical Group Corp. GO was purchased from Nanjing XFNANO Materials Tech. Co., Ltd. (Nanjing, China). A DSN kit containing 100 U DSN and 10Â DSN reaction buffer was purchased from Evrogen (Moscow, Russia). Fluorescent aptamer (D205) and target RNA were synthesized by Sangon Biotechnology Co. Ltd. (Shanghai, China) and puried through high-performance liquid chromatography (all sequences used in this investigation show in Table 1). The 1Â Tris-HCl buffer solution (20 mmol L À1 Tris-HCl, 100 mmol L À1 NaCl, 10 mmol L À1 MgCl 2 $6H 2 O, pH ¼ 7.2) was prepared using DEPC water. Fluorescence spectra were obtained from Hitachi F-7000 spectrophotometer (Hitachi Ltd., Japan). The samples placed in quartz cuvettes were excited at a wavelength of 480 nm and all emission spectra were collected at wavelengths ranging from 500 to 600 nm at room temperature.

Maximization of enzyme action
In order to promote the optimal function of the DSN enzyme, the various conditions which including the order of GO addition, the amount of DSN, the temperature of the reaction solution and the time required for the reaction were separately optimized.
For the exploration of the operation steps, two methods of operation were tried respectively. Method one, D205 (100 nmol L À1 ) was added to 10 mg mL À1 of GO solution for 10 minutes, then miR-205 (50 nmol L À1 ) and DSN (0.3 and 0.6 U) was added into react mixture at 52 C for 3.5 hours. Method two, DSN (0.3 and 0.6 U) was added to buffer containing D205 (100 nmol L À1 ) and miR-205 (50 nmol L À1 ) at 52 C for 3.5 hours, and nally GO (10 mg mL À1 ) was added for 10 minutes.
As for the determination of the detection temperature, DSN (0.3 U) was added to buffer containing D205 (100 nmol L À1 ) and miR-205 (50 nmol L À1 ) at different temperature (49, 52, 55 and 58 C) for 3.5 hours, and nally GO (10 mg mL À1 ) was added for 10 minutes.

miRNA-205 detection
Various concentrations of miRNA-205 (0-150 nmol L À1 ) were added to 100 nmol L À1 D205, followed by 0.3 U DSN and a quantity of 10Â DSN buffer were added. Aer incubation at 52 C for 3.5 hours, 10 mg mL À1 of GO was added for 10 minutes. The uorescence intensity was excited at 480 nm and measured at 520 nm.

Specicity experiments
Three mismatches and two different target sequences were designed, which including single base mismatch targets (Rm1), double base mismatch targets (Rm2), three base mismatch targets (Rm3), NC (non-complementary RNA), miR-21, miR-141 and Let-7a. The RNA sequence and D205 were mixed while reacting with DSN for 3.5 hours, then GO was added for 10 minutes, and the uorescence intensity at 520 nm was recorded, and the F/F 0 ratio was compared. [29][30][31][32] Results and discussion  heteroduplexes were formed as the substrate for DSN. DSN selectively hydrolyzed DNA strands in DNA/RNA heteroduplexes, resulting in the release of miR-205 and the recovery of uorescence signal. Because miRNA-205 remained intact during the hydrolytic process, it could return to the solution and then react with another DNA, which trigger the next round of DSN-based hydrolytic process. Such a cycle started anew, leading to the continuous cleavage of DNA/RNA heteroduplexes, which greatly accumulated the uorescence signal for miRNA-205 detection. [33][34][35] As a consequence, the change of uorescence intensity with the concentration of miR-205 could be measured correspondingly. It was speculated by us that the purposed method was suitable for using to determination of miR-205.

Feasibility analysis
In order to initially evaluate the feasibility of the strategy (Fig. 1), four react conditions were performed and results can be denitely observed by the naked eye. Tube 1 is 100 nmol L À1 D205, tube 2 is mixed with 100 nmol L À1 D205 in 10 mg mL À1 GO solution, tube 3 add 50 nmol L À1 target miR205 to GO (10 mg mL À1 ) containing 100 nmol L À1 D205, tube 4 was further added with 0.3 U of DSN enzyme in a GO (10 mg mL À1 ) solution containing 100 nmol L À1 of D205 and 50 nmol L À1 of miR-205. It is easy to nd that the uorescence intensity of tube 2 is almost total quenched by GO. Furthermore, the uorescence intensity varies greatly in the absence and the presence of DSN. In the absence of DSN (tube 3), the uorescence intensity is slightly restored, which owing to the uorescence only contributed by the concentration of D205. On the other hand, the uorescence intensity obvious recovers to the initial state under the action of DSN (tube 4). Because DSN specically digest DNA in DNA/RNA duplexes which induce RNA cyclic hybridize to complementary DNA probe, uorescence signal would quickly accumulate and remarkably enhance. These results indicated that the DSNbased GO sensor can achieve signal amplication for detection applications.

Optimization of the assay conditions
To optimize the degree of amplication of the sensor signal, various DSN reaction conditions were tested. Considering that GO possesses signicantly different adsorption affinity for ssDNA and DNA/RNA heteroduplexes, the effect of different sequence of add GO to the reaction is preferentially investigated. As shown in Fig. 2A, it is clear that the degree of signal   amplication is signicantly higher in the case which GO was added in the last step. In addition, regardless of the amount of DSN was 0.3 U or 0.6 U, same signal amplied tendency were observed. Consequently, the strategy that GO was put into reaction mixture in the last step was used in the following RNA detection studies.
The enzymatic amplication efficiency is closely related to the amount of DSN. Therefore, the detection sensitivity of miR-205 is inevitably affected by the enzyme content in the system. The effect of different concentrations of DSN (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 U) system on the uorescence response signal was investigated in this experiment. As can be seen from Fig. 2B, as the amount of enzyme increases, the F/F 0 ratio increases rapidly, and then maintains a relatively stable trend. When the amount of DSN is more than 0.3 U, further increase in the amount of the enzyme does not cause a markedly signal change. It is demonstrated that 0.3 U DSN is enough to amplify uorescence signal in the designed detecting platform. As a consequence, 0.3 U was selected as the optimum DSN amount in this experiment.
With the purpose to increase the efficiency of detection, the temperature of digest reaction was also investigated. According to the product specication, the optimum temperature of the DSN is 45-60 C. Either temperature is too high or too low, the activity of the enzyme will be signicantly reduced. Considering the melting temperature (T m value) of D205 probe and miR-205 hybrid double-stranded structure, it was optimized at 49-58 C to ensure efficient hybridization-DSN digestion cycle amplication. As shown in Fig. 2C, when the temperature is 52 C, the F/F 0 ratio reaches the maximum value. However, the signal of the sample system dramatic decline when the temperature over 52 C. It is speculated by us that D205 and miR205 is difficult to format a stable heteroduplexes resulting from temperature is very close to the T m value. The enzymatic cleavage effect observably decreases because there is not enough substrate to perform the digest reaction. Therefore, 52 C was selected as the detecting temperature in this study. Finally, reaction time was another key factor affecting the enzymatic cleavage reaction and was tested (Fig. 2D). As the reaction time prolonged, the F/F 0 ratio gradually increased and revealed that 3.5 hours was the optimal time for the reaction.

Sensitivity of assay miR-205 by DSN amplied signal
The results of the proposed method for detecting miR-205 under optimal optimization conditions are shown in Fig. 3. The Fig. 3A is the uorescence spectrum corresponding to different concentrations of the target miR-205. It is clear to know that almost no uorescence signal is generated in the absence of miR-205, and the uorescence intensity of the system gradually enhance when the concentration of miR-205 is increasing. The maximum uorescence intensity is always exhibited at 520 nm. When the concentration of miR-205 is in the range from 5 to 40 nmol L À1 , the uorescence intensity at 520 nm is linear with the concentration (Fig. 3B). The regression equation is F ¼ 53.24 Â [miR-205] nmol L À1 + 933.59, R 2 ¼ 0.954, where F is the uorescence intensity at 520 nm. The detection limit is 132 pmol L À1 (3s/slope), where s represents the standard deviation of the control group and the slope represents the slope of the linear regression curve. The detection limit is lower than that of the ordinary GO detection method, 36 indicating that the combination of DSN and GO sensor can realize uorescence signal amplication and high sensitivity detection of miR-205.

Specicity of miR-205 detection by DSN
DSN is a very specic cleavage enzyme that distinguishes base mismatches in double-stranded nucleic acid structures. Based on the superior discriminating power of DSN enzymes, we also examined the specicity of the method. Under the same experimental conditions, when the probe is present, the same concentration of different sequences of miRNAs are detected, including NC, Let-7a, miR-21, miR-141, Rm3, Rm2, Rm1 and miR-205. Interference with RNA detection of miR-205, mismatched target was hybridized to D205 and digested with DSN for 3.5 hours. Aer 5 minutes of GO was added in reaction, the uorescence spectrum was recorded and the uorescence intensity at 520 nm was compared with the uorescence intensity obtained with miR-205. As shown in Fig. 4, F/F 0 decreases as the number of mismatched bases increases, and RNA with one base mismatch has only half of the uorescent signal change relative to the target RNA, and RNA with more than three base mismatches does not cause signicant uorescence signal changes, which conrms the superior selectivity of our method.

Conclusions
In summary, aer investigating the stability and specicity of molecular probe targeting miR-205 and optimizing the hybridization reaction conditions, a sensitive detection method for miR-205 using the DSN to trigger uorescence signal cascade amplication is constructed. Through a series of verication, the established method exhibited excellent selectivity, good accuracy and satisfactory sensitivity (LOD, 132 pmol L À1 ), which is expected to provide valuable information for prediction of radioresistance and early clinical diagnosis of NPC.

Conflicts of interest
There are no conicts to declare.