KR101995895B1 - Isothermal detection method of DNA using graphene oxide and RNase H - Google Patents

Abstract

The present invention provides an isothermal detecting method of a target DNA. The method comprises: a step (a) of reacting RNA probe having a florescent dye on an end portion thereof with graphene oxide to obtain a first reactant; a step (b) of adding a sample expected to contain a target DNA in the first reactant obtained in the step (a) and obtaining a second reactant to make the RNA probe and the target DNA form a dimer; a step (c) of adding RNase H to the second reactant obtained in the step (b) and obtaining a third reactant decomposing the RNA probe; and a step (d) of measuring florescence of the third reactant obtained in the step (c). The isothermal detecting method of DNAs using graphene oxides and RNase H may be useful as a detecting method of simple, specific, sensitive DNAs in clinical diagnosis, cancer immune therapy, food safety, forensic medicine and precision medicine fields.

Description

[0002] Isothermal detection method of DNA using graphene oxide and RNase H [

The present invention relates to a method for detecting DNA at a single temperature, and more particularly to a method for detecting isothermal detection of target DNA using graphene oxide and RNase H.

There has been a continuing effort to detect small amounts of DNA or RNA in a very specific and simple manner in the fields of clinical diagnostics, cancer immunotherapy, food safety, forensics and precision medicine. Traditional analytical methods rely on polymerase chain reaction (PCR) in thermocycler to amplify the target gene. In recent years, a thermal cycling technique for performing PCR in a relatively small device compared to the initial device of the PCR machine has been greatly developed. However, there is an increasing need to develop new assays for detecting specific sequences in a way that has simple yet excellent sensitivity and specificity, available in resource-constrained countries and small clinics.

To date, there has been a growing interest in nucleic acid-sequence-based amplification, loop-mediated amplification, rolling-circle amplification and helicase-dependent amplification Methods for detecting nucleic acids without thermocycler (isothermal gene amplification) have been extensively studied. These methods use amplification products that are specific for polymerases and primers similar to conventional PCR under isothermal conditions. The performance comparable to the performance was shown.

Another isothermal detection method that is simpler than isothermal gene amplification is to directly amplify the detection signal from a particular target gene. Isothermal signal amplification can be accomplished by digesting nuclease-labeled probes, such as RNase H, exonuclease III, nicking endonuclease, and duplex specific nuclease Lt; RTI ID = 0.0 > DNA < / RTI > Isothermal signal amplifying methods (ISAM) require enzyme-specific structures in the target DNA-probe hybridization. For example, ISAM by exonuclease III forms a hybridization with the 3'blunt end of the probe, and the cleavage endonuclease ISAM contains a specific sequence for cleavage of the probe. Therefore, careful probe design is required for the formation of the enzyme substrate. In contrast, RNase H has the advantage of less restriction on the detection sequence because it only shows specificity for RNA in the form of target DNA-RNA duplexes.

Since the first use of fluorescence expression after probe DNA cleavage using target DNA re-use and fluorescent donor-quencher pairs, ISAM using RNase H called cycling probe technology (CPT) has greatly developed. Surface plasmon resonance (SPR) imaging was performed in CPT for 2 hours at room temperature, using a preliminary document (TT Goodrich, HJ Lee and RM Corn, J. Am. Chem. Soc. , 2004, 126, 4086-4087) Lt; RTI ID = 0.0 > 1 < / RTI > fM of the synthetic oligonucleotide. In another prior art (JH Kim, RA Estabrook, G. Braun, BR Lee and NO Reich, Chem. Commun. , 2007, 0, 4342-4344) The probe was immobilized on a gold nanoparticle (AuNPs) as a fluorescence quencher and was applied to the sensitive and rapid analysis of protease activity. However, since the gold nanoparticles are easily aggregated or immobilized on a disulfide-bonded reducing agent such as dithiothreitol (DTT), an RNA probe is desorbed, which is disadvantageous in that there is a restriction on the use of an RNase H enzyme requiring a disulfide bonded reducing agent . Since at least two hybridized bases as a substrate for RNase H can cause false positive signal amplification, the 2D structure of the oligonucleotide and nucleic acid ligase is applied to the ISAM to solve this lack of specificity (Prior Art JH Kim, Analyst , 2016, 141, 6381-6386). However, since this method also has difficulties in increasing the reuse of the target DNA while specifically controlling the formation of the hybrid of the RNA probe-target DNA, the RNA probe It is required to develop a new method that can increase the specificity by increasing the number.

Korean Registered Patent No. 10-1554173 (2015.09.18.) Korean Patent Publication No. 10-2018-0065748 (June 18, 2018).

Xiang Li et al., Science of Advanced Materials 2014, Vol. 6, 1-7.

In the present invention, a new method for overcoming the problems of the RNase H auxiliary ISAM using the oxidized graphene has been developed. Similar to pure gold nanoparticles, oxidized graphene is known to have selective affinity for single stranded DNA / RNA and can quench the fluorescence of dyes linked to the adsorbed nucleic acid. In contrast, functional groups such as epoxy, carbonyl and hydroxyl groups of oxidized graphene enable stable dispersion in aqueous solutions containing various polar molecules without the need for stabilizing ligands such as polyethylene glycols or oligonucleotides. Using these properties, the target RNA using the enzyme can be sensitively detected. Recently, it has been reported that double stranded DNA in which a single base is mismatched interacts with oxidized graphene. However, single base discrimination interactions have not yet been applied to nuclease based ISAMs.

Thus, we used oxidized graphene as a method to control sequence-specific degradation of dye-labeled RNA probes for simple, specific, sensitive detection of target DNA. As a result, the present invention, in which the graphene oxide was added before the RNase H enzyme reaction, was able to detect a 26-base-long target DNA in an amount as low as 20 attomoles within 30 minutes, and found a single base inconsistency regardless of the type of the incompatible base And thus the non-specificity, which is a disadvantage of using RNase H, could be significantly lowered. It is therefore an object of the present invention to provide a method for isothermal detection of DNA using graphene oxide and RNase H.

According to an aspect of the present invention, there is provided a method for producing a nucleic acid comprising the steps of: (a) reacting an RNA probe containing a fluorescent dye at its end with an oxidized graphene to obtain a first reactant; (b) adding a specimen expected to contain the target DNA to the first reactant obtained in step (a) to obtain a second reactant for forming a duplex between the RNA probe and the target DNA; (c) adding RNase H to the second reactant obtained in step (b) to obtain a third reactant for degrading the RNA probe; And (d) measuring the fluorescence of the third reactant obtained in step (c).

In one embodiment, the third reactant from which the RNA probe of step (c) has been degraded may be transferred to step (b) and repeatedly performed.

In one embodiment, the graphene oxide may be at a concentration of 1 to 50 [mu] g / ml.

In one embodiment, the RNA probe may be at a concentration of 0.1-500 nM.

In one embodiment, the RNase H concentration may be 0.01 to 5 U / [mu] l.

In one embodiment, steps (a) through (c) may be performed at a temperature of 20-70 ° C.

According to the present invention, it has been found that the isothermal detection method of DNA using graphene oxide and RNase H shows excellent specificity to the target DNA when the graphene oxide is added before the RNase H enzyme reaction. In addition, the isothermal detection method of the DNA can be performed rapidly within 30 minutes and is performed at a mild temperature of 37 ° C, which can effectively overcome the complexity and high cost of conventional molecular diagnostics.

Therefore, the isothermal detection method of DNA using the graphene oxide and RNase H can be used as a simple, specific and sensitive DNA detection method in clinical diagnosis, cancer immunotherapy, food safety, forensic medicine and precision medicine.

1 is a schematic diagram showing isothermal control of RNA probe-target DNA interaction using oxidized graphene and RNase H (Examples and Comparative Examples).
Figure 2 shows the fluorescence spectra of RNA probes in the presence and absence of oxidized graphene and target DNA.
Figure 3 shows the results (a, relative fluorescence intensity of the example, b, of the comparative example) of optimizing the amount of oxidized graphene mixed with the probe in the presence of the target DNA (100 nM) or non-complementary DNA Relative fluorescence intensity c, signal-to-noise ratio (SN ratio) of the embodiment, d, SN ratio of the comparative example).
Figure 4 shows the results (a, SN ratio of the examples, b, comparison) of optimizing the concentration of RNase H in the example or comparative example using 100 nM RNA probe, 100 nM target DNA and 10 쨉 g / SN ratio).
FIG. 5 shows the fluorescence intensity (FI) at 520 nm of a sample reacted according to the concentration of RNase H in the Example or Comparative Example using 100 nM RNA probe, 100 nM target DNA and 10 μg / (A in the example, FI in the embodiment, FI in the comparative example). The blue line in (a) shows the case where the target DNA is present in the examples, and the orange line in (b) shows the case in which the target DNA exists in the comparative example. The black lines in (a) and (b) show the case where the target DNA is not present in the examples and the comparative examples.
Figure 6 shows the results (a, fluorescence spectrum; B, SN ratio) showing the sensitivity of target DNA detection in the examples using optimized amounts of target DNA of 0-100 nM, optimized RNase H and oxidized graphene. The fluorescence intensity at 520 nm was used for SN ratio measurements. (a) shows the fluorescence spectrum of the target DNA of less than 10 pM, and the graph of (b) shows the SN ratio of the result of the target DNA of 0 to 0.1 nM.
FIG. 7 shows the results (a, fluorescence spectrum, b, SN ratio) showing the sensitivity of the target DNA detection in the comparative example using the amount of target DNA of 0-100 nM, the optimized RNase H and the oxidized graphene. The fluorescence intensity at 520 nm was used for SN ratio measurements. (a) shows the fluorescence spectrum of the target DNA of less than 10 pM, and the graph of (b) shows the SN ratio of the result of the target DNA of 0 to 0.1 nM.
Fig. 8 shows the results (a / c, fluorescence spectrum; b) showing the sensitivity of detection of target DNA in the examples / comparative examples using the amount of target DNA of 0-100 nM in the absence of RNase H and the optimized graphene oxide / d, SN ratio). The fluorescence intensity at 520 nm was used for SN ratio measurements. The graphs inserted in (a) and (b) are the fluorescence spectra obtained by enlarging the results for the target DNA.
Fig. 9 shows the results of a standard curve of fluorescence intensity for an RNA probe.
Fig. 10 shows the results (a, 100 nM RNA probe, oxidized graphene and four kinds of DNA sequences (wild type and three single bases) which were carried out according to the example, showing the specificity in detection of target DNA according to the order of addition of graphene oxide SN ratio in the reaction of mismatches (T [M_T], G [M_G] and C [M_C]); b, reaction of 100 nM RNA probe, oxidized graphene and four kinds of DNA sequences SN ratio). The graphs inserted in (a) and (b) show the SN ratios in which the results for the three mismatched DNAs are expanded.
Figure 11 shows the relative SN ratio results of RNA probes in the presence of wild type target DNA and three single base mismatched DNAs (T [M_T], G [M_G] and C [M_C]). The relative SN ratio is a value obtained by dividing the SN ratio of the reacted sample by the SN ratio of the reacted sample according to the comparative example.

(A) reacting an RNA probe containing a fluorescent dye at its end with oxidized graphene to obtain a first reactant; (b) adding a specimen expected to contain the target DNA to the first reactant obtained in step (a) to obtain a second reactant for forming a duplex between the RNA probe and the target DNA; (c) adding RNase H to the second reactant obtained in step (b) to obtain a third reactant for degrading the RNA probe; And (d) measuring the fluorescence of the third reactant obtained in step (c).

The detection method of the present invention includes a step (that is, step (a)) of reacting an RNA probe containing a fluorescent dye at its terminal end with an oxidized graphene to obtain a first reactant. In step (a), the oxidized graphene has strong affinity for single-stranded DNA and RNA and has fluorescence quenching capability, so that the fluorescence of the dye labeled with the RNA probe is quenched by the oxidative graphene in the absence of the target DNA.

The detection method of the present invention comprises the steps of adding a specimen expected to contain a target DNA to the first reactant obtained in step (a) to obtain a second reactant for forming a duplex between the RNA probe and the target DNA , Step (b)]. In step (b), the RNA / target DNA adsorbed to the oxidized graphene is protected from the DNA degrading enzyme and is released from the oxidized graphene by forming a duplex with the target DNA.

The detection method of the present invention includes the step of adding a RNase H to the second reactant obtained in step (b) to obtain a third reactant for allowing the RNA probe to be degraded (i.e., step (c)). The degradation of the RNA probe by RNase H in step (c) causes the target DNA to adsorb on the oxidized graphene without thermocycling to form a new duplex with the uninjured RNA probe, and the RNA probe of step (c) The third reactant may be moved to step (b) and repeatedly performed.

The detection method of the present invention includes the step of measuring the fluorescence of the third reactant obtained in step (c) (i.e., step (d)). In step (d), fluorescence is recorded using a microplate reader at an excitation wavelength of 480 nm and a wavelength of 500 to 700 nm.

In one embodiment, the oxidized graphene may be in a concentration of 1 to 50 μg / ml, preferably 5 to 20 μg / ml, most preferably 10 μg / ml, and the RNA probe may be in a concentration of 0.1-500 nM, Preferably 10 to 200 nM, most preferably 100 nM, and the RNase H concentration may be 0.01 to 5 U / μl, preferably 0.1 to 3 U / μl, and most preferably 2 U / have.

In one embodiment, steps (a) to (c) can be performed at a temperature of 20 to 70 캜, preferably 30 to 40 캜, most preferably 37 캜.

To evaluate the role of the oxidized graphene, two different methods (Examples and Comparative Examples) were compared according to the order in which the oxidized graphene was added to the RNase H enzyme reaction. As a result, the fluorescence of the RNA probe was observed in the presence of oxidized graphene And the fluorescence labeled RNA probe was adsorbed to the oxidized graphene. In addition, the optimum amount of graphene oxide and RNase H was secured, respectively, to confirm excellent sensitivity and excellent specificity of 20 attomole to target DNA. Therefore, the isothermal detection method using DNA graphene and RNase H is performed at a mild temperature of 37 ° C, thus effectively overcoming the complexity and high cost of conventional molecular diagnostics, while at the same time having excellent sensitivity and excellent specificity, DNA analysis can be usefully used.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

<Examples>

1. Materials and Methods

(1) Material

All oligonucleotides used in the present invention (see Table 1) were obtained from IDT, Inc. (Illinois, USA). RNase H (60 Units / μl) in storage buffer (25 mM Tris-HCl, 30 mM sodium chloride, 0.5 mM EDTA, 1 mM DTT, 50% glycerol, pH 7.5) was purchased from Takara Bio Inc. . 10 × reaction buffer (750 mM potassium chloride, 500 mM Tris-HCl, 30 mM magnesium chloride, 100 mM DTT, pH 8.3) was obtained from New England BioLabs Inc. (Ipswich, USA).

All other compounds were purchased from Sigma-Aldrich and used without further purification. Ultrapure DNase / RNase-free distilled water (DW) processed using a Biopak ^® grinder from Milli-Q ^® water purification system (Millipore, MA, USA) Were used for all experiments.

(2) Preparation of oxidized graphene or oxidized graphene-probe complex

A solution of oxidized graphene (0.1 mg / ml) dispersed in D.W. was sonicated in a thermostatic bath sonicator (Branson, USA) for 1 hour to obtain a stable oxidized graphene suspension. The brown dispersion was centrifuged at 4000 rpm for 10 minutes and then the remaining pellets were dispersed in D.W. The quality of the oxidized graphene suspension was analyzed by measuring the size and concentration of the oxidized graphene using a nanoparticle analyzer (SZ-100, Horiba, Japan) and a UV-visible spectrophotometer (Carry 5000, Agilent, USA).

The oxidized graphene-RNA probe complex was prepared by adding 100 μl of the oxidized graphene solution (0.1 mg / ml) to a mixture of 100 μl of RNA probe (1 μM) and 100 μl of 10 × reaction buffer to 700 μl of DW After vigorous stirring for 30 seconds, the cells were incubated at room temperature for 1 hour. The graphene oxide-probe complex obtained by centrifuging the RNA probe-oxidized graphene mixture obtained by centrifugation at 12,000 rpm for 20 minutes at 20,000 rpm was vigorously stirred for 1 to 3 minutes and ultrasonicated and dispersed in a new buffer. Respectively.

(3) Evaluation of optimum amount of oxidized graphene and RNase H

To determine the optimum amount of oxidized graphene for target DNA detection analysis, the oxidized graphene-RNA probe complex prepared by mixing 100 nM RNA probe with various amounts of oxidized graphene was incubated at 37 &lt; 0 &gt; DNA or non-complementary DNA. Fluorescence of the reacted mixture was recorded using a microplate reader (Infinite M200 Pro, Tecan, Switzerland) at excitation wavelength of 480 nm.

A DNA detection assay was performed with an optimized amount of graphene oxide, 100 pM of target DNA, 100 nM of RNA probe and various amounts of RNase H to determine the optimal amount of RNase H for analysis. Before DNA detection analysis, the target DNA and RNA probes were denatured at 90 DEG C for 10 minutes and slowly cooled to room temperature. After the enzymatic reaction with RNase H, the enzyme activity was stopped by heat treatment at 90 ° C for 10 minutes, and after cooling at room temperature, the fluorescence spectrum was measured.

(4) DNA detection analysis

100 nM RNA probe or RNA probe-oxidized graphene complex was reacted in 20 μl of DNA buffer at various concentrations in the presence or absence of RNase H at 37 ° C. To 700 nm. &Lt; tb &gt; &lt; TABLE &gt; In the case of RNase H-added samples, the sample was heat-treated at 90 ° C for 10 minutes to stop the enzyme activity, and after cooling to room temperature, the fluorescence spectrum was measured.

(5) Method of forming oxidized graphene-probe complex before enzyme reaction (Example)

As the RNA probe is adsorbed on the oxidized gaffin surface, the fluorescence of the labeled dye in the RNA probe is quenched by the oxidative graphene. When the target DNA is present, the RNA probe is desorbed from the oxidized graphene as a duplex of RNA probe-target DNA (substrate of RNase H) is formed. RNase H degrades the RNA probe from the duplex and the target DNA is separated from the duplex and forms a new duplex with the RNA probe adsorbed to the intact oxidative graphene. In this way, the RNA probe was multi-isolated and digested with the target DNA. When the RNA probe is degraded by this method, the dye labeled on the probe is far away from the oxidized graphene, and fluorescence is amplified as a result of the multiple degradation cycle of the RNA probe. Therefore, the amplified fluorescence is measured to detect a single target DNA (See FIG. 1).

(6) Method of adding graphene oxide after enzyme reaction (Comparative Example)

Since the oxidized graphene is added to the reaction after the enzymatic reaction with RNase H is completed, the oxidized graphene does not affect the interaction of the enzyme or RNA probe-target DNA, and the fluorescence quenching of the remaining undamaged RNA probe after the enzyme reaction (See FIG. 1).

2. Results

(1) Evaluation of fluorescence quenching in detection of target DNA of oxidized graphene

To investigate the specificity of RNA probes and the role of oxidative graphenes on multiple degradation, oxidative graphene was used in two different ways (Example and Comparative Example) according to the order in which they were added to the RNase H reaction ). In the examples, the RNase H enzyme reaction was carried out in the presence of oxidized graphene. Since oxidized graphene has a strong affinity for single stranded DNA and RNA and has the ability to extinguish fluorescence, the fluorescence of the dye labeled with the RNA probe was quenched by oxidative graphene in the absence of the target DNA. The RNA / DNA adsorbed to the oxidized graphene is protected from the DNA degrading enzyme, and the single RNA probe can decompose the RNA probe only after it is released from the oxidized graphene by forming a duplex with the target DNA. RNase H dissociates the RNA probe to dissociate the target DNA to form another duplex with the unimpaired RNA probe adsorbed onto the oxidized graphene without thermal cycling. During the isothermal reaction, the labeled dye of the degraded probe is away from the oxidized graphene and is recovered as a result of the reaction. As a result, fluorescence can be amplified through multiple degradation cycles of RNA probes per single target DNA, allowing sensitive detection of target DNA. If the oxidized graphene participates in the specific formation of RNA probe-target DNA duplexes, high specificity can be expected with a simple linear RNA probe structure alone. In the comparative example, the oxidized graphene was added to the analytical sample after performing the enzymatic reaction. At this time, the oxidized graphene acts only as a fluorescence scavenger of the remaining intact RNA probe after the reaction.

The adsorption of single strand RNA probes and subsequent desorption was tested in the presence of the target DNA. As shown in Fig. 2, the fluorescence of the RNA probe is remarkably reduced in the presence of the oxidized graphene, which is a result of well showing the adsorption of the fluorescent dye-labeled RNA probe to the oxidized graphene (the apparent fluorescence quenching efficiency of the oxidized graphene Almost 100%). When the target DNA was added to the RNA probe-oxidized graphene mixture, 15.14% of the original fluorescence of the RNA probe was restored (see Table 2). Partial recovery of fluorescence means that not all RNA probes hybridize to the target DNA. A relatively large amount of oxidized graphene will interfere with the hybridization between the RNA probe and the target DNA. On the other hand, a small amount of oxidized graphene can not effectively adsorb RNA probes, resulting in a high background signal.

(2) Evaluation of optimum amount of graphene oxide

To achieve a low background signal due to hybridization of high efficiency probe-target DNA, the concentration of the RNA probe was fixed at 100 nM and then analyzed using two analytical methods (Examples and Comparative Examples Respectively.

Since the RNA probe can be desorbed from the oxidized graphene by competitive adsorption with non-complementary (NCD) DNA that induces a false-positive signal, the amount of oxidized graphene is sufficient to maintain adsorption of the RNA probe and NCD should. Thus, the optimal amount of graphene oxide was determined to maximize fluorescence increase after addition of target DNA, and to minimize fluorescence increase after addition of NCD. In the examples, the target DNA and NCD were added to the oxidized graphene-RNA probe complex, and in the comparative example, the oxidized graphene was added to the mixture of the RNA probe and the target DNA or the mixture of the RNA probe and the NCD.

As shown in Figure 3, fluorescence of RNA probes according to the amount of oxidized graphene added in the presence of 100 nM of target DNA and NCD, respectively, decreased with increasing amounts of oxidized graphene in both methods . Fluorescence in the presence of the target DNA was higher than fluorescence of the target or control without NCD. Increased fluorescence in the presence of the target DNA was maximized at 10 / / ml and 5 / / ㎖ of oxidized graphene, respectively, in the Examples and Comparative Examples. NCD increased fluorescence due to competitive adsorption with RNA in oxidized graphene. The false positive signal decreased as the amount of graphene oxide increased, and became equal to that of the control group in amounts of 20 μg / ml and 10 μg / ml of oxidized graphene for each of the Examples and Comparative Examples. However, in the presence of the target DNA in the amount of 20 μg / ml oxidized graphene of the example, fluorescence was hardly increased. In the comparative example, the fluorescence caused by the target DNA at the 5 ug / ml oxidative graphene concentration was maximized, but the fluorescence increased in the presence of NCD was relatively high. Thus, the optimal amount of graphene oxide was selected to be 10 [mu] g / ml in both methods.

(3) Evaluation of optimal amount of RNase H

The optimal amount of RNase H was determined by varying the enzyme concentration from 0 to 5 U / μl in both Example and Comparative Example. As shown in FIG. 4, the increase of relative fluorescence intensity was confirmed by increasing the amount of RNase H in the presence of the target DNA (100 pM) as compared with the control. In both methods, RNase H was maximized at 2 U / . Fluorescence was increased within the limited reaction time since the increased amount of RNA probes doubled with the target DNA was degraded by an increased amount of RNase H. However, in the presence of the target DNA at an RNase H concentration exceeding 2 U / μl, the rate of fluorescence increase was slow (see FIG. 5). Instead, the rate of fluorescence increase was maintained at RNase H concentrations higher than 2 U / μl in the absence of target DNA. Thus, 2 U / μl was determined as the optimal amount of RNase H in both methods. The reaction buffer solution of RNase H contained 10 mM DTT. ( J. JH Kim, RA Estabrook, G. Braun, BR Lee and NO Reich, Chem. Commun. , 2007, 0, 4342-4344 and DH Tsai, MP Shelton, FW Del Rio, S. Elzey, S. Guha, MR Zachariah and VA Hackley, Anal. Bioanal. Chem. , 2012, 404, 3015-3023) have shown that dye-labeled RNA probes immobilized on AuNPs are separated by high DTT concentrations, resulting in high background signals. However, in the present invention, even when 10 mM of DTT was present, instability of the RNA probe adsorbed on the oxidized graphene was not observed.

(4) Sensitivity evaluation according to the order of addition of graphene oxide

After optimizing the amount of graphene oxide and RNase H, the detection sensitivities according to the order of addition of graphene oxide were measured by using different amounts of target DNA (0-100 nM), the optimum amounts of oxidized graphene and RNase H, And evaluated by measuring the strength. Fig. 6 (Example) and Fig. 7 (Comparative Example) show fluorescence measured at 500 nm to 700 nm, and the intensity of the fluorescence increased with increasing amount of target DNA.

(공시료 분석시그널의 표준편차의 3배)에 근거하여, 실시예 및 비교예에 대한 검출한계를 각각 72 fM과 31 fM으로 추정하였고, 이는 각각 1.04 attomole와 620 zeptomole에 해당한다. 신호대 잡음비(SN ratio) 2에 기초한 검출된 표적 DNA의 최저 농도는 실시예 및 비교예 각각에 대해 20 attomole 및 2 attomole이었다. 그러나, RNase H 없이 수행된 분석에서 검출된 표적 DNA의 최저 농도는 실시예 및 비교예에 대해 각각 10 nM 및 1 nM인 것으로 확인되었다(도 8 참조). AuNP를 이용한 CPT의 SN ratio 2에 근거한 검출한계는 100 attomole이었다. 본 발명에서 제안하는 검출 방법은 AuNP를 이용한 CPT보다 적어도 5배 더 민감한 것이다. 선행문헌(Y.-H. Wu, C.-M. Jiang, and G.-X. Yang, Eur. Rev. Med. Pharmacol. Sci. 2016, 20, 1775-1780. 및 C. Hong, A. Baek, S. S. Hah, W. Jung, and DE. Kim, Anal. Chem., 2016, 88, 2999-3003.)에서는 각각 2 femtomole과 20 attomole의 검출한계를 갖는 산화그래핀과 효소 보조 ISAM을 이용한 RNA 검출을 보고하였다. 그러나 선행문헌에서의 검출한계는 2시간 이상의 반응에서 여러 단계를 거쳐야 달성될 수 있었다.For each of the Examples and Comparative Examples, a target DNA concentration as low as 1 pM and 100 fM was detectable when signal to noise ratio (SN ratio) was 2 and 3, respectively. As shown in Figs. 6 (b) and 7 (b), excellent linear relationships of 10 ³ and 10 ⁴ times or more were observed for each of the examples and the comparative examples. Based on detection limit (LOD)

(Three times the standard deviation of the blob analysis signal), the detection limits for the Examples and Comparative Examples were estimated at 72 fM and 31 fM, respectively, which correspond to 1.04 attomole and 620 zeptomole, respectively. The lowest concentration of detected target DNA based on the signal-to-noise ratio (SN ratio) 2 was 20 attomoles and 2 attomoles for each of the Examples and Comparative Examples, respectively. However, the lowest concentration of target DNA detected in the assay performed without RNase H was confirmed to be 10 nM and 1 nM, respectively, for the Examples and Comparative Examples (see Fig. 8). The detection limit based on SN ratio 2 of CPT using AuNP was 100 attomole. The detection method proposed in the present invention is at least 5 times more sensitive than CPT using AuNP. Y.-H. Wu, C.-M. Jiang, and G.-X. Yang, Eur. Rev. Med. Pharmacol. Sci., 2016 , 20, 1775-1780, and C. Hong, A. RNA using two-femtomole and enzyme-assisted ISAM with detection limits of 20 attomole and 20 femtomole, respectively, were used in this study (Baek, SS Hah, W. Jung, and DE Kim, Anal. Chem., 2016, 88, 2999-3003. Detection. However, the limit of detection in the preceding literature could be achieved by several steps in response of more than 2 hours.

In addition, we estimated the number of RNA probes degraded per single target DNA according to the concentration of RNase H from the standard curve of fluorescence intensity for RNA probes (see FIG. 9). As shown in Table 3, it was confirmed that up to 8419 and 9725 RNA probes per single target DNA were degraded by RNase H for each of the examples and the comparative examples.

6, 7, and 8, it was confirmed that RNase H amplifies fluorescence and significantly improves sensitivity in isothermal control of hybridization of the target DNA-RNA probe. Depending on the order in which the graphene grafts are added, the difference in the detection limit of the target DNA can be attributed to the RNase H adsorbed on the oxidized graphene. On the other hand, DNA-RNA-DNA chimera probes were used in conventional CPT because RNase H lacked specificity.

(5) Evaluation of specificity according to the order of addition of graphene oxide

The specificity of the examples and comparative examples was evaluated with a simple RNA probe and three DNA sequences (a DNA sequence having only one mismatch base in the middle of the sequence). As shown in FIG. 10, dependence on specificity was observed to be significant depending on the order of addition of the graphene oxide, and it was estimated that the graphene graphex helps to regulate the specific formation of the substrate.

In both methods, a small increase was observed in the fluorescence of mismatched DNA as compared to the perfectly matched DNA (wild-type target DNA), but unlike the comparative example, the mismatched DNA sequence in the examples was the control (In the blank experiment in which the DNA sequence itself was not added), fluorescence was not increased. The effect of the mismatch base on specificity in both methods was negligible. Different specificities depending on the order of the oxidative graphene addition in the enzymatic reaction clearly demonstrated the role of oxidative graphene in the regulation of substrate specific formation. In the example in which the RNA probe was adsorbed to the oxidized graphene, the oxidized graphene influenced the formation of a duplex between the adsorbed RNA and the target DNA, and in the comparative example in which the oxidized graphene was added after the enzyme reaction, Oxidative graphene did not affect specificity. Since RNase H only degrades RNA in the DNA-RNA duplex, the reduction in fluorescence for the mismatched DNA in the comparative example would have resulted from the dependence of enzyme activity on the number of base equivalents of the substrate. As previously observed, the graphene graphenes involved in the enzymatic reaction in the examples appeared to contribute to the reduced fluorescence due to mismatched DNA. Increased fluorescence in the Examples and Comparative Examples was compared to investigate fluorescence reduction by mismatched DNA. As shown in FIG. 11, the relative SN ratio of fluorescence to mismatched DNA was smaller than that of wild-type target DNA, and it was confirmed that the specificity was not caused by inhibition of RNase H activity by graphene.

Thus, we propose two models for describing complexes formed by target DNA and RNA probes in oxidized graphene. In one model, if oxidized graphene-single stranded DNA or oxidized graphene-single stranded RNA complexes are more stable than DNA-RNA duplexes, single-stranded DNA or single-stranded RNAs will form substrates and, Should be adsorbed preferentially. In this case, only the fully hybridized form of the duplex can destroy the stability of the adsorbed target and probe. In another model, an incomplete duplex of an RNA probe and a mismatched DNA sequence is attached to the oxidized graphene by two mismatched bases in the duplex. In the latter, the number of bases in the two hybridized arms of the duplex is sufficient to serve as a substrate for RNase H and allows for enzyme access. However, as a result, since the fluorescence was not significantly increased in the examples, the mechanism of the electronic model more accurately describes the role of oxidative graphene in regulating the formation of specific target DNA-RNA probes.

(6) Conclusion

In conclusion, we have successfully demonstrated isothermal control of target DNA-RNA probe interactions using RNase H and oxidized graphene to sensitively and specifically detect the target DNA in a simple and rapid manner. Target DNA amounts as low as 20 attomoles and 2 attomoles were detected by the methods of the Examples and Comparative Examples, respectively. In two methods, RNase H dissociates the RNA bound to the target DNA to dissociate the target DNA to form a substrate, allowing reuse of single target DNA molecules up to 10,000 times.

Fluorescence from the dye coupled to the probe adsorbed on the oxidized graphene was effectively quenched by the oxidized graphene itself. Once the RNA probe is degraded by RNase H, fluorescence is emitted from the label dye as it moves away from the effective extinction distance of the graphene oxide. Sensitivity in the absence of RNase H was reduced by ~ 10 ^4- fold, while multiple cycles of enzymatic desorption of target DNA by RNase H and efficient fluorescence quenching with oxidative graphene enhanced sensitivity. Although the involvement of oxidized graphene in the enzymatic reaction reduced sensitivity by a factor of 10, single base mismatches of DNA sequences could be distinguished using linear and pure RNA probes, regardless of the type of mismatched base. In addition, high sensitivity and specificity were achieved in a single step reaction within 30 minutes by isothermal control of the interaction between the target DNA and RNA probe with oxidized graphene and RNase H.

Above all, the present invention does not require a precise and costly thermocycling meter to control RNA probe-target DNA interactions. Isothermal analysis performed at a fixed mild temperature can effectively overcome the complexity and cost of conventional molecular diagnostics. Because of these excellent properties, the present invention can be usefully used as a new target DNA detection method with clinically relevant performance in the biological and medical field, particularly where field testing is required.

(7) Summary

In the present invention, a method for controlling the interaction between an RNA probe using oxidized graphene and RNase H and a target DNA for highly sensitive and specific detection of a target DNA without a thermocycler has been developed. The present invention uses oxidized graphene for selective coupling between RNA probe-target DNA hybrids and RNase H to detect target DNA bound with fluorescently labeled RNA probes. Oxidized graphene and RNase H showed strong affinity and selectivity for single-stranded DNA or RNA and for RNA in DNA-RNA duplexes, respectively. The RNA probe bound to the perfectly matched target DNA was degraded by RNase H and the target DNA was dissociated, allowing for the formation of multiple hybrids. Due to the high efficiency fluorescence quenching by the oxidative graphene, fluorescence was expressed as the labeled RNA probe was degraded in the presence of the target DNA, whereas fluorescence was not observed in the mismatched DNA sequence even with a single base. The amounts of oxidized graphene and RNase H were optimized to maximize the number of fluorescent amplified and degraded RNA probes (10 μg / ml and 2 U / μl, respectively). Optimized control of the interaction of the oxidized graphene and RNase H is achieved by reuse of the target DNA within 30 minutes without thermocycler and degradation of about 10,000 RNA probes per single target DNA since the reaction of the present invention is carried out at 37 & . As a result, the present invention was able to detect as little as 20 attomoles of the target DNA, thus confirming excellent sensitivity, and showed excellent specificity for the target DNA perfectly matched as compared with the mismatched DNA sequence.

Claims (6)

(a) reacting an RNA probe containing a fluorescent dye at its end with oxidized graphene to obtain a first reactant;
(b) adding a specimen expected to contain the target DNA to the first reactant obtained in step (a) to obtain a second reactant for forming a duplex between the RNA probe and the target DNA;
(c) adding RNase H to the second reactant obtained in step (b) to obtain a third reactant for degrading the RNA probe; And
(d) measuring the fluorescence of the third reactant obtained in step (c)
/ RTI &gt; of the target DNA. The isothermal detection method of claim 1, wherein the third reactant in which the RNA probe of step (c) has been degraded is transferred to step (b) and repeatedly performed. The isothermal detection method of claim 1, wherein the graphene oxide is contained at a concentration of 1 to 50 占 퐂 / ml. The isothermal detection method of claim 1, wherein the RNA probe is contained at a concentration of 0.1 to 500 nM. The isothermal detection method of claim 1, wherein the RNase H is contained at a concentration of 0.01 to 5 U / μl. The method according to claim 1, wherein the steps (a) to (c) are carried out at a temperature of 20 to 70 ° C.

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