Endonuclease dynamics measurement device based on magnetic sensor, and manufacturing method therefor

Abstract

Provided in an embodiment of the present invention is an endonuclease dynamics measurement device comprising: a reaction unit having at least one substrate which is connected to magnetic nanoparticles through one side, and which reacts with an endonuclease so as to cause a cleavage reaction; a sensor unit having at least one magnetic sensor connected through the other side of each substrate; and a dynamics measurement unit which receives, from the magnetic sensor, a magnetic signal corresponding to the result of the reaction between the substrate and the endonuclease, when the endonuclease is introduced into the reaction unit, and which generates the result of measurement of the cleavage dynamics of the substrate according to the endonuclease on the basis of the magnetic signal.

Description

Endonuclease dynamics measurement device based on a magnetic sensor and a method for manufacturing the same

[0001] The present invention relates to a dynamics measuring device and a method for manufacturing the same, and more particularly, to a technical idea for measuring the dynamics of an endonuclease using a magnetic sensor.

[0002] Endonucleases, or DNA restriction enzymes, cut specific parts of genes and are therefore increasingly used in gene editing technology.

[0003] However, since the activity (or efficiency) and specificity of these endonucleases are determined by the binding of the endonuclease to DNA and the reaction that breaks the covalent bond of DNA, they are affected by the base sequence of the DNA recognition site of the enzyme and environmental changes such as temperature, pH, salt concentration, and ionic strength.

[0004] Therefore, to utilize endonuclease-based gene scissors systems for gene editing and other purposes under various in vivo conditions (e.g., relatively acidic cancer microenvironments), evidence regarding the precise reaction speed and safety of DNA restriction enzyme activity is required.

[0005] In fact, the problem of reduced specificity of the gene scissors, such as off-target effect (DNA site that should not react) cutting, has not yet been overcome in DNA restriction enzyme-based gene editing tools such as the CRISPR-Cas system, which is the most actively studied gene scissors system recently.

[0006] To address this, there has been a persistent need for an understanding of the kinetics of the CRISPR-Cas system. However, a sensor platform that can recognize multiple DNA targets and effectively quantify activation, efficiency, and specificity under buffer conditions that mimic the biological environment does not yet exist.

[0007] In particular, conventional optical-based sensors that have been widely used for measuring enzyme kinetics have a disadvantage in that they use fluorescent proteins as signal generating substances, the intensity of which is affected by buffer conditions such as temperature, pH, and salt concentration, making it difficult to properly measure the activity of endonuclease, which is the target of measuring kinetic parameters.

[0008] Therefore, there is a need to develop a sensor that can measure only the speed at which endonuclease cuts DNA without being affected by various environmental changes.

[0009] The present invention aims to provide a kinetic measurement device and a manufacturing method thereof capable of more accurately measuring the enzyme kinetics of various endonucleases in a biomimetic environment based on a magnetic sensor that is less affected by the surrounding environment such as temperature and buffer composition.

[0010] In addition, the present invention aims to provide a dynamic measurement device and a manufacturing method thereof that can more easily confirm the reactivity of endonuclease to various DNA base sequences.

[0011] In addition, the present invention provides information necessary for the development of precise drugs for gene editing, and also provides a dynamic measurement device and a manufacturing method thereof that can minimize the side effects of editing unwanted DNA when applying gene editing technology by confirming whether reactivity varies depending on the DNA base sequence.

[0012] A dynamic measurement device according to one embodiment of the present invention may include a reaction unit having at least one substrate that is connected to a magnetic nanoparticle through one end and reacts with an endonuclease to cause a cleavage reaction, a sensor unit having at least one magnetic sensor connected through the other end of each of the substrates, and a dynamic measurement unit that receives a magnetic signal corresponding to the reaction result between the substrate and the endonuclease from the magnetic sensor when an endonuclease is introduced into the reaction unit, and generates a measurement result of the cleavage kinetics of the substrate by the endonuclease based on the magnetic signal.

[0013] According to one side, the sensor unit can be attached to each magnetic sensor, each substrate having a different base sequence.

[0014] According to one aspect, the substrate may comprise at least one of double-stranded DNA, single-stranded DNA, and RNA.

[0015] According to one aspect, the substrate may include probe DNA that is connected to a magnetic sensor and target DNA that binds to the probe DNA and to which magnetic nanoparticles are attached.

[0016] According to one side, the probe DNA can be attached to each magnetic sensor through at least one of a dispenser, a stamp, and a microchannel.

[0017] According to one side, the substrate has at least one attachment substance among biotin and sulfur attached to one side thereof, and can be connected to the magnetic nanoparticles through the attachment substance.

[0018] According to one side, when a distance difference occurs between the magnetic sensor and the magnetic nanoparticles due to the substrate being cut off by the reaction with the endonuclease, the sensor part can output a magnetic signal corresponding to the distance difference.

[0019] According to one side, the magnetic sensor may include at least one sensor among a giant magneto-resistance (GMR) sensor, a tunnel magnetic resistance (TMR) sensor, an anisotropic magnetic resistance (AMR) sensor, and a Hall sensor.

[0020] According to one side, the dynamic measurement device may further include a temperature control unit that controls and compensates the temperature of an area corresponding to each magnetic sensor.

[0021] A method for manufacturing a kinetic measurement device according to an embodiment of the present invention comprises the steps of: preparing a sensor unit having at least one magnetic sensor and being connected to a kinetic measurement unit that generates a measurement result of the cleavage kinetics of at least one substrate in which a cleavage reaction occurs by reacting with an endonuclease; attaching substrates constituting the reaction unit to each of the magnetic sensors; and injecting at least one magnetic nanoparticle to connect the magnetic nanoparticle to the substrate via an attachment material formed on one side of the substrate, wherein the kinetic measurement unit can receive a magnetic signal corresponding to the reaction result of the substrate and the endonuclease from the magnetic sensor when an endonuclease is introduced into the reaction unit, and generate a measurement result of the cleavage kinetics of the substrate by the endonuclease based on the magnetic signal.

[0022] According to one side, the step of attaching the substrate to each magnetic sensor can attach each substrate having a different base sequence to each magnetic sensor.

[0023] According to one aspect, the substrate may comprise at least one of double-stranded DNA, single-stranded DNA, and RNA.

[0024] According to one aspect, the step of attaching the substrate to each magnetic sensor may include the step of attaching probe DNA to each magnetic sensor via at least one of a dispenser, a stamp, and a microchannel, and the step of binding target DNA complementary to each probe DNA to each corresponding probe DNA.

[0025] According to one side, the step of binding to each probe DNA may include injecting target DNA corresponding to each probe DNA attached to each magnetic sensor onto the magnetic sensor through at least one of a dispenser, a stamp, and a microchannel.

[0026] According to one embodiment, the present invention can more accurately measure the enzymatic kinetics of various endonucleases in a biomimetic environment based on a magnetic sensor that is less affected by the surrounding environment such as temperature and buffer composition.

[0027] According to one embodiment, the present invention can more easily confirm the reactivity of an endonuclease to various DNA base sequences.

[0028] According to one embodiment, the present invention provides information necessary for the development of precise drugs for gene editing, and also confirms whether reactivity varies depending on DNA base sequence, thereby minimizing the side effects of editing unwanted DNA when applying gene editing technology.

[0029] Figure 1 is a drawing for explaining a dynamic measurement device according to one embodiment.

[0030] FIGS. 2A to 2D are drawings for explaining a first embodiment of a dynamic measurement device according to one embodiment.

[0031] Figure 3 is a drawing for explaining the reaction rate of a substrate and an enzyme in a dynamic measurement device according to one embodiment.

[0032] Figure 4 is a drawing for more specifically explaining the temperature control unit of a dynamic measurement device according to one embodiment.

[0033] Figures 5 to 9 are drawings for explaining the dynamic measurement results of the dynamic measurement device according to the first embodiment.

[0034] FIGS. 10A to 10C are drawings for explaining a second embodiment of a dynamic measurement device according to one embodiment.

[0035] Fig. 11 is a drawing for explaining the dynamic measurement results of the dynamic measurement device according to the second embodiment.

[0036] Figure 12 is a drawing for explaining a method for manufacturing a dynamic measurement device according to one embodiment.

[0037] Specific structural or functional descriptions of embodiments according to the concept of the present invention disclosed in this specification are merely illustrative for the purpose of explaining embodiments according to the concept of the present invention, and embodiments according to the concept of the present invention may be implemented in various forms and are not limited to the embodiments described in this specification.

[0038] Embodiments according to the concept of the present invention may have various modifications and take various forms, and thus, embodiments are illustrated in the drawings and described in detail in this specification. However, this is not intended to limit embodiments according to the concept of the present invention to specific disclosed forms, but rather includes modifications, equivalents, or alternatives that fall within the spirit and technical scope of the present invention.

[0039] While terms such as "first" or "second" may be used to describe various components, the components should not be limited by the terms. Terms are used solely to distinguish one component from another. For example, a first component may be referred to as a "second component," and similarly, a second component may also be referred to as a "first component," without departing from the scope of the invention.

[0040] When a component is referred to as being "connected" or "connected" to another component, it should be understood that it may be directly connected or connected to that other component, but that there may be other components in between. Conversely, when a component is referred to as being "directly connected" or "directly connected" to another component, it should be understood that there are no other components in between. Expressions that describe relationships between components, such as "between," "immediately between," or "directly adjacent to," should be interpreted similarly.

[0041] The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the present invention. The singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, it should be understood that the terms "comprises" or "has" are intended to specify the presence of a described feature, number, step, operation, component, part, or combination thereof, but do not preclude the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0042] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and will not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0043] Hereinafter, embodiments will be described in detail with reference to the attached drawings. However, the scope of the patent application is not limited or restricted by these embodiments. The same reference numerals in each drawing represent the same components.

[0044] Figure 1 is a drawing for explaining a dynamic measurement device according to one embodiment.

[0045] Referring to FIG. 1, a dynamic measurement device (100) according to one embodiment can more accurately measure the enzyme dynamics of various endonucleases in a biomimetic environment based on a magnetic sensor that is less affected by the surrounding environment such as temperature and buffer composition.

[0046] In addition, the dynamic measurement device (100) can more easily confirm the reactivity of endonuclease to various DNA base sequences.

[0047] In addition, the dynamic measurement device (100) provides information necessary for the development of precise drugs for gene editing, and can also confirm whether reactivity varies depending on the DNA base sequence, thereby minimizing the side effects of editing unwanted DNA when applying gene editing technology.

[0048] Specifically, the dynamic measurement device (100) is a magnetic sensor-based measurement platform (e.g., a sensor chip) that detects a target molecule through a change in resistance caused by an external magnetic field induced by magnetic nanoparticles by applying magnetic nanoparticles that can stably generate signals even under changes in various buffer conditions (e.g., environmental conditions such as temperature, pH, and salt concentration), and exhibits characteristics more suitable than existing biosensors for measuring the dynamics of endonuclease, i.e., reaction speed and characteristics according to reaction speed, under various conditions imitating a biological environment.

[0049] To this end, the dynamic measurement device (100) may include a reaction unit (110), a sensor unit (120), and a dynamic measurement unit (130).

[0050] A reaction unit (110) according to one embodiment may be connected to a magnetic nanoparticle through one end and may include at least one substrate that reacts with an endonuclease to cause a cleavage reaction.

[0051] For example, the substrate may include at least one of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA.

[0052] The sensor unit (120) according to one embodiment may be provided with at least one magnetic sensor connected through each other side end of the substrate.

[0053] According to one side, if the substrate is double-stranded DNA, it may include a probe DNA that is connected to a magnetic sensor and a target DNA that binds to the probe DNA and to which magnetic nanoparticles are attached.

[0054] Additionally, if the substrate is single-stranded DNA (or RNA), magnetic nanoparticles can be attached through one end of the single-stranded DNA (or RNA), and a magnetic sensor can be attached through the other end.

[0055] Additionally, the magnetic sensor may include at least one of a giant magneto-resistance (GMR) sensor, a tunnel magnetic resistance (TMR) sensor, an anisotropic magnetic resistance (AMR) sensor, and a Hall sensor.

[0056] According to one side, the substrate may be attached to one end with at least one of biotin and sulfur as an attachment material, and may be connected to magnetic nanoparticles through the attachment material, and the magnetic nanoparticles may be coated with streptavidin, a material that binds to biotin, or gold, a material that binds to sulfur, but is not limited thereto, and may be implemented as a combination of protein A and IgG, or a combination of a tag protein and its corresponding antibody (e.g., HA and anti-HA, FLAG and anti-FLAG).

[0057] For example, when the substrate is double-stranded DNA, the attachment material is formed on the target DNA, and the target DNA combined with the probe DNA can be attached to the magnetic nanoparticles through the attachment material.

[0058] In other words, DNA and magnetic nanoparticles attached to the surface of the magnetic sensor can bind to each other through biotin-streptavidin bonding, but are not limited thereto, and can bind to each other through sulfide bonding by attaching sulfur to the end of DNA. In addition, DNA and magnetic nanoparticles can also bind to each other through any one of DNA-DNA bonding, IgG Fc-protein A / G bonding, and antigen-antibody bonding.

[0059] According to one side, the sensor unit (120) can be attached to each magnetic sensor corresponding to the probe DNA through at least one means of a dispenser, a stamp, and a microchannel.

[0060] Specifically, when the probe DNA is delivered through at least one means, the sensor unit (120) can be attached to each corresponding magnetic sensor through at least one method among physisorption, charge interaction, and covalent bonding.

[0061] In addition, the sensor unit (120) may be attached to a probe DNA having an amine group (NH2) attached to the other end and a magnetic sensor having a carboxyl group (COOH) formed on the surface, based on NHS-EDC.

[0062] According to one side, the sensor unit (120) may dispense probe DNA to each magnetic sensor through dispenser equipment, and may introduce multiple target DNAs complementary to each probe DNA by mixing them all at once.

[0063] Specifically, since each probe-target set must be independent and not affect other sets in order for the target DNA to not bind to unwanted probes, the sensor unit (120) can be designed with probe-target designs as independent sets by utilizing the thermodynamic properties of DNA, and through this, even if multiple target DNAs are mixed at once and injected into multiple magnetic sensors, each probe DNA can bind more easily to complementary target DNA.

[0064] In addition, the sensor unit (120) dispenses probe DNA to each magnetic sensor through the dispenser device, and dispenses target DNA complementary to each probe DNA to each corresponding magnetic sensor through the dispenser device, so that each probe DNA and complementary target DNA can be more accurately combined.

[0065] Depending on the embodiment, the sensor unit (120) may be formed by attaching the probe DNA and target DNA to the magnetic sensor in a state in which they are externally bound. However, in this case, if multiple parts of the DNA are bound to the sensor surface, a situation may occur in which the DNA does not fall off the sensor surface even if cut by an enzyme.

[0066] Meanwhile, when an endonuclease (i.e., an enzyme) is introduced into the reaction portion (110), the sensor portion (120) can generate a magnetic signal corresponding to the reaction result between the substrate and the endonuclease from the magnetic sensor.

[0067] According to one side, when a distance difference occurs between the magnetic sensor and the magnetic nanoparticles due to the substrate being cut off by the reaction with the endonuclease, the sensor unit (120) can output a magnetic signal corresponding to the distance difference.

[0068] For example, the sensor unit (120) is injected under changed buffer conditions when the endonuclease is injected into the reaction unit (110), or additionally Na is added to change the composition in the state where the endonuclease is injected. + or Mg 2+ It can output magnetic signals in real time or at preset intervals depending on changes in experimental conditions, such as injecting a back or injecting an inhibitor such as EDTA.

[0069] According to one embodiment, a dynamic measurement unit (130) can receive a magnetic signal generated from a sensor unit (120) and generate a measurement result of the cleavage dynamics of a substrate according to an endonuclease based on the magnetic signal.

[0070] Specifically, the magnetic nanoparticles are cleaved by the endonuclease reaction, causing the substrate to move away from the magnetic sensor, thereby reducing the magnetic field applied to the magnetic sensor.

[0071] That is, the sensor unit (120) provides a magnetic signal corresponding to the intensity of the magnetic field reduced due to the reaction of the endonuclease, and since the amount of change in the magnetic signal provided at this time well represents the reaction speed of the endonuclease, the dynamics measurement unit (130) can measure the dynamics of the endonuclease, i.e., the reaction speed, more accurately and easily by utilizing the magnetic signal provided from the sensor unit (120).

[0072] According to one side, magnetic nanoparticles that do not settle can be used because when the substrate is cut, the magnetic signal should be reduced by moving away from the magnetic sensor surface together with the cut substrate, and for this purpose, the magnetic nanoparticles can be formed with a diameter of 100 nm or less.

[0073] Meanwhile, the dynamic measurement device (100) may further include a temperature control unit that controls and corrects the temperature of an area corresponding to each magnetic sensor.

[0074] FIGS. 2A to 2D are drawings for explaining a first embodiment of a dynamic measurement device according to one embodiment.

[0075] Referring to FIGS. 2A to 2D, reference numerals 210 to 240 are drawings illustrating a dynamics measurement device based on double-stranded DNA (dsDNA), and exemplify a method for measuring the dynamics of an endonuclease using a multiple probe-target oligonucleotide set.

[0076] Specifically, the dynamic measurement device of drawing numbers 210 to 240 uses EcoRI, which recognizes and reacts to the base sequence of GAATTC as an endonuclease (i.e., enzyme), and Cas12a, which is attracting attention as a next-generation genetic scissors, and can use a giant magnetoresistance sensor, which has been developed a lot as a biosensing technology among various magnetic sensors.

[0077] Additionally, the dynamic measurement device can be designed such that a total of 80 magnetic sensors manufactured in a 10×8 array are formed on one chip, and DNA having a different base sequence is attached to each magnetic sensor.

[0078] According to drawing reference numeral 210, when a dynamic measurement device attaches a probe DNA on a magnetic sensor and then injects a complementary target DNA, the target DNA binds only to the corresponding probe DNA, thereby forming a double-stranded DNA (dsDNA).

[0079] According to the drawing reference numeral 220, the dynamic measurement device has biotin attached to one end of target DNA complementary to probe DNA, so that the target DNA can bind to magnetic nanoparticles coated with streptavidin through one end, and in this case, the magnetic sensor can generate a magnetic signal by recognizing an external magnetic field generated from magnetic nanoparticles captured near the sensor surface by the biotin-streptavidin bond.

[0080] According to drawings 230 and 240, the dynamic measurement device can introduce a restriction enzyme that recognizes a specific sequence and cuts DNA, such as EcoRI, or an endonuclease that cuts regardless of the sequence, such as trans-cleavage of CRISPR / Cas12a.

[0081] In this case, the dynamic measurement device can measure and quantify the dynamics such as the activity and cleavage rate of the restriction enzyme that has discovered the recognition site and cuts a specific site of the DNA, and as a result, the magnetic nanoparticles on the magnetic sensor surface can also fall off from the sensor surface.

[0082] Figure 3 is a drawing for explaining the reaction rate of a substrate and an enzyme in a dynamic measurement device according to one embodiment.

[0083] Referring to FIG. 3, reference numeral 300 illustrates a substrate (e.g., double-stranded DNA or single-stranded DNA) (S) attached to the surface of a magnetic sensor and an enzyme reaction kinetics.

[0084] In the drawing symbol 300, k1 is the rate of formation of the complex (ES) of enzyme (E, enzyme) and substrate (S), k -1 k is the rate at which the enzyme-substrate complex (ES) dissociates back into the enzyme (E) and substrate (S). cat refers to the rate (turnover number) at which an enzymatic reaction (DNA cleavage in the present invention) occurs in an enzyme-substrate complex to produce a product (P).

[0085] According to drawing symbol 300, in the case of a reaction on the surface of a magnetic sensor, the reaction model can be expressed as in Equation 1 below, and a dynamic measurement device according to one embodiment can determine the effective reaction speed by finding a parameter using Equation 1.

[0086] [Formula 1]

[0087]

[0088] Specifically, when measuring the dynamics of DNA restriction enzymes (i.e., endonucleases), the surface enzyme kinetics equation based on the Michelis-Menten enzyme kinetics is basically used, and existing optical-based sensors measure 3D-based dynamics in a floating state in a liquid buffer without being affected by the position or direction of the target DNA that the endonuclease must cut, which may be different from the situation when gene editing is performed.

[0089] On the other hand, the dynamic measurement device according to one embodiment recognizes and cuts target DNA attached to the sensor surface, so it can measure the cutting activity of an endonuclease that is most similar to the actual situation within a cell during gene editing.

[0090] Figure 4 is a drawing for more specifically explaining the temperature control unit of a dynamic measurement device according to one embodiment.

[0091] Referring to FIG. 4, the dynamic measurement device may include a reaction unit (400-1, 400-2) having at least one substrate that is connected to a magnetic nanoparticle through one end and reacts with an endonuclease to cause a cleavage reaction, and a sensor unit (hatched area at the bottom of drawing symbol 400-1) having at least one magnetic sensor that is connected through the other end of each of the substrates.

[0092] Additionally, the dynamic measurement device may further include a temperature control unit that controls and compensates the temperature of an area corresponding to the magnetic sensor.

[0093] That is, since the reactivity of enzymes is affected by temperature, the kinetic measurement device is equipped with a temperature control unit that can automatically control and compensate for the temperature, so that the kinetics can be measured more precisely through accurate temperature control.

[0094] According to one side, the dynamic measurement device may have a plurality of magnetic sensors (e.g., 80) arranged on a chip, and the temperature control unit may include a resistance temperature detector (RTD) and a thermoelectric cooler (TEC) arranged below the sensor unit, thereby controlling and maintaining each of the plurality of magnetic sensors at a preset temperature.

[0095] Specifically, the temperature control unit is a system that controls and compensates for the temperature on its own, and can be implemented in a form mounted on a sensor reader. Here, the sensor reader has a resistance temperature detector (RTD) and a thermoelectric cooler (TEC) placed inside a cartridge that surrounds a magnetic sensor, and can control and compensate for the temperature by communicating with a computer through a DAQ board. Through this, the kinetic measurement device can maintain the reaction temperature of the endonuclease uniformly or change it as needed.

[0096] Figures 5 to 9 are drawings for explaining the dynamic measurement results of the dynamic measurement device according to the first embodiment.

[0097] Referring to FIGS. 5 to 9, (a) of FIG. 5 shows the measurement results of the dynamics (i.e., the presence or absence of a specific reaction and the speed) for DNAs (P1, P2, P3, P4, P5, P6, P7 and NP) having different base sequences attached to each magnetic sensor (wherein, △MR means a change in the magnetic signal), and (b) of FIG. 5 shows the results of curve fitting the measured signal to the surface enzyme kinetics equation based on the Michelis-Menten enzyme kinetics (i.e., the results of fitting through the above-described Equation 1).

[0098] In (a) of Fig. 5, BSA can be used as a negative control to check whether there is no non-specific binding signal, and if the signal is low, to check whether the experiment was successful.

[0099] Figure 6 (a) shows the results of verifying the cross-reactivity for a combination of probe-target, i.e., a DNA pair that includes the recognition site of EcoRI, an endonuclease, but is designed independently from each other, and Figure 6 (b) shows the effective reaction rate parameter (k) measured by changing the temperature using a temperature control unit according to an embodiment. eff ) are shown, and (c) and (d) of Fig. 6 show the effective reaction rate parameter (k) according to the concentration of EcoRI. eff ) shows the measurement results. Here, (c) of Fig. 6 shows the signal of the GMR sensor decreasing in real time according to the reaction time of the endonuclease, and since all signals are divided by the signal immediately before the endonuclease is injected, the signal for each concentration starts from 1.

[0100] In addition, each of (a) to (d) of Figure 7 is an effective reaction rate parameter (k) measured by sequentially changing the salt concentration, ionic strength, pH, and concentration of enzyme activity inhibitor (EDTA). eff ) shows the measurement results, and Fig. 7 (e) and (f) show the results of measuring the change in magnetic signal according to the change in the composition of the buffer in real time by adding a high concentration of enzyme activity inhibitor (EDTA), which is an inhibitor of endonuclease activity, during the middle of the activity.

[0101] Each of (a) to (d) of Figure 8 shows the effective reaction rate parameter (k) of EcoRI measured while sequentially changing the metal cofactor, pH, salt concentration, and ionic strength. eff ) shows the measurement results.

[0102] Here, PT1 to PT6 refer to a magnetic sensor that attaches P1 to P6 probe DNA to the GMR sensor surface and adds complementary target DNA, T1 to T6, to generate double-stranded DNA (dsDNA).

[0103] That is, PT1 to PT6 each contain the sequence of GAATTC, which EcoRI generally cleaves (PT1), or the sequence known to be cut when star activity occurs (PT2 to PT6). Since the star activity of the enzyme appears specifically depending on the base sequence of the recognition site, each bar in the graph represents the reaction rate for each different probe-target set. In addition, the graph indicated by * represents a probe-target set for which the enzyme's cleavage reaction could not be confirmed.

[0104] Figures 9 (a) and (b) illustrate the results of verifying that various buffers and saliva / serum do not contain components that cut or disrupt the probe-target set.

[0105] In addition, Fig. 9 (c) shows the results of measuring the enzyme activity parameters of EcoRI under biomimetic buffer conditions, and Fig. 9 (d) shows the results of measuring the enzyme activity parameters of EcoRI under biomimetic buffer conditions in the presence of magnesium ions (Mg 2+ ) was added to the buffer without EcoRI, and then the buffer was added to the reaction section, and magnesium ions (Mg 2+ ) are shown in the experimental results, and Fig. 9 (e) and (f) show the results of the experiment in which magnesium ions (Mg 2+ ) is shown as a comparison of the results of inserting it in the middle and the results of inserting it from the beginning.

[0106] According to FIG. 5, the dynamic measurement device uses a sensor chip including multiple magnetic sensors when measuring the reaction speed of an endonuclease, and attaches DNA having a different sequence to each sensor, thereby measuring the presence or absence of a specific reaction and speed of the endonuclease to the DNA sequence.

[0107] Additionally, the dynamic measurement device can determine whether the platform is measuring normally by adding DNA sequences that can never be cut by endonucleases, and can also derive previously unknown reactivity by adding various sequences.

[0108] In this way, the dynamic measurement device measures the change in the magnetic sensor signal according to the reaction of the endonuclease, and then performs curve fitting on the mathematical model to obtain the effective reaction rate parameter (k) of the enzyme. eff ) can be easily calculated.

[0109] Specifically, the kinetic measurement device can derive more easily quantifiable kinetic measurements of endonuclease (i.e., enzyme) such as whether the endonuclease (i.e., enzyme) cleaves DNA (i.e., substrate) and, if so, how quickly it cleaves (i.e., cleavage time; speed) using a magnetic sensor based on magnetic nanoparticles.

[0110] Additionally, the dynamic measurement device can easily determine whether the endonuclease simultaneously cuts DNA having different base sequences by attaching DNA having different base sequences to each of the multiple magnetic sensors, and if so, how quickly it cuts it.

[0111] According to Fig. 6, the dynamic measurement device can more easily confirm that each probe-target combination is independent and that non-corresponding DNA pairs do not bind to each other by observing the signal generated when only one target DNA is added in a situation where all probe DNAs are loaded onto each magnetic sensor. Specifically, it can be confirmed that T1 binds only to P1 and does not bind to P2, P3, etc., and similarly, T2 binds only to P2 and does not bind to P1, P3, etc.

[0112] According to FIGS. 7 and 8, the dynamic measurement device can more easily measure changes in reaction rates as environmental factors (changes in salt concentration, pH, ionic strength, and concentration of enzyme activity inhibitor (EDTA)) change.

[0113] According to one side, the dynamics measuring device can stop the endonuclease reaction by injecting an enzyme activity inhibitor (EDTA) during the dynamics measuring process of endonuclease and re-perform the dynamics measuring process by changing the buffer conditions in real time, and even in this case, the dynamics of endonuclease can be stably measured.

[0114] According to Fig. 9, the signal of the GMR sensor can stably derive dynamic measurement results in buffers that mimic the in vivo environment, such as plasma, artificial saliva, DMEM, HBSS, and cell culture media, and Mg in real time. 2+ Even in the process of changing the environment by adding ions, it is possible to easily derive measurement results of signal changes and enzyme activity and dynamics.

[0115] FIGS. 10A to 10C are drawings for explaining a second embodiment of a dynamic measurement device according to one embodiment.

[0116] According to FIGS. 10a to 10c, reference numerals 1010 to 1030 are drawings illustrating a dynamic measurement device based on single-stranded DNA (ssDNA).

[0117] Specifically, drawing reference numeral 1010 is a drawing illustrating a method for measuring the endonuclease (enzyme) activity and dynamics of a representative gene scissors system, CRISPR / Cas12, applied to gene editing technology, in which CRISPR RNA (crRNA) and complementary target DNA can be assembled to activate trans-cleavage of the endonuclease (i.e., enzyme) CRISPR / Cas12a.

[0118] Additionally, drawings 1020 and 1030 illustrate examples of activated CRISPR / Cas12a indiscriminately cleaving single-stranded DNA of different base sequences attached to the sensor surface.

[0119] Specifically, according to the drawings 1020 and 1030, when single-stranded DNA (ssDNA) is attached on a magnetic sensor, magnetic nanoparticles can be attached to the side of the single-stranded DNA (ssDNA) that is not attached to the magnetic sensor, and when CRISPR / Cas12a is then introduced, CRISPR / Cas12a cuts a specific portion of the DNA, and as a result, the magnetic nanoparticles on the surface of the magnetic sensor can also be detached from the sensor surface, thereby reducing the signal, and the dynamics such as the activity and cutting speed of the restriction enzyme can be measured and quantified using this magnetic signal.

[0120] Fig. 11 is a drawing for explaining the dynamic measurement results of the dynamic measurement device according to the second embodiment.

[0121] Referring to FIG. 11, (a) of FIG. 11 shows a decrease in signal that occurs as single-stranded DNA (ssDNA) cut by CRISPR / Cas12a exhibiting indiscriminate cleavage activity and magnetic nanoparticles attached to the side ends of these single-stranded DNA (ssDNA) move away from the sensor surface.

[0122] In addition, the * mark in (b) of Fig. 10 indicates that the CRISPR / Cas12a complex assembled by removing one Cas12a enzyme, CRISPR RNA (crRNA), and complementary target DNA (TS) required for the enzyme activity of the CRISPR-Cas system is inactive, and thus the effective reaction rate parameter (k) of the enzyme is low. eff ) was not measured.

[0123] Additionally, Fig. 10 (c) shows that the cleavage kinetic parameters of CRISPR / Cas12a are affected by the length of the target DNA (TS).

[0124] Figure 12 is a drawing for explaining a method for manufacturing a dynamic measurement device according to one embodiment.

[0125] In other words, FIG. 12 is a drawing explaining a method for manufacturing a dynamic measurement device according to an embodiment described through FIGS. 1 to 11. In the following, any explanation that overlaps with the explanation through FIGS. 1 to 11 among the contents described through FIG. 12 will be omitted.

[0126] Referring to FIG. 12, in step 1210, the manufacturing method can prepare a sensor unit having at least one magnetic sensor and connected to a kinetic measurement unit that generates a measurement result of the cleavage kinetics of at least one substrate in which a cleavage reaction occurs by reacting with an endonuclease.

[0127] Next, in step 1220, the manufacturing method can attach the substrate constituting the reaction section to each magnetic sensor.

[0128] According to one side, in step 1220, the manufacturing method can attach each substrate having a different base sequence to each magnetic sensor, wherein the substrate can include at least one of double-stranded DNA, single-stranded DNA, and RNA.

[0129] According to one side, in step 1220, the manufacturing method can attach probe DNA to each magnetic sensor through at least one means of a dispenser, a stamp, and a microchannel, and then bind target DNA complementary to each probe DNA to each corresponding probe DNA.

[0130] Specifically, in step 1220, the manufacturing method can attach probe DNA to each magnetic sensor, and then mix multiple target DNAs complementary to each probe DNA at once and inject them into the probe DNA.

[0131] Additionally, in step 1220, the manufacturing method may attach probe DNA to each magnetic sensor, and then inject target DNA corresponding to each probe DNA attached to each magnetic sensor onto the magnetic sensor through at least one of a dispenser, a stamp, and a microchannel.

[0132] Next, in step 1230, the manufacturing method can inject at least one magnetic nanoparticle so as to connect the magnetic nanoparticle to the substrate via an attachment material formed on one side of the substrate.

[0133] Meanwhile, when an endonuclease is introduced into the reaction section of the dynamic measurement device generated through steps 1210 to 1230, the dynamic measurement section receives a magnetic signal corresponding to the reaction result between the substrate and the endonuclease from the magnetic sensor, and can generate a measurement result of the cleavage dynamics of the substrate according to the endonuclease based on the magnetic signal.

[0134] Ultimately, the present invention allows for more accurate measurement of the enzymatic kinetics of various endonucleases in a biomimetic environment based on a magnetic sensor that is less affected by the surrounding environment, such as temperature and buffer composition.

[0135] In addition, by using the present invention, the reactivity of endonuclease to various DNA base sequences can be more easily confirmed.

[0136] In addition, the present invention provides information necessary for the development of precise drugs for gene editing, and also confirms whether reactivity varies depending on the DNA base sequence, thereby minimizing the side effects of editing unwanted DNA when applying gene editing technology.

[0137] Although the embodiments described above have been described with limited drawings, those skilled in the art will recognize that various modifications and variations are possible based on the above description. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described devices, structures, apparatuses, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0138] Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the claims described below.

[0139] The present invention can be applied in various fields of industry related to gene editing technology.

Claims

1. A reaction unit having at least one substrate that is connected to a magnetic nanoparticle through one side and reacts with an endonuclease to cause a cleavage reaction; A sensor unit having at least one magnetic sensor connected through each of the other side ends of the above substrates, and When the endonuclease is introduced into the reaction section, a kinetic measurement section receives a magnetic signal corresponding to the reaction result of the substrate and the endonuclease from the magnetic sensor, and generates a measurement result of the cleavage kinetics of the substrate according to the endonuclease based on the magnetic signal. A dynamic measuring device including:

2. In paragraph 1, The above sensor part, Each substrate having a different base sequence is attached to each of the magnetic sensors. Dynamic measuring device.

3. In paragraph 1, The above-mentioned temperament is, Containing at least one of double-stranded DNA, single-stranded DNA, and RNA Dynamic measuring device.

4. In paragraph 3, The above-mentioned temperament is, Probe DNA connected to the above magnetic sensor and Containing target DNA that binds to the probe DNA and to which the magnetic nanoparticles are attached Dynamic measuring device.

5. In paragraph 4, The above probe DNA is, Attached to each of the magnetic sensors by means of at least one of a dispenser, a stamp and a microchannel. Dynamic measuring device.

6. In paragraph 1, The above-mentioned temperament is, At least one of biotin and sulfur is attached to one side, and is connected to the magnetic nanoparticle through the attachment material. Dynamic measuring device.

7. In paragraph 1, The above sensor part, When the substrate is cut off due to the reaction with the endonuclease, a distance difference occurs between the magnetic sensor and the magnetic nanoparticle, and the magnetic signal corresponding to the distance difference is output. Dynamic measuring device.

8. In paragraph 1, The above magnetic sensor, A sensor comprising at least one of a GMR (giant magneto-resistance) sensor, a TMR (tunnel magnetic resistance) sensor, an AMR (anisotropic magnetic resistance) sensor, and a Hall sensor. Dynamic measuring device.

9. In paragraph 1, A temperature control unit that controls and compensates the temperature of the area corresponding to each of the above magnetic sensors. A dynamic measuring device further comprising:

10. A step of preparing a sensor unit having at least one magnetic sensor and connected to a kinetic measurement unit that generates a measurement result of the cleavage kinetics of at least one substrate that reacts with an endonuclease to cause a cleavage reaction; A step of attaching the substrate constituting the reaction section to each of the magnetic sensors; and A step of injecting at least one magnetic nanoparticle and connecting the magnetic nanoparticle to the substrate through an attachment material formed on one side of the substrate. Including, The above dynamic measurement unit, When the endonuclease is introduced into the reaction section, a magnetic signal corresponding to the reaction result of the substrate and the endonuclease is received from the magnetic sensor, and a measurement result of the cleavage dynamics of the substrate according to the endonuclease is generated based on the magnetic signal. A method for manufacturing a dynamic measuring device including a .

11. In paragraph 10, The step of attaching the above substrate to each of the above magnetic sensors is: Attaching each substrate having a different base sequence to each of the magnetic sensors. Method for manufacturing a dynamic measuring device.

12. In paragraph 10, The above-mentioned temperament is, Containing at least one of double-stranded DNA, single-stranded DNA, and RNA Method for manufacturing a dynamic measuring device.

13. In paragraph 10, The step of attaching the above substrate to each of the above magnetic sensors is: A step of attaching probe DNA to each of the magnetic sensors through at least one means of a dispenser, a stamp, and a microchannel; and A step of binding target DNA complementary to each of the above probe DNAs to each of the corresponding probe DNAs. A method for manufacturing a dynamic measuring device including a .

14. In paragraph 13, The step of binding to each of the above probe DNAs is: Injecting the target DNA corresponding to each of the probe DNAs attached to each of the magnetic sensors onto the magnetic sensor through at least one of the dispenser, the stamp and the microchannel. Method for manufacturing a dynamic measuring device.

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