Method and platform for dna programmable manipulation based on dynamic magnetic torque

Abstract

The application relates to the technical field of biotechnology, and particularly relates to an in-vivo DNA programmed manipulation method and platform based on dynamic magnetic moment, which comprises the following steps: (a) providing a magnetic micro motor with magnetic anisotropy and a target double-stranded DNA, wherein one end of the target double-stranded DNA is anchored to a substrate; (b) positioning the magnetic micro motor at the free end of the target double-stranded DNA, and combining the magnetic micro motor with the target double-stranded DNA through chemical coupling to realize mechanical transmission between the magnetic micro motor and the target double-stranded DNA; (c) applying a rotating magnetic field to the magnetic micro motor, so that the magnetic micro motor generates a dynamic magnetic moment under the driving of the rotating magnetic field; and (d) using the dynamic magnetic moment to act on the target double-stranded DNA, so that the target double-stranded DNA is subjected to programmed mechanical unwinding, and precise manipulation of the DNA is realized. The application applies controllable torque to the double-stranded DNA through programmable dynamic magnetic moment, realizes accurate and adjustable programmed manipulation of different stability DNA molecules, breaks through the bottleneck that the force output distance of traditional magnetic tweezers is limited, greatly improves the action space range, overcomes the defect that the time resolution of conventional DNA manipulation means is insufficient in the body, and finally realizes efficient and accurate spatiotemporal control of the DNA in the body.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to a method and platform for programmed manipulation of DNA in vivo based on dynamic magnetic torque. Background Technology

[0002] Conformationally programmable DNA nanostructures provide important molecular tools for precision nanomedicine. However, existing strategies for regulating DNA conformation largely rely on passive environmental factors or chemical stimuli such as temperature, pH, and nucleic acid strands. Under the complex physiological homeostasis of living organisms, these methods face bottlenecks such as unstable reaction kinetics, response lag (requiring several hours), and a severe lack of spatial precision, making it difficult to meet the requirements for real-time, spatially precise positioning, and active programmable manipulation. Recent studies have shown that the mechanical forces generated by enzymes play a crucial role in DNA unwinding, revealing the unique mechanical properties of DNA and providing new insights for achieving in vivo spatiotemporal unwinding of DNA.

[0003] Single-molecule mechanical manipulation techniques (such as optical tweezers and atomic force microscopy) provide powerful tools for studying force-induced DNA manipulation, but their application in vivo is limited by insufficient tissue penetration and the need for physical contact with probes. Magnetic tweezers are widely used in DNA mechanical property research due to their low cost, ease of implementation, and remote spatiotemporal control. However, traditional magnetic tweezers techniques mainly employ a combination of isotropic magnetic beads and gradient magnetic fields, failing to fully utilize the advantage of anisotropic micromotors generating strong magnetic torque in rotating magnetic fields. Furthermore, the magnetic field frequency parameter, a key determinant of torque generation, has not received due attention in molecular manipulation research. More importantly, traditional static magnetic field application methods based on the combination of magnetic beads and gradient magnetic fields suffer from magnetic field strength decaying with the cube of the distance (1 / r). 3 The physical limitations of traditional methods make it difficult to achieve precise force output at centimeter-level working distances, severely restricting their ability to penetrate biological tissues and their translational potential in clinical applications. Summary of the Invention

[0004] To address the aforementioned issues, the purpose of this application is to provide a method and platform for programmed manipulation of DNA based on dynamic magnetic torque. This method can apply controllable torque to double-stranded DNA through programmable dynamic magnetic torque, enabling precise and adjustable programmed manipulation of DNA molecules with different stabilities. This overcomes the bottleneck of limited force output distance of traditional magnetic tweezers, significantly improving the spatial range and temporal accuracy of the operation.

[0005] This application discloses a programmed DNA unwinding method based on dynamic magnetic torque, comprising the following steps: (a) A magnetic micromotor with magnetic anisotropy and a target double-stranded DNA, wherein one end of the target double-stranded DNA is anchored to a substrate; (b) Position the magnetic micromotor at the free end of the target double-stranded DNA and bind the magnetic micromotor to the target double-stranded DNA via chemical coupling to achieve mechanical transfer between the magnetic micromotor and the target double-stranded DNA; (c) Apply a rotating magnetic field to the magnetic micromotor, so that the magnetic micromotor generates a dynamic magnetic torque under the drive of the rotating magnetic field; (d) The target double-stranded DNA is subjected to the dynamic magnetic torque to cause the target double-stranded DNA to undergo programmed unwinding.

[0006] In a preferred embodiment, the target double-stranded DNA consists of two complementary DNA strands, including a ligand strand and a complementary strand.

[0007] In a preferred embodiment, the magnetic micromotor may be spherical, rod-shaped, block-shaped, triangular, or other shapes.

[0008] In a preferred embodiment, the magnetic micromotor is a rod-shaped magnetic motor with a length-to-diameter ratio of 3:1.

[0009] In a preferred embodiment, the frequency of the rotating magnetic field is 0-15 Hz; more preferably, the frequency of the rotating magnetic field is 0-5 Hz.

[0010] In a preferred embodiment, the strength of the rotating magnetic field is 20-270 mT; more preferably, the strength of the rotating magnetic field is 270 mT.

[0011] In a preferred embodiment, the material of the magnetic micromotor is a ferromagnetic or superparamagnetic material, including iron(II,III) oxide, iron oxide, cobalt ferrite, nickel ferrite, etc.

[0012] In a preferred embodiment, step (a), anchoring one end of the target double-stranded DNA to the substrate, includes: One end of the target double-stranded DNA with the first binding group is brought into contact with the substrate surface coated with the binding molecule; The target double-stranded DNA is anchored to the substrate surface through the specific interaction between the first binding group and the binding molecule.

[0013] In a preferred embodiment, the first binding group is a biotin group.

[0014] In a preferred embodiment, the binding molecule is a tetrameric protein; more preferably, the binding molecule is streptavidin.

[0015] In a preferred embodiment, the first binding group is located on the complementary chain; more preferably, the first binding group is located at the 5' end of the complementary chain.

[0016] In a preferred embodiment, step (b), positioning the magnetic micromotor at the free end of the target double-stranded DNA, comprises: The magnetic micromotor with a first chemical linker group on its surface is added to the substrate on which the target double-stranded DNA is anchored, and the magnetic micromotor diffuses in the solution to approach the free end of the target double-stranded DNA. The second chemical linker group disposed at the free end of the target double-stranded DNA undergoes a click chemical reaction with the first chemical linker group to form a covalent link.

[0017] In a preferred embodiment, the magnetic micromotor is linked to the target double-stranded DNA via a click chemistry reaction and incubated at 25-40°C for 30-90 minutes.

[0018] In a preferred embodiment, the first chemical linking group is a azircyclic octylene (DBCO) group, and the second chemical linking group is an azide group.

[0019] In a preferred embodiment, the second chemical linker group is located on the ligand chain; more preferably, the second chemical linker group is located at the 3' end of the ligand chain.

[0020] In a preferred embodiment, when preparing the conjugate of the target double-stranded DNA and the magnetic micromotor, the concentration of the target double-stranded DNA is 100 nM, and the concentration of the magnetic micromotor is 0-100 µg / mL, measured in Fe.

[0021] In a preferred embodiment, the substrate is one of the following: a glass sheet, a quartz sheet, a polymer surface, a hydrogel, a phospholipid bilayer, a magnetic microsphere, or a microfluidic chip surface.

[0022] In a preferred embodiment, the method unwinds target double-stranded DNA with different mechanical unwinding thresholds.

[0023] In a preferred embodiment, the mechanical unwinding threshold of the target double-stranded DNA is at least one of 12 pN, 23 pN, 33 pN, 43 pN, or 54 pN.

[0024] In a preferred embodiment, the method unwinds target double-stranded DNA with different GC contents.

[0025] In a preferred embodiment, the target double-stranded DNA with different GC contents includes: 22%, 77%, and 100%.

[0026] In a preferred embodiment, the method is applied as a precise magnetically controlled molecular switch in at least one of DNA computing, molecular logic gates, and information processing systems.

[0027] In a preferred embodiment, it also includes: (e) Fluorescence detection is performed on the target double-stranded DNA, wherein, The 5' end of the ligand strand is modified with a fluorescence quenching group, and the 3' end of the complementary strand is modified with a fluorescent group. When the ligand strand and the complementary strand of the target double-stranded DNA are complementary, the fluorescence of the fluorescent group is quenched. When the target double-stranded DNA is unwound, the fluorescence of the fluorescent group is restored.

[0028] In a preferred embodiment, the fluorescence quenching group is BHQ2 and the fluorescent group is Cy3.

[0029] In a preferred embodiment, the fluorescence quenching group has a quenching range of 480-580 nm.

[0030] In a preferred embodiment, the emission wavelength of the fluorescent group is 480-580 nm; more preferably, the emission wavelength of the fluorescent group is 570 nm.

[0031] This application also discloses a DNA programmed manipulation platform based on dynamic magnetic torque, including: A substrate, the surface of which is configured to anchor one end of a target double-stranded DNA; A magnetically anisotropic magnetic micromotor is configured to connect to the free end of the target double-stranded DNA via chemical coupling, so as to transfer the magnetic torque generated by the magnetically anisotropic magnetic micromotor to the target double-stranded DNA. The rotating magnetic field generating component is configured to generate a rotating magnetic field acting on the magnetic micromotor under preset parameters, so that the magnetic micromotor generates a dynamic magnetic torque under the drive of the rotating magnetic field.

[0032] In a preferred embodiment, a control device is further included, which is electrically connected to the rotating magnetic field generating component and is used to provide a drive current to the rotating magnetic field generating component and control the on / off state, frequency, magnetic field strength and / or phase of the rotating magnetic field to programmatically regulate the dynamic magnetic torque applied to the magnetic anisotropic micromotor.

[0033] In a preferred embodiment, an imaging and feedback unit is further included, comprising an inverted fluorescence microscope and an image acquisition and processing module electrically connected thereto. This unit is used to observe the fluorescence signal changes in the region where the target double-stranded DNA is located in real time while the rotating magnetic field is applied, and to provide feedback information characterizing the DNA unwinding state to the control device, so that the operator can adjust the parameters of the rotating magnetic field according to the feedback information through the control device.

[0034] In a preferred embodiment, In the embodiments of this application, a magnetic anisotropic micromotor is used to output a programmable dynamic magnetic torque under the drive of a rotating magnetic field. The torque is effectively transmitted to the target double-stranded DNA anchored to the substrate through chemical coupling, thereby achieving precise external field control of the DNA unwinding process. This method can not only apply a consistent and adjustable mechanical torque to double-stranded DNA at the single-molecule scale to achieve high-precision and repeatable programmed unwinding, but also overcome the limitations of traditional magnetic tweezers, such as short operating distance, weak torque output, and low throughput. It significantly improves the effective spatial range and operational efficiency of mechanical manipulation, thus providing a new controllable manipulation strategy for DNA mechanical regulation, molecular logic manipulation, and related bioengineering applications. Attached Figure Description

[0035] Figure 1 A TEM image of the morphology of a rod-shaped magnetic micromotor according to one embodiment of this application; Figure 2 This is a schematic diagram of the construction process according to one embodiment of this application; Figure 3 The results are fluorescence imaging of DNA unwinding under different dynamic magnetic torque conditions according to one embodiment of this application. Figure 4 This is a schematic diagram of a platform structure according to one embodiment of this application; Figure 5 This is a schematic diagram illustrating the dynamic real-time imaging and comparison of the unwinding of the same DNA according to one embodiment of this application; Figure 6 A cellular-level DNA manipulation strategy according to one embodiment of this application Figure 7 An in vivo DNA manipulation strategy according to one embodiment of this application Detailed Implementation

[0036] In the following description, many technical details are presented to help the reader better understand this application. However, those skilled in the art will understand that the technical solutions claimed in this application can be implemented even without these technical details and various variations and modifications based on the following embodiments.

[0037] Compared with the prior art, this application has the following beneficial effects: 1. The magnetically controlled rod-shaped magnetic micromotor has a 3:1 aspect ratio. This asymmetric geometry enhances the magnetic anisotropy of the shape, constraining the magnetization direction along the long axis to form a directional magnetic dipole moment. Compared to a spherical structure, the rod-shaped structure can generate stronger coupling with an external magnetic field, thereby achieving enhanced magnetic torque response and excellent magnetic control performance.

[0038] 2. The rotating magnetic field dynamically programmed magnetic torque overcomes the limitation of the existing static magnetic field where the magnetic field strength decreases sharply with distance. Its precise control of magnetic field frequency can increase the operating distance of traditional magnetic tweezers from the millimeter level to the centimeter level, which is more conducive to the remote and precise control of DNA manipulation ability and efficiency.

[0039] 3. The rotating magnetic field combined with confocal inverted fluorescence microscopy can observe the magnetically induced DNA strand unwinding process in real time, providing a high-resolution experimental platform for studying the mechanical properties and molecular mechanisms of DNA unwinding.

[0040] The present application will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0041] The first embodiment of this application discloses an in vivo programmed DNA unwinding method based on dynamic magnetic torque, comprising the following steps: (a) Provides a magnetic micromotor with magnetic anisotropy and a target double-stranded DNA, wherein one end of the target double-stranded DNA is anchored to a substrate; (b) Position the magnetic micromotor at the free end of the target double-stranded DNA and bind the magnetic micromotor to the target double-stranded DNA through chemical coupling to achieve mechanical transfer between the magnetic micromotor and the target double-stranded DNA; (c) Apply a rotating magnetic field to the magnetic micromotor so that the magnetic micromotor generates dynamic magnetic torque under the drive of the rotating magnetic field; (d) Using dynamic magnetic torque to act on the target double-stranded DNA, the target double-stranded DNA undergoes programmed mechanical unwinding.

[0042] In some alternative embodiments, the magnetically anisotropic micromotor is preferably composed of rod-shaped or long rod-shaped magnetic micro / nanoparticles with a morphology exhibiting significant magnetic moment directionality, in order to generate controllable magnetic torque in an applied rotating magnetic field. For example... Figure 1 Figure a shows a TEM image of the morphology of a rod-shaped magnetic micromotor according to an embodiment of this application. The magnetic micromotor has a regular rod-shaped structure with a length of about micrometers and obvious long and short axis directions. Figure 1 b shows the dimensional statistics of the magnetic micromotor, illustrating the distribution of its major and minor axes. The average size of the major axis is approximately 2 μm, and the average size of the minor axis is approximately 0.6 μm, with a length-to-diameter ratio of approximately 3:1, which is beneficial for generating dynamic magnetic torque in a rotating magnetic field.

[0043] In some alternative embodiments, the frequency of the rotating magnetic field is 0-15 Hz; preferably, the frequency of the rotating magnetic field is 0-5 Hz.

[0044] In some alternative embodiments, the strength of the rotating magnetic field is 0-270 mT; preferably, the strength of the rotating magnetic field is 270 mT.

[0045] In some alternative embodiments, step (a), anchoring one end of the target double-stranded DNA to the substrate, includes: One end of the target double-stranded DNA with the first binding group is brought into contact with the substrate surface coated with the binding molecule; The target double-stranded DNA is anchored to the substrate surface through the specific interaction between the first binding group and the binding molecule.

[0046] In some alternative embodiments, the first binding group is a biotin group.

[0047] In some alternative embodiments, the binding molecule is a tetrameric protein; more preferably, the binding molecule is streptavidin.

[0048] In some alternative embodiments, the first binding group is located on the complementary chain; more preferably, the first binding group is located at the 5' end of the complementary chain.

[0049] In some alternative embodiments, positioning the magnetic micromotor at the free end of the target double-stranded DNA includes: A magnetic micromotor with a first chemical linker group on its surface is added to a substrate with the target double-stranded DNA already anchored, and the magnetic micromotor diffuses in the solution to approach the free end of the target double-stranded DNA. The second chemical linker group located at the free end of the target double-stranded DNA undergoes a click chemical reaction with the first chemical linker group to form a covalent link.

[0050] In some optional embodiments, the first chemical linking group is a azircyclic octylene (DBCO) group, and the second chemical linking group is an azide group.

[0051] In some alternative embodiments, the second chemical linker group is located on the ligand chain; more preferably, the second chemical linker group is located at the 3' end of the ligand chain.

[0052] In some optional embodiments, when preparing the conjugate of the target double-stranded DNA and the magnetic micromotor, the concentration of the target double-stranded DNA is 100 nM, and the concentration of the magnetic micromotor is 0-100 µg / mL, measured in Fe.

[0053] In some alternative embodiments, the substrate is one of the following: a glass sheet, a quartz sheet, a polymer surface, a hydrogel, a phospholipid bilayer, a magnetic microsphere, or a microfluidic chip surface.

[0054] In some alternative embodiments, the method unwinds target double-stranded DNA with different mechanical unwinding thresholds.

[0055] Preferably, the mechanical unwinding threshold of the target double-stranded DNA is at least one of 12 pN, 23 pN, 33 pN, 43 pN, or 54 pN.

[0056] In some alternative embodiments, the method is applied as a precise magnetically controlled molecular switch in at least one of DNA computing, molecular logic gates, and information processing systems.

[0057] In some optional embodiments, it also includes: (e) Fluorescence detection of the target double-stranded DNA, wherein, The 5' end of the ligand strand is modified with a fluorescence quenching group, and the 3' end of the complementary strand is modified with a fluorescent group. When the ligand strand and the complementary strand of the target double-stranded DNA are complementary, the fluorescence of the fluorescent group is quenched. When the target double-stranded DNA is unwound, the fluorescence of the fluorescent group is restored.

[0058] In some optional embodiments, the fluorescence quenching group is BHQ2 and the fluorescent group is Cy3.

[0059] In some optional embodiments, the fluorescence quenching group has a quenching range of 480-580 nm.

[0060] In some optional embodiments, the emission wavelength of the fluorescent group is 480-580 nm; more preferably, the emission wavelength of the fluorescent group is 570 nm.

[0061] The second embodiment of this application relates to a DNA programmed manipulation platform based on dynamic magnetic torque, including a substrate whose surface is configured to anchor one end of a target double-stranded DNA. A magnetically anisotropic magnetic micromotor is configured to connect to the free end of the target double-stranded DNA via chemical coupling, so as to transfer the magnetic torque generated by the magnetically anisotropic magnetic micromotor to the target double-stranded DNA. The rotating magnetic field generating component is configured to generate a rotating magnetic field acting on a magnetic micromotor under preset parameters, so that the magnetic micromotor generates a dynamic magnetic torque under the drive of the rotating magnetic field.

[0062] In some alternative embodiments, a control device is also included, electrically connected to the rotating magnetic field generating component, for providing drive current to the rotating magnetic field generating component and controlling the on / off state, frequency, magnetic field strength and / or phase of the rotating magnetic field to programmatically regulate the dynamic magnetic torque applied to the magnetically anisotropic micromotor.

[0063] In some optional embodiments, an imaging and feedback unit is also included, comprising an inverted fluorescence microscope and an image acquisition and processing module electrically connected thereto, for real-time observation of fluorescence signal changes in the region where the target double-stranded DNA is located while a rotating magnetic field is applied, and for providing feedback information characterizing the DNA unwinding state to a control device so that the operator can adjust the parameters of the rotating magnetic field according to the feedback information through the control device.

[0064] The following detailed description of the various technical details for achieving the objectives of this application will enable those skilled in the art to implement them after reading this application. It should be understood that these descriptions are not intended to limit this application. For those skilled in the art, all equivalent substitutions, improvements, or modifications made without departing from the spirit and substance of this application should be considered to fall within the scope of protection claimed in this application.

[0065] First aspect: Fabrication method of magnetic micromotor The first aspect of this application provides a method for fabricating a magnetic micromotor, comprising the following steps: (1) Preparation of anisotropic magnetic micromotors (MRs): Ferric nitrate nonahydrate and glucose were weighed and dissolved in 75 mL of ethylene glycol and stirred thoroughly for 30 min until completely dissolved; the amount of ferric nitrate nonahydrate was fixed at 0.76 g and the amount of glucose was 0.5 g. The above mixed solution was transferred and sealed in a 100 mL reactor and reacted at 220 ℃ for 12 h. After the reaction was completed, the sample was collected after cooling to room temperature, centrifuged at 9500 rpm for 15 min, and then washed three times with anhydrous ethanol and deionized water respectively. After centrifugation, magnetic microparticle precursors under different reaction conditions were obtained. After drying in an oven at 65 ℃ for 24 h, the powder was collected and stored. The above powder was placed in a tube furnace at 350 ℃ and annealed under an argon and hydrogen atmosphere. The powder was collected after reacting for 4 h. The argon flow rate was 140 mL / min, the hydrogen flow rate was 60 mL / min, the heating rate was 5 ℃ / min, and the maximum temperature was 350 ℃. The powder was collected after annealing and cooling to room temperature.

[0066] (2) DBCO functionalization modification of MRs surface: MRs surface amino modification was performed. 30 mg of the above powder was weighed and dispersed in 40 mL of 75% ethanol solution and ultrasonically dispersed evenly. 916.8 μL of 3-aminopropyltriethoxysilane was dissolved in 2 mL of N,N-dimethylformamide (DMF) and transferred to the above solution and mixed evenly. The reaction was stirred at room temperature for 2 h and then allowed to stand at 4℃ for 48 h. After the modification was completed, the mixture was centrifuged at 9500 rpm for 15 min, washed 3 times each with anhydrous ethanol and deionized water, and the functionalized MRs were collected and dispersed in deionized water and stored at 4℃. MRs were modified with DBCO groups on their surface. 4.4 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was added to 1 mL of PBS buffer (pH 6.0), along with 1.0 mg of DBCO-COOH and 6.6 mg of N-hydroxysuccinimide (NHS). The mixture was stirred at room temperature for 1 h to activate the carboxyl groups in DBCO-COOH. 0.5 mg of an amino-modified rod-shaped micromotor was added to the mixture and ultrasonically dispersed. The mixture was then ultrasonically broken up for 2 h, followed by standing at 4 °C for 48 h. After the reaction, the mixture was purified by magnetic separation, washed three times with deionized water, and finally dispersed in deionized water and stored at 4 °C.

[0067] After completing the above preparation and functionalization modification, a magnetically anisotropic micromotor with good magnetic response performance and the ability to click with the free ends of DNA can be obtained. This application utilizes the covalent connection structure formed at the ends of this magnetic micromotor and the target double-stranded DNA to achieve precise transmission of dynamic magnetic torque under the drive of an external rotating magnetic field, thereby enabling programmed mechanical manipulation of DNA.

[0068] The second aspect: the construction method of the control platform Specifically, as in the second aspect of this application, a method for constructing a control platform is provided, comprising the following steps: (1) Binding of the magnetic micromotor to the target double-stranded DNA: The DNA sequence consists of two complementary DNA strands. The 3' end of the ligand strand is modified with an azide group (N3), which can be used to undergo a click chemical reaction with azacyclic octylene (DBCO) for the modification of the magnetically controlled micromotor. Biotin is located at the 5' end and is used to bind to the streptavidin (SA) on the substrate, thereby anchoring the DNA to the substrate.

[0069] (2) Construction of the mechanical system to be driven: The DNA strand modified with azide group was incubated with the substrate at a concentration of 100 nM for 30 min at room temperature to fix the DNA to the substrate, and washed three times. The DBCO-functionalized rod-shaped magnetic micromotor prepared in the first aspect was added to the DNA-modified substrate at a concentration of 100 µg / mL (calculated based on Fe concentration), and incubated at 37 °C for a total of 60 min, followed by washing three times.

[0070] (3) Apply a rotating magnetic field (RMF) to the above magnetic micromotor. The magnetic micromotor generates dynamic magnetic torque under the drive of the RMF, which produces a higher magnetomechanical force output than the static magnetic field motion mode.

[0071] like Figure 4 As shown, the control device, serving as the main control unit, is electrically connected to the rotating magnetic field generator (RMF). This control device is equipped with LabVIEW software for setting the magnetic field parameters of the RMF and executing control algorithms. The operator can input motion commands via a human-machine interface (such as a joystick), and the control program receives this input and generates corresponding drive signals. These drive signals are output from a high-speed data acquisition card (DAQ), amplified by a voltage amplifier, and then transmitted to multiple electromagnetic coils. Upon receiving the drive signals, the electromagnetic coils generate a spatially controllable magnetic field distribution, thereby producing magnetic torque on the magnetic micromotor or magnetic material within the RMF's active region, achieving motion drive and trajectory control in three-dimensional space.

[0072] Third aspect: Rotating magnetic field generating component Accordingly, a third aspect of this application provides a rotating magnetic field generating assembly.

[0073] The rotating magnetic field generating component of this application uses two rotating permanent magnets to construct the magnetic field source. The magnets are driven by a motor to achieve adjustable frequency rotational motion, thereby generating a controllable rotating magnetic field. The maximum magnetic field strength can reach 270 mT, and the operating frequency range is 0-15 Hz, meeting the experimental requirements for programmed manipulation of DNA mechanics.

[0074] The rotating magnetic field generating component may include: (1) Rotating rotor carrying permanent magnets. The permanent magnets are fixed on the rotating rotor and can be evenly distributed along the circumference of the rotor to form a uniform two-dimensional planar rotating magnetic field when the rotor rotates. This magnetic field is constructed by the magnetism of the permanent magnets themselves.

[0075] (2) An electric motor for driving a rotating rotor to generate a rotating magnetic field. The output shaft of the motor can be directly connected to the rotating rotor via a coupling, or via a transmission mechanism such as a belt or gear, so as to drive the rotor to rotate smoothly, thereby forming a rotating magnetic field acting on the magnetic micromotor, so that the magnetic micromotor generates a dynamic magnetic torque under the drive of the rotating magnetic field.

[0076] It should be noted that the rotating magnetic field generating component itself can be implemented using an electromagnetic coil array drive system already available in the field, and is not the focus of this application. The innovation of this application focuses on the technical solution of realizing programmatic manipulation of double-stranded DNA through programmable dynamic magnetic torque.

[0077] The mechanism by which this application generates dynamic magnetic torque is briefly explained below: The rod-shaped magnetic micromotor used in this application exhibits significant magnetic anisotropy. When a rotating magnetic field is applied externally, the direction of the magnetic field changes continuously over time, and the magnetic moment inside the magnetic micromotor tends to align with the direction of the applied magnetic field. As the magnetic field direction rotates continuously, the magnetic micromotor needs to continuously adjust its orientation to follow the changes in the magnetic field. During this process, the magnetic micromotor experiences torque generated by the interaction between the magnetic moment and the magnetic field. The magnitude and direction of this torque change dynamically with the magnetic field, thereby forming a continuous, stable, and programmable dynamic magnetic torque.

[0078] The dynamic magnetic torque generated by the above mechanism is transmitted to the DNA molecule through the chemical coupling interface between the magnetic micromotor and the target double-stranded DNA, thereby achieving a mechanical torsion of the DNA double strand with consistent direction and controllable amplitude, thus driving it to undergo programmed mechanical unwinding.

[0079] Fourth aspect: Visualization methods for DNA manipulation To verify the successful manipulation of the target double-stranded DNA, the fourth aspect of this application provides a visualization method for DNA manipulation: modifying the 5' end of the ligand strand with a fluorescent quencher group BHQ2 and modifying the 3' end of the complementary strand with a fluorescent group Cy3, wherein the quenching range of BHQ2 is 480–580 nm, and the emission wavelength of Cy3 is approximately 570 nm. Figure 2 Figure c shows the fluorescence emission spectra of Cy3, BHQ2, and MRs. It can be seen that Cy3 has a significant fluorescence emission peak at approximately 570 nm, while BHQ2 and MRs show almost no fluorescence signal in the visible light region. This further proves that BHQ2 mainly exists as a quenching group of Cy3 in this system, and the magnetic micromotor itself does not introduce additional fluorescence background, which is beneficial for subsequent visualization and monitoring of DNA manipulation processes through changes in Cy3 fluorescence.

[0080] After the double-stranded structure is formed, the spatial distance between Cy3 and BHQ2 is close, causing the fluorescence energy of Cy3 to be absorbed by BHQ2 located at the 5' end of the ligand strand, thus putting it into a fluorescence quenching state. If the target double-stranded DNA that has formed the quenching state is connected to a magnetic micromotor in the aforementioned manner, and a rotating magnetic field is applied to drive the magnetic micromotor to generate dynamic magnetic torque, when the double-stranded DNA undergoes local or overall unwinding under the action of torque, the spatial distance between Cy3 and BHQ2 increases, thus reducing the quenching efficiency.

[0081] The fluorescence intensity of Cy3 is monitored using a confocal microscope or other fluorescence imaging device; when mechanical unwinding occurs, the fluorescence of Cy3 recovers from the quenched state, thereby enabling the visual detection of the mechanical manipulation process of the target double-stranded DNA.

[0082] Fifth aspect: Visualized control devices for DNA manipulation To observe whether the target double-stranded DNA as described in the fourth aspect of this application has been successfully manipulated and regulated accordingly, the fifth aspect of this application provides a visualization and regulation device for DNA manipulation. It achieves precise driving and visualization control of a micromotor through the coordinated operation of a control device, a rotating magnetic field generating component, and an imaging and feedback unit as described in the third aspect of this application.

[0083] This device is equipped with an inverted confocal fluorescence microscope (such as the Nikon Eclipse Ti2) to monitor changes in fluorescence intensity in real time during DNA manipulation. The microscope captures the fluorescence signal of Cy3 and its spatial distribution, and has high temporal resolution for continuously recording fluorescence quenching and recovery phenomena during DNA manipulation. The acquired fluorescence signal is converted into quantifiable intensity data by a signal acquisition and amplification module.

[0084] During DNA mechanical unwinding, the distance between Cy3 and BHQ2 increases, leading to a rise in observable fluorescence intensity. This provides real-time visual feedback on the DNA unwinding state, and the acquired images and fluorescence data are fed back to the master control unit in real time. The master control unit can analyze the position, rotation behavior, and degree of DNA unwinding of the magnetic micromotor, and adjust the rotation magnetic field parameters based on the feedback results to achieve closed-loop control of the manipulation behavior or data acquisition.

[0085] Through the coordinated operation of the above modules, this device can monitor and quantify the manipulation process of DNA in real time while applying dynamic magnetic torque, thus constructing a visual measurement and control platform that can be used for DNA mechanical research, molecular switch operation, and DNA logic calculation.

[0086] To make the objectives, technical solutions, beneficial effects, and significant advancements of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the embodiments of this application. Obviously, all the described embodiments are only some embodiments of this application, and not all embodiments; based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0087] Example 1 This embodiment provides a dynamic magnetic torque DNA programmed manipulation technique and its selective DNA manipulation, as detailed below: (1) DNA structure design: The DNA sequence consists of two complementary DNA strands. The ligand strand is modified with a quenching group BHQ2 at the 5' end, with a quenching range of 480-580 nm, and an azide group (N3) at the 3' end, which can be used to perform click chemistry with azacyclic octylene (DBCO) for modification of magnetically controlled micromotors. In the complementary strand, Cy3 is a fluorescent reporter group located at the 3' end, with an emission wavelength of 570 nm, and Biotin is located at the 5' end, used to bind to SA. When it is complementary to the ligand strand, the fluorescence of Cy3 is absorbed by the quenching group BHQ2 distributed at the 5' end of the ligand strand, thereby quenching its fluorescence. Biotin is mainly used to bind to streptavidin (SA) on the substrate to anchor the DNA to the substrate. Table 1 (2) Preparation of rod-shaped magnetic micromotor: Ferric nitrate nonahydrate and glucose were weighed and dissolved in 75 mL of ethylene glycol and stirred thoroughly for 30 min until completely dissolved; the amount of ferric nitrate nonahydrate was fixed at 0.76 g and the amount of glucose was 0.5 g. The above mixed solution was transferred and sealed in a 100 mL reaction vessel and reacted at 220 ℃ for 12 h; after the reaction was completed, the sample was collected after cooling to room temperature, centrifuged at 9500 rpm for 15 min, and then washed three times with anhydrous ethanol and deionized water respectively. After centrifugation, magnetic microparticle precursors under different reaction conditions were obtained; after drying in a 65 ℃ oven for 24 h, the powder was collected and stored, and then the morphology was characterized by TEM as follows. Figure 1As shown. The above powder was placed in a tube furnace at 350℃ and annealed under an argon and hydrogen atmosphere. The powder was collected after 4 h of reaction. The argon flow rate was 140 mL / min, the hydrogen flow rate was 60 mL / min, the heating rate was 5℃ / min, and the maximum temperature was 350℃. The surface of the annealed rod-shaped micromotor was modified with amino groups. 30 mg of the above powder was weighed and dispersed in 40 mL of 75% ethanol solution and ultrasonically dispersed evenly. 916.8 μL of 3-aminopropyltriethoxysilane was dissolved in 2 mL of N,N-dimethylformamide (DMF) and transferred to the above solution and mixed evenly. The mixture was stirred at room temperature for 2 h and then allowed to stand at 4℃ for 48 h. After modification, the mixture was centrifuged at 9500 rpm for 15 min, washed three times each with anhydrous ethanol and deionized water, and the functionalized modified MRs were collected and dispersed in deionized water and stored at 4℃. Subsequently, 4.4 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was added to 1 mL of PBS buffer (pH 6.0), along with 1.0 mg of DBCO-COOH and 6.6 mg of N-hydroxysuccinimide (NHS). The mixture was stirred at room temperature for 1 h to activate the carboxyl group in DBCO-COOH. 0.5 mg of an amino-modified rod-shaped micromotor was added to the mixture and ultrasonically dispersed. The mixture was then stirred using an ultrasonic homogenizer for 2 h, followed by standing at 4 °C for 48 h. After the reaction was completed, the mixture was purified by magnetic separation, washed three times with deionized water, and finally dispersed in deionized water and stored at 4 °C.

[0088] (3) Construction of the dynamic magnetic moment DNA manipulation platform: The DNA strand prepared in step (1) was incubated with the substrate at a concentration of 100 nM for 30 min at room temperature to immobilize the DNA on the substrate, and washed three times. The rod-shaped magnetic micromotor prepared in step (2) was added to the DNA-modified substrate at a concentration of 100 µg / mL (calculated based on Fe concentration), and incubated at 37°C for a total of 60 min, followed by washing three times to obtain the dynamic magnetic moment DNA manipulation platform, as shown below. Figure 2 As shown, Figure 2 Figure 'a' illustrates the construction process: a DNA strand (red) containing Biotin and Cy3 is immobilized on a substrate coated with streptavidin (SA) via Biotin–SA interaction; subsequently, a complementary DNA strand (blue) with an azide group (N3) at the 5' end and a quencher group BHQ2 at the 3' end is introduced and hybridized with the anchor strand to form a double-stranded structure; finally, rod-shaped magnetic micromotors (MRs) with DBCO groups modified on their surface are added to the system, and the magnetic micromotors are connected to the free ends of the double-stranded DNA via a DBCO–N3 click chemistry reaction, thus completing the platform for subsequent dynamic magnetic torque manipulation.

[0089] (4) Further, the dynamic magnetic torque DNA manipulation platform prepared in step (3) is placed under a rotating magnetic field and treated with different rotation frequencies for 15 min. The output magnetic torque is adjusted by changing the frequency to regulate the manipulation rate and capability of DNA. The DNA manipulation efficiency is observed by confocal microscopy imaging or the DNA manipulation situation is observed in real time by a magnetic field confocal coupling system. Figure 2 Figure b shows confocal fluorescence images of Cy3, BHQ2, and MRs in different channels. Scattered red fluorescent dots can be observed in the Cy3 channel, corresponding to Cy3-labeled DNA immobilized on the substrate surface. There are basically no obvious fluorescence signals in the BHQ2 and MRs channels, indicating that BHQ2 itself and the magnetic micromotor do not produce observable fluorescence under these excitation / emission conditions and will not interfere with the fluorescence detection of Cy3.

[0090] For DNA manipulation efficiency statistics: The dynamic magnetic moment DNA manipulation platform was placed under a 40 mT rotating magnetic field, with the magnetic field rotation frequency adjusted to 2-5 Hz, and treated for 15 min. After washing the substrate, Cy3 fluorescence recovery was observed under a confocal microscope. The degree of fluorescence recovery was statistically analyzed, and the relationship between different frequencies and DNA manipulation efficiency was established, such as... Figure 3 The image shows fluorescence imaging results of DNA manipulation under different dynamic magnetic torque conditions. It can be seen that when no rotating magnetic field is applied or only a weak rotating magnetic field is applied, the number of fluorescent spots in the field of view is small. As the rotating magnetic field parameters (such as frequency) increase, the number of fluorescent spots gradually increases and the brightness increases, indicating that more double-stranded DNA is manipulated by the dynamic magnetic torque to unwind and transition from the quenched state to the fluorescence-recovered state. Furthermore, according to... Figure 3 It can also be seen that this application can selectively manipulate double-stranded DNA with different mechanical thresholds (pN level) by adjusting the frequency of the rotating magnetic field, so as to achieve programmed manipulation and hierarchical regulation that matches the torque magnitude with the mechanical stability of DNA.

[0091] For real-time observation of DNA manipulation: The dynamic magnetic moment DNA manipulation platform prepared in (3) is placed on a magnetic field-driven and confocal coupled real-time imaging platform (e.g. Figure 4 (As shown). A rotating magnetic field of 20 mT and 5 Hz was applied, and the fluorescence recovery caused by DNA unwinding was observed in real time by confocal microscopy, as shown. Figure 5 As shown, Figure 5 Image a shows the fluorescence change of a single 12 pN TGTs DNA over time under the influence of a rotating magnetic field. The left side (BF) shows the corresponding bright-field image for localization; the right side (0–5 min) shows the time-series fluorescence images of the Cy3 channel. From 0 min to 5 min, the red fluorescence signal gradually increases from almost invisible to a distinct bright spot, indicating that under the continuous action of the dynamic magnetic torque, the Cy3-labeled DNA, originally in a quenched state, is gradually unwound, and the fluorescence changes from an off state to an on state. Figure 5 Figure b shows the case where MRs are driven by a rod-shaped magnetic anisotropic micromotor, and MBs are the control group driven by spherical magnetic beads. Within 0–5 min of applying the rotating magnetic field, the fluorescence intensity of the MRs group continuously increased, indicating that it could effectively transmit torque and gradually unwind DNA; while the fluorescence intensity of the MBs group remained essentially unchanged, indicating that the spherical magnetic beads could not generate sufficient torque to unwind DNA at the same mechanical threshold. This further demonstrates the significant advantages of the dynamic magnetic torque actuation based on rod-shaped magnetic micromotors in DNA manipulation presented in this application.

[0092] Example 2 This embodiment provides a method for programmed DNA manipulation based on dynamic magnetic torque, and focuses on disclosing the verification process of achieving precise DNA manipulation at the cellular level at a macroscopic working distance of 4 cm. The specific implementation steps are as follows: (1) DNA structure design: The DNA consists of two complementary DNA strands. The 5' end of the magnetic DNA element is modified with a quenching group BHQ2, which has a quenching range of 480-580 nm, and the 3' end is modified with an azide group (N3), which can be used to undergo a click chemical reaction with azacyclic octylene (DBCO) for modification of magnetically controlled micromotors. In the force-responsive DNA sequence, Cy3 is a fluorescent reporter group located at the 3' end, with an emission wavelength of 570 nm, while cholesterol is located at the 5' end and is used for binding to the cell membrane. When it is complementary to the ligand strand, the fluorescence of Cy3 is absorbed by the quenching group BHQ2 distributed at the 5' end of the ligand strand, thereby quenching its fluorescence. Table 2 (2) Preparation of rod-shaped micromotor: Ferric nitrate nonahydrate and glucose were weighed and dissolved in 75 mL of ethylene glycol and stirred thoroughly for 30 min until completely dissolved; the amount of ferric nitrate nonahydrate was fixed at 0.76 g and the amount of glucose was 0.5 g. The above mixed solution was transferred and sealed in a 100 mL reaction vessel and reacted at 220 ℃ for 12 h; after the reaction was completed, the sample was collected after cooling to room temperature, centrifuged at 9500 rpm for 15 min, and then washed three times with anhydrous ethanol and deionized water respectively. After centrifugation, magnetic microparticle precursors under different reaction conditions were obtained; after drying in a 65 ℃ oven for 24 h, the powder was collected and stored, and then the morphology was characterized by TEM as follows. Figure 1As shown in Figure a. The above powder was placed in a tube furnace at 350℃ and annealed under an argon and hydrogen atmosphere. The powder was collected after 4 h of reaction. The argon flow rate was 140 mL / min, the hydrogen flow rate was 60 mL / min, the heating rate was 5℃ / min, and the maximum temperature was 350℃. The surface of the annealed rod-shaped micromotors was modified with amino groups. 30 mg of the above powder was weighed and dispersed in 40 mL of 75% ethanol solution and ultrasonically dispersed evenly. 916.8 μL of 3-aminopropyltriethoxysilane was dissolved in 2 mL of N,N-dimethylformamide (DMF) and transferred to the above solution and mixed evenly. The mixture was stirred at room temperature for 2 h and then allowed to stand at 4℃ for 48 h. After modification, the mixture was centrifuged at 9500 rpm for 15 min, washed three times each with anhydrous ethanol and deionized water, and the functionalized rod-shaped micromotors (MMs) were collected and dispersed in deionized water for storage at 4℃. Subsequently, 4.4 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was added to 1 mL of PBS buffer (pH 6.0), along with 1.0 mg of DBCO-COOH and 6.6 mg of N-hydroxysuccinimide (NHS). The mixture was stirred at room temperature for 1 h to activate the carboxyl group in DBCO-COOH. 0.5 mg of an amino-modified rod-shaped micromotor was added to the mixture and ultrasonically dispersed. The mixture was then stirred using an ultrasonic homogenizer for 2 h, followed by standing at 4 °C for 48 h. After the reaction was completed, the mixture was purified by magnetic separation, washed three times with deionized water, and finally dispersed in deionized water and stored at 4 °C.

[0093] (3) The dynamic magnetic moment DNA manipulation platform is used to achieve precise DNA manipulation at a macroscopic working distance of 4 cm at the cellular level: The single strand of the cholesterol-containing force-responsive DNA sequence prepared in step (1) is incubated with 4T1 cells at 4 degrees Celsius for 30 min at a concentration of 200 nM, so that the DNA can be inserted into the cell membrane through the hydrophobic interaction of cholesterol, and washed three times. The rod-shaped magnetic micromotor prepared in step (2) is incubated with 200 nM magnetic DNA elements at a concentration of 100 µg / mL (calculated based on Fe concentration) at 37 degrees Celsius for 60 min to connect the DNA strand with the rod-shaped magnetic micromotor, followed by magnetic washing three times. The rod-shaped magnetic micromotor with a modified magnetic element DNA strand of 100 µg / mL (calculated based on Fe concentration) is added to the above 4T1 cells. The rod-shaped magnetic micromotor is coupled to the cell membrane surface through DNA base programming, and the Cy3 fluorescence carried by the force-responsive DNA sequence is quenched by the BHQ2 group coupled to the magnetic DNA element. Furthermore, by applying a rotating magnetic field at a distance of 4 cm from the cell surface with a magnetic field strength of approximately 20 mT, magnetic torque was output for manipulating the DNA strand, thereby releasing the magnetic DNA element DNA strand and restoring the fluorescence on the surface of 4T1 cells. Figure 6The diagram shows a schematic of DNA manipulation at the cellular level. Figure 6 a is a schematic diagram of programmed DNA manipulation based on dynamic magnetic torque at the cellular level. This application utilizes the hydrophobic intercalation of cholesterol molecules to anchor target double-stranded DNA to the surface of living cell membranes. Figure 6 b shows typical laser confocal fluorescence images of 4T1 cells, demonstrating that the target double-stranded DNA remained stable after the addition of a magnetic micromotor. Subsequently, a programmed rotating magnetic field (20 mT, 5 min) was applied at a macroscopic working distance of 4 cm. When the rotation frequency exceeded 2 Hz, the dynamic magnetic torque generated by the magnetic micromotor drove the DNA to mechanically unwind, resulting in a significant recovery of the specific Cy3 fluorescence signal, achieving precise manipulation of cell surface DNA. Corresponding statistical results ( Figure 6 c) shows that under the action of a programmed magnetic field, over 94% of the DNA-modified 4T1 cells exhibited significant mechanounwind fluorescence signals. These results demonstrate that the dynamic magnetic torque manipulation strategy based on magnetic micromotors in this application possesses good biocompatibility and successfully achieves highly efficient spatiotemporal precise programmed manipulation of DNA at the cellular level.

[0094] Example 3 This embodiment provides a DNA in vivo spatiotemporal manipulation method based on dynamic magnetic torque. The specific implementation steps are as follows: (1) DNA structure design: The DNA consists of two complementary DNA strands. The 5' end of the magnetic DNA element is modified with a quenching group BHQ2, which has a quenching range of 480-580 nm, and the 3' end is modified with an azide group (N3), which can be used to undergo a click chemical reaction with azacyclic octylene (DBCO) for modification of magnetically controlled micromotors. In the force-responsive DNA sequence, Cy3 is a fluorescent reporter group located at the 3' end, with an emission wavelength of 570 nm, while cholesterol is located at the 5' end and is used for binding to the cell membrane. When it is complementary to the ligand strand, the fluorescence of Cy3 is absorbed by the quenching group BHQ2 distributed at the 5' end of the ligand strand, thereby quenching its fluorescence. Table 2 (2) Preparation of rod-shaped micromotor: Ferric nitrate nonahydrate and glucose were weighed and dissolved in 75 mL of ethylene glycol and stirred thoroughly for 30 min until completely dissolved; the amount of ferric nitrate nonahydrate was fixed at 0.76 g and the amount of glucose was 0.5 g. The above mixed solution was transferred and sealed in a 100 mL reaction vessel and reacted at 220 ℃ for 12 h; after the reaction was completed, the sample was collected after cooling to room temperature, centrifuged at 9500 rpm for 15 min, and then washed three times with anhydrous ethanol and deionized water respectively. After centrifugation, magnetic microparticle precursors under different reaction conditions were obtained; after drying in a 65 ℃ oven for 24 h, the powder was collected and stored, and then the morphology was characterized by TEM as follows. Figure 1 As shown in Figure a. The above powder was placed in a tube furnace at 350℃ and annealed under an argon and hydrogen atmosphere. The powder was collected after 4 h of reaction. The argon flow rate was 140 mL / min, the hydrogen flow rate was 60 mL / min, the heating rate was 5℃ / min, and the maximum temperature was 350℃. The surface of the annealed rod-shaped micromotors was modified with amino groups. 30 mg of the above powder was weighed and dispersed in 40 mL of 75% ethanol solution and ultrasonically dispersed evenly. 916.8 μL of 3-aminopropyltriethoxysilane was dissolved in 2 mL of N,N-dimethylformamide (DMF) and transferred to the above solution and mixed evenly. The mixture was stirred at room temperature for 2 h and then allowed to stand at 4℃ for 48 h. After modification, the mixture was centrifuged at 9500 rpm for 15 min, washed three times each with anhydrous ethanol and deionized water, and the functionalized rod-shaped micromotors (MMs) were collected and dispersed in deionized water for storage at 4℃. Subsequently, 4.4 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was added to 1 mL of PBS buffer (pH 6.0), along with 1.0 mg of DBCO-COOH and 6.6 mg of N-hydroxysuccinimide (NHS). The mixture was stirred at room temperature for 1 h to activate the carboxyl group in DBCO-COOH. 0.5 mg of an amino-modified rod-shaped micromotor was added to the mixture and ultrasonically dispersed. The mixture was then stirred using an ultrasonic homogenizer for 2 h, followed by standing at 4 °C for 48 h. After the reaction was completed, the mixture was purified by magnetic separation, washed three times with deionized water, and finally dispersed in deionized water and stored at 4 °C.

[0095] (3) Spatiotemporal manipulation of DNA in vivo based on dynamic magnetic torque: A rod-shaped magnetic micromotor with a concentration of 1 mg / mL was first co-incubated with a 10 µM magnetic DNA element at 37 °C for 1 hour. After magnetic separation and washing, the above complex was incubated with 10 µM of a cholesterol-modified force-responsive DNA sequence at room temperature for 30 minutes to couple the DNA strands with the rod-shaped magnetic micromotor. Subsequently, magnetic separation and washing were performed to remove unbound free DNA strands, finally yielding a functionalized rod-shaped magnetic micromotor with the target double-stranded DNA.

[0096] 1×10 6 Four T1 cells (resuspended in 100 µL PBS) were subcutaneously injected into the right ventral region of 6-week-old female BALB / c mice. The tumors were allowed to grow to an average size of approximately 300 mm. 3 Mice were randomly divided into four groups (n=3 per group): control group, Cy3 fluorescence group, magnetic micromotor group (MMs group), and magnetic field group (5 Hz group). Each group of mice underwent intratumoral injection: the control group received 50 µL of physiological saline; the Cy3 fluorescence group received 50 µL of force-responsive DNA sequence (10 µM); and the MMs and 5 Hz groups received the prepared functionalized rod-shaped magnetic micromotors at a dose of 10 mg / kg. After incubation for 20 minutes (allowing the magnetic micromotors to insert into the living tumor cell membrane via cholesterol-mediated hydrophobic intercalation), an external rotating magnetic field (magnetic field strength 270 mT, rotation frequency 5 Hz) was applied to the tumor region of the 5 Hz group mice for 5 minutes. In vivo fluorescence imaging was performed immediately after the magnetic field treatment ended; subsequently, the mice were euthanized and the tumor tissue was dissected for in vitro imaging and section analysis. Figure 7 The image shows the verification results of DNA spatiotemporal manipulation based on dynamic magnetic torque in vivo. Figure 7 Figure a is a schematic diagram of in vivo fluorescence imaging monitoring the mechanical unwinding process of double-stranded DNA in the tumor target area. The results show that simple intratumoral injection of Cy3-labeled DNA produces a detectable fluorescence signal in the tumor region; however, injection of a functionalized magnetic micromotor (internally coupled with a BHQ2 quenching group) results in significant fluorescence quenching in the tumor region, confirming that the complex probe maintains high structural integrity even in the complex in vivo tumor microenvironment. Subsequently, as... Figure 7 As shown in b, after applying a 5 Hz rotating magnetic field for 5 minutes, the Cy3-specific fluorescence signal in the tumor region showed a significant response and recovery. Further in vitro imaging and quantitative fluorescence analysis of the dissected tumor were performed. Figure 7 c. Figure 7d) shows that the 5 Hz magnetic field group with the applied procedure exhibits a significant Cy3 fluorescence signal, and its fluorescence intensity is comparable to that of the Cy3-DNA injection group alone. Furthermore, confocal fluorescence imaging and quantitative analysis of tumor tissue sections ( Figure 7 e Figure 7 f) Clearly revealed the recovery of cell membrane-related specific fluorescence signals in the 5 Hz magnetic field group. These results demonstrate that the dynamic magnetic torque DNA manipulation platform based on a rod-shaped magnetic micromotor, as described in this application, can manipulate DNA within 5 minutes in a complex in vivo environment. This system successfully overcomes the physical limitations of centimeter-level macroscopic distances, inducing efficient programmed unwinding of target double-stranded DNA in vivo, thus providing a robust and highly controllable innovative technology platform for precise spatiotemporal manipulation of DNA structures in vivo.

[0097] It should be noted that in this patent application, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one" does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this patent application, if it refers to performing an action according to an element, it means performing the action at least according to that element, including two cases: performing the action only according to that element, and performing the action according to that element and other elements. Expressions such as "multiple," "repeatedly," and "various" include two, two times, two kinds, and more than two, more than two times, and more than two kinds.

[0098] It should be understood that the numbering used in describing the steps of a method does not inherently limit the order of these steps. For example, a step with a higher number does not necessarily have to be executed after a step with a lower number; it can be executed first and then second, or even in parallel, as long as this execution order is reasonable to those skilled in the art. Similarly, multiple steps with consecutively numbered sequences (e.g., step a, step b, step c, etc.) do not restrict other steps from being executed between them; for example, there can be other steps between step a and step b.

[0099] All documents mentioned in this application are considered to be incorporated in their entirety into the disclosure of this application so that they can serve as a basis for modifications if necessary. Furthermore, it should be understood that after reading the foregoing disclosure of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope of protection claimed in this application.

Claims

1. A method for programmed manipulation of DNA based on dynamic magnetic torque, characterized in that, Includes the following steps: (a) A magnetic micromotor with magnetic anisotropy and a target double-stranded DNA, wherein one end of the target double-stranded DNA is anchored to a substrate; (b) Position the magnetic micromotor at the free end of the target double-stranded DNA and bind the magnetic micromotor to the target double-stranded DNA via chemical coupling to achieve mechanical transfer between the magnetic micromotor and the target double-stranded DNA; (c) Apply a rotating magnetic field to the magnetic micromotor, so that the magnetic micromotor generates dynamic magnetic torque under the drive of the rotating magnetic field; (d) The target double-stranded DNA is subjected to the dynamic magnetic torque to cause the target double-stranded DNA to undergo programmed unwinding.

2. The method as described in claim 1, characterized in that, The frequency of the rotating magnetic field is 0-15 Hz; preferably, the frequency of the rotating magnetic field is 0-5 Hz.

3. The method as described in claim 1, characterized in that, In step (a), anchoring one end of the target double-stranded DNA to the substrate includes: One end of the target double-stranded DNA with the first binding group is brought into contact with the substrate surface coated with the binding molecule; The target double-stranded DNA is anchored to the substrate surface through the specific interaction between the first binding group and the binding molecule.

4. The method as described in claim 1, characterized in that, In step (b), positioning the magnetic micromotor at the free end of the target double-stranded DNA includes: The magnetic micromotor with a first chemical linker group on its surface is added to the substrate on which the target double-stranded DNA is anchored, and the magnetic micromotor diffuses in the solution to approach the free end of the target double-stranded DNA. The second chemical linker group disposed at the free end of the target double-stranded DNA undergoes a click chemical reaction with the first chemical linker group to form a covalent link.

5. The method as described in claim 1, characterized in that, In preparing the conjugate of the target double-stranded DNA and the magnetic micromotor, the concentration of the target double-stranded DNA was 100 nM, and the concentration of the magnetic micromotor was 0-100 µg / mL, measured in Fe.

6. The method as described in claim 1, characterized in that, The substrate is one of the following: glass sheet, quartz sheet, polymer surface, hydrogel, phospholipid bilayer, magnetic microspheres, or microfluidic chip surface.

7. The method as described in claim 1, characterized in that, The method unwinds target double-stranded DNA with different mechanical unwinding thresholds.

8. The method according to any one of claims 1-7, characterized in that, The method is applied as a precise magnetically controlled molecular switch in at least one of DNA computing, molecular logic gates, and information processing systems.

9. The method as described in claim 1, characterized in that, Also includes: (e) Fluorescence detection is performed on the target double-stranded DNA, wherein, The 5' end of the ligand strand is modified with a fluorescence quenching group, and the 3' end of the complementary strand is modified with a fluorescent group. When the ligand strand and the complementary strand of the target double-stranded DNA are complementary, the fluorescence of the fluorescent group is quenched. When the target double-stranded DNA is unwound, the fluorescence of the fluorescent group is restored.

10. A DNA programmed manipulation platform based on dynamic magnetic torque, characterized in that, include: A substrate, the surface of which is configured to anchor one end of the target double-stranded DNA; A magnetically anisotropic magnetic micromotor is configured to connect to the free end of the target double-stranded DNA via chemical coupling, so as to transfer the magnetic torque generated by the magnetically anisotropic magnetic micromotor to the target double-stranded DNA. The rotating magnetic field generating component is configured to generate a rotating magnetic field acting on the magnetic micromotor under preset parameters, so that the magnetic micromotor generates a dynamic magnetic torque under the drive of the rotating magnetic field.

Images