A DNA nanostructure scale, its preparation method and application
The fluorescently labeled standards constructed using DNA origami technology solve the accuracy and repeatability problems of fluorescence structure measurement in existing technologies, enabling accurate microscope calibration and SI traceability, and improving the reliability of measurement results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-07-20
- Publication Date
- 2026-03-10
AI Technical Summary
The lack of stable fluorescent labeling standards in current technologies makes it impossible to accurately measure and compare fluorescent structures under different microscopes and methods, resulting in insufficient accuracy and repeatability of measurement results.
DNA nanostructure rulers were constructed using DNA origami technology. Through fluorescent molecule modification and DNA tetrahedral hybridization, rulers traceable to SI units were formed. The length and fluorescence color of the nanoscale rulers were precisely controlled, and measurements were performed using photoelectric combined technology.
It enables accurate calibration of fluorescence microscopes, improves the calibration accuracy and repeatability of microscope instruments, and ensures the traceability of measurement results in the International System of Units (SI).
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of DNA nanotechnology, and relates to a DNA nanostructure ruler and a preparation method and application thereof. BACKGROUND
[0002] The analysis and detection of trace active substances in living organisms are of great significance for obtaining chemical and biological information in the life process, understanding the relationship between biological molecules and their structure and function, and explaining the mechanism of life activities and disease diagnosis. It is also an important research content of chemical biology. With the rapid development of life science, people's observation and research of life phenomena have reached the level of single cell, single molecule and single base of nucleic acid. It is urgent to obtain relevant biochemical information in situ, in vivo and in real time on a more microscopic scale. Many traditional and conventional biological analytical chemistry methods and means are facing great challenges.
[0003] With the development of biology from descriptive qualitative research to quantitative research, such as describing the number of organisms by measuring concentration and molecular abundance, and the kinetic rate of metabolic and developmental processes, the measurement results are closely related to disease, disease probability and prognosis of disease progression. The measurement results will have a profound impact on the treatment of patients, so the accuracy of the measurement results must be guaranteed, which requires that the measurement results should be traced back to the International System of Units (SI).
[0004] The emergence of super-resolution fluorescence microscopy breaks the limit of optical diffraction, enabling scientists to explore the biological world with an unprecedented perspective. This technology aims to reveal the relationship between biological quantitative information and molecular parameters and structural characteristics. This technology can be divided into three categories: methods based on focal modulation such as STED, methods based on single molecule localization such as PALM / STORM, and statistical super-resolution methods based on the blinking characteristics of fluorescent probes such as SOFI / 3B, etc. With the emergence of various methods, technologies and their changes, there is a need to determine the standard of fluorescent structures, so that people can directly and fairly compare the advantages and disadvantages of various methods and super-resolution technologies by measuring fixed fluorescent structures.
[0005] In summary, there is a need for stable fluorescent marker standards with strict stoichiometric control of the number of fluorescent dye molecules and stability, so that they can exist stably and have good portability and repeatability in different modes and different uses, in order to compare the performance differences of different microscopes and different methods when testing the same structure. SUMMARY
[0006] In view of the deficiencies of the prior art and actual needs, the present application provides a DNA nanostructure ruler and a preparation method and application thereof, the present application utilizes DNA origami technology, can precisely and controllably assemble various DNA nanostructure patterns, since each main chain has a known position in the nanostructure, so the position of the inserted modification is also fixed, so that the DNA nanostructure becomes a very suitable fluorescence microscope standard sample, and a DNA nanostructure ruler traceable to SI unit is constructed.
[0007] In order to achieve the above object, the present application adopts the following technical scheme:
[0008] In a first aspect, the present application provides a DNA nanostructure ruler, the DNA nanostructure ruler comprising a DNA origami structure; the DNA origami structure is modified with a fluorescent molecule, and / or the DNA origami structure is hybridized with at least two DNA tetrahedron molecules through the principle of base complementary pairing.
[0009] In the present application, a DNA nanostructure ruler traceable to SI unit is constructed by utilizing DNA origami technology, the ruler is hybridized with staples to form a DNA origami structure through a scaffold chain, and a DNA tetrahedron molecule is hybridized or connected on different regions of the DNA origami structure, for example, a structural schematic diagram is shown in Figure 1 The structure is composed of three regions A, B and C, in the region A of the structure, a DNA sequence is extended on the staple chain, the extended sequence is hybridized with a DNA tetrahedron molecule modified with a fluorescent molecule, the distance between the centers of the two DNA tetrahedron molecules is the length of the DNA nano ruler, the length change of the nano ruler can be precisely controlled by controlling the position of the sequence extended on the staple chain, the ruler can change between 20nm and 100nm, compared with connecting metal nanoparticles (such as quantum dots), the size of the DNA tetrahedron structure is precisely controlled (the length of the tetrahedron edge can be controlled according to the length of the base sequence, and can be varied between 7bp and 37bp), the position of hybridization can be precisely controlled by adjusting the position of the extended sequence, thereby effectively improving the accuracy of the nano ruler. In addition, the fluorescent molecules can be modified on the staple chain in the regions B and C of the triangle to construct nano rulers of different scales and different colors, and the multi-scale and multi-color ruler can more accurately improve the accuracy of the calibration of the super-resolution microscope instrument.
[0010] In the present application, the DNA nanostructure ruler can be characterized by atomic force microscopy (AFM), transmission electron microscopy (TEM), super-resolution microscopy and other means to characterize the morphology of the ruler and the distance of the ruler. The ruler modified by tetrahedral DNA molecules can be effectively measured by super-resolution microscopy and atomic force microscopy through photoelectric combination, and the distance between the two tetrahedrons on the ruler can be accurately obtained, so that the distance of the DNA nano-ruler can be traced back to the international system of units (SI) meter. Both origami and DNA tetrahedron are composed of DNA, and the distance between the double helix of DNA is relatively fixed, so the distance of the ruler can be inferred by theoretical calculation. Comprehensive analysis of the theoretical value and the measured value can obtain more accurate assignment, and the microscope can be calibrated more accurately.
[0011] Preferably, the DNA origami structure comprises a triangular DNA origami structure, a rectangular origami structure or a square origami structure.
[0012] Preferably, the triangular DNA origami structure is formed by hybridization of a scaffold chain and a staple chain according to the principle of base complementary pairing.
[0013] Preferably, the staple chain has an extension sequence.
[0014] Preferably, the DNA tetrahedral molecule is hybridized on the extension sequence of the staple chain according to the principle of base complementary pairing.
[0015] Preferably, the DNA nanostructure ruler further comprises a fixed connection molecule.
[0016] Preferably, the fixed connection molecule comprises biotin.
[0017] Preferably, the side length of the triangular DNA origami structure is 50-200nm, including but not limited to 51nm, 52nm, 55nm, 60nm, 70nm, 80nm, 90nm, 100nm, 120nm, 150nm, 160nm, 170nm, 180nm, 190nm, 192nm, 195nm, 196nm, 198nm or 199nm.
[0018] Preferably, the distance between the DNA tetrahedral molecules is 20-100nm, including but not limited to 21nm, 25nm, 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 91nm, 92nm, 93nm, 95nm, 96nm, 98nm or 99nm.
[0019] In this invention, all fluorescent molecules commonly used for fluorescent labeling in the art are applicable to the present invention and are not subject to any special limitations.
[0020] Preferably, the fluorescent molecule can be selected from any one or at least a combination of two of AlexaFluor647, AlexaFluor488, Cy5, atto488, or atto647N.
[0021] Preferably, the nucleic acid sequence of the scaffold chain includes the sequence shown in SEQ ID NO.1.
[0022] Preferably, the nucleic acid sequence of the staple chain includes the sequences shown in SEQ ID NO.2 to SEQ ID NO.247.
[0023] Preferably, the DNA tetrahedral molecule is modified with a fluorescent molecule.
[0024] Preferably, the nucleic acid sequence of the DNA tetrahedral molecule includes the sequences shown in SEQ ID NO.248 to SEQ ID NO.251.
[0025] In a second aspect, the present invention provides a method for preparing a DNA nanostructure scale as described in the first aspect, characterized in that the preparation method comprises:
[0026] Synthesize fluorescently modified DNA origami structures, and / or,
[0027] A DNA tetrahedral molecule modified with fluorescent molecules was synthesized, and the DNA origami structure was hybridized with the DNA tetrahedral molecule modified with fluorescent molecules to obtain the DNA nanostructure scale.
[0028] In this invention, the fluorescently modified DNA origami structure is formed by hybridization of scaffold chains and staple chains according to the principle of complementary base pairing; the fluorescently modified DNA tetrahedron is formed by hybridization of four DNA single strands according to the principle of complementary base pairing; and the DNA nanostructure ruler is formed by hybridization of the DNA origami structure and the DNA tetrahedron according to the principle of complementary base pairing.
[0029] Thirdly, the present invention provides the application of the DNA nanostructure scale described in the first aspect in the preparation of calibrators for super-resolution fluorescence microscopes, transmission electron microscopes, or atomic force microscopes.
[0030] Fourthly, the present invention provides a super-resolution fluorescence microscope, transmission electron microscope, or atomic force microscope calibrator, wherein the super-resolution fluorescence microscope, transmission electron microscope, or atomic force microscope calibrator includes the DNA nanostructure scale described in the first aspect.
[0031] Fifthly, the present invention provides the application of the DNA nanostructure scale described in the first aspect in super-resolution fluorescence microscopy, transmission electron microscopy or atomic force microscopy imaging.
[0032] Sixthly, the present invention provides a microscope imaging method, the microscope imaging method comprising:
[0033] The scale of the DNA nanostructure described in the first aspect was measured using a microscope.
[0034] Preferably, the microscope includes a super-resolution microscope, a transmission electron microscope, or an atomic force microscope.
[0035] Compared with the prior art, the present invention has the following beneficial effects:
[0036] In this invention, DNA origami technology is used to form a DNA origami structure by hybridizing scaffold chains and staple chains. Different regions on the structure can be modified with fluorescent molecules or hybridized with DNA tetrahedral molecules to construct a DNA nanostructure scale that can be directly traced to SI units through photoelectric technology, which can more accurately improve the accuracy of microscope instrument calibration. Attached Figure Description
[0037] Figure 1 This is a schematic diagram of the DNA nanostructure scale bar of the present invention;
[0038] Figure 2 An atomic force microscope image of the DNA nanostructure scale in Example 2;
[0039] Figure 3 This is a transmission electron microscope image of the DNA nanostructure scale bar in Example 2;
[0040] Figure 4 This is an atomic force microscope image of region A of the DNA nanostructure scale in Example 3;
[0041] Figure 5 This is an atomic force microscope image of gold nanoparticles modified with the scale B region of the DNA nanostructure in Example 4.
[0042] Figure 6 This is a transmission electron microscope image of gold nanoparticles modified with the scale B region of the DNA nanostructure in Example 4.
[0043] Figure 7 This is a super-resolution fluorescence image of the DNA nanostructure scale bar in Example 5;
[0044] Figure 8 This is a super-resolution fluorescence scintillation intensity diagram of the DNA nanostructure scale in Example 5. Detailed Implementation
[0045] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.
[0046] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.
[0047] Example 1
[0048] This embodiment provides a DNA nanostructure ruler, the preparation method of which includes:
[0049] The triangular DNA origami structure is formed by hybridization of scaffold strands (SEQ ID NO.1) and staple strands (SEQ ID NO.2~SEQ ID NO.247) according to the principle of complementary base pairing. The specific experimental procedure is to hybridize the scaffold strands and staple strands at a ratio of 1:10 in 1×TAE (containing 12.5mM Mg). 2+ Mix in the solution. Anneal using a programmed annealing process: hold at 95°C for 15 min, then decrease the temperature by 1°C every min until it reaches 5°C. Finally, concentrate by centrifugation through a 100kD ultrafiltration tube to remove unreacted staple strands, thus forming a triangular DNA origami structure.
[0050] The DNA tetrahedron is formed by hybridization of four DNA single strands (SEQ ID NO.248~SEQ ID NO.251) through the principle of complementary base pairing. The specific experimental procedure is to mix the four DNA single strands in equal proportions in TM (20mM Tris and 50mM Mg). 2 + In a solution, the final concentration of each single strand is 1 μM. Annealing is performed using a programmed process: hold at 95°C for 10 min, then rapidly decrease to 4°C and hold for at least 15 min, until DNA tetrahedra are formed.
[0051] The DNA nanostructure scale is formed by hybridization of triangular DNA origami structures and DNA tetrahedrons through the principle of complementary base pairing. The specific experimental steps are as follows: the triangular DNA origami structures and DNA tetrahedrons are mixed at a ratio of 1:10 and reacted at 25°C for 12 hours. Then, unreacted DNA tetrahedrons are removed by ultracentrifugation, thus forming the triangular DNA origami structure.
[0052] Example 2
[0053] This embodiment characterizes the morphology of the DNA nanostructure ruler prepared in Example 1. The morphology of the triangular ruler prepared in Example 1 was characterized by atomic force microscopy (AFM) and transmission electron microscopy (TEM).
[0054] The AFM characterization process is as follows: A 1 nM triangular origami structure is deposited on a fresh mica sheet for 5 min, and imaged using an AFM (Multimude 8) in peak force QNM mode with an AFM tip (Bruker).
[0055] The TEM characterization process is as follows: A 1 nM triangular origami structure was dropped onto an ultrathin carbon film for 10 min. The sample was then removed with filter paper and stained with 1% uranium acetate for 1 min. The droplet was removed again with filter paper and the carbon film was rinsed three times with MilliQ water. Finally, the carbon film was dried under an infrared lamp at 35 °C for 30 min and imaged by a 120 kV TEM (Talo L120C G2).
[0056] Atomic force microscopy results as follows Figure 2 As shown, the transmission electron microscopy results are as follows: Figure 3 As shown, the results indicate that the triangular DNA origami structure was successfully synthesized with good uniformity and dispersion. Compared to transmission electron microscopy, atomic force microscopy, due to its higher resolution and imaging of the triangular structure in the liquid phase, more completely preserves the fine morphology of the triangular structure.
[0057] Example 3
[0058] This embodiment performs AFM measurements on region A of the DNA nanostructure scale prepared in Example 1, including: imaging the 1 nM triangular scale in AFM liquid phase mode, selecting a small field of view (1 μm) for imaging, which reveals fine DNA tetrahedra on the DNA nanostructure scale, as shown in the results. Figure 4 As shown, on the triangular scale, the two white dots, which are the two DNA tetrahedrons, are clearly visible.
[0059] Example 4
[0060] This embodiment performs AFM and TEM measurements on the B region modified gold nanoparticles of the DNA nanostructure scale prepared in Example 1, including replacing the original tetrahedral positions of the DNA nanostructure scale with 5 nm AuNPs, and performing AFM and TEM characterization respectively. The AFM characterization process involves measuring the 1 nM AuNP-modified triangular scale in liquid phase mode. The TEM characterization process involves dropping the 1 nM AuNP-modified triangular scale onto an ultrathin carbon film for measurement. The results are as follows... Figure 5 and Figure 6As shown, each triangular origami has two AuNPs, and the AuNPs are spaced close together.
[0061] Example 5
[0062] This embodiment describes the super-resolution microscopy measurement of the DNA nanostructure scale prepared in Example 1. A Nikon N-STORM super-resolution microscope was used for the measurement. The parameters were set as follows: a 647 nm laser (6 mW) was used to record the scintillation of fluorescent molecules on the DNA origami, and a 561 nm laser was used to monitor the drift of the fluorescent sphere. The Andor DU-897CCD readout bandwidth was set to 10 MHz, 14 bits, and 3x conversion gain. The EM gain was set to 100. The exposure time was selected as 50–100 ms. A total of 15,000 to 20,000 frames were acquired for further analysis and super-resolution reconstruction. The testing procedure included first washing the reaction dish three times with 500 μL PBS; then incubating the reaction dish with 200 μL of 0.5 mg / mL BSA-biotin solution for 5 min; after incubation, the BSA-biotin solution was removed, and 200 μL of neutralvidin solution was added for incubation for 5 min; finally, excess neutralvidin solution was removed, and 5 μL of filtered DNA nanostructure scale was added to the reaction dish and incubated for 5 min. Use 500 μL of 1×TAE-Mg 2+ After rinsing the chamber three times, 1 μL of orange fluorescent microspheres (Life Technologies) were added. The fluorescent microspheres served as photostability drift markers. Finally, 300 μL of imaging buffer (50 mM Tris, 10 mM NaCl, 10% glucose, 10% catalase / glucose, 100 mM MEA) was added to the reaction dish for imaging measurements. Results are as follows: Figure 7 and Figure 8 As shown, from Figure 7 As can be seen in the field of view, there are many fluorescent dots with two points of equal distance between them. The distance between the two dots is about 50 nm, which is the fluorescent nanoscale bar of this invention. Figure 8 This represents the fluorescence intensity of fluorescent molecules flashing on a fluorescent nanoscale; super-resolution reconstruction can only be performed when the flashing intensity is sufficient.
[0063] In summary, this invention utilizes DNA origami technology to form a triangular DNA origami structure by hybridizing scaffold chains and staple chains. Different regions on this structure can be modified with fluorescent molecules and hybridized DNA tetrahedral molecules, thus constructing a DNA nanostructure scale traceable to SI units, which can more accurately improve the calibration accuracy of microscope instruments.
[0064] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.
Claims
1. A DNA nanostructure ruler, characterized in that, The DNA nanostructure ruler comprises a DNA origami structure and a DNA tetrahedron molecule, the DNA origami structure is a triangular DNA origami structure, the triangular DNA origami structure is composed of three regions A, B and C, and the DNA tetrahedron molecule is modified with a fluorescent molecule; The DNA origami structure is hybridized with at least two DNA tetrahedron molecules by base complementary pairing principle, and the distance between the centers of the two DNA tetrahedron molecules is the length of the DNA nanostructure ruler; The triangular DNA origami structure is formed by hybridization of a scaffold chain and a staple chain by base complementary pairing principle; The staple chain has an extension sequence; In the A region of the triangular DNA origami structure, the DNA tetrahedron molecule is hybridized on the extension sequence of the staple chain by base complementary pairing principle; The DNA origami structure is modified with a fluorescent molecule, specifically, the fluorescent molecule is modified on the staple chain in the B region and the C region of the triangular structure, The side length of the triangular DNA origami structure is 50-200 nm; The distance between the DNA tetrahedron molecules is 20-100 nm.
2. The DNA nanostructure ruler of claim 1, wherein, The DNA nanostructure ruler further comprises a fixed linker molecule.
3. The DNA nanostructure ruler of claim 2, wherein, The fixed linker molecule comprises biotin.
4. The DNA nanostructure ruler of claim 1, wherein, The fluorescent molecule comprises any one or a combination of at least two of Alexa Fluor 647, Alexa Fluor 488, Cy5, atto488 or atto647N.
5. The DNA nanostructure ruler of claim 1, wherein, The nucleic acid sequence of the DNA tetrahedron molecule comprises the sequence shown in SEQ ID NO. 248-SEQ ID NO.
251.
6. The DNA nanostructure ruler of claim 1, wherein, The nucleic acid sequence of the scaffold chain comprises the sequence shown in SEQ ID NO.
1.
7. The DNA nanostructure ruler of claim 1, wherein, The nucleic acid sequence of the staple chain comprises the sequence shown in SEQ ID NO. 2-SEQ ID NO.
247.
8. The method of claim 1-7, wherein, The preparation method comprises: synthesizing a fluorescent molecule modified DNA origami structure, and synthesizing a fluorescent molecule modified DNA tetrahedron molecule, hybridizing the DNA origami structure with the fluorescent molecule modified DNA tetrahedron molecule to obtain the DNA nanostructure ruler.
9. A super-resolution fluorescence microscope, transmission electron microscope or atomic force microscope calibration artifact, characterized in that, The calibration standard comprises the DNA nanostructure ruler according to any one of claims 1-7.
10. Use of the DNA nanostructure ruler according to any one of claims 1-7 in super-resolution fluorescence microscopy, transmission electron microscopy or atomic force microscopy imaging.