A method for functionalizing engineered DNA crystals after crystallization and its application

Abstract

The present invention discloses a method and application for functionalization of engineered DNA crystals after crystallization. The present invention constructs a rigid triangular tensile structural unit, including three different DNA component chains, a total of seven chains, the side chains extending in three double helix directions, and the central chain with three repeating sequences is located in the center of the structural unit. Each double helix structure has two sticky ends with two bases at both ends, which interact with each other to form a very ordered 3D crystal network. T4 DNA ligase is used to connect the sticky ends to obtain stable DNA crystals. The crystal structure of the present invention is stable, the target central chain can be added with complex modifications, and it has universality. The crystal has a wide range of uses in medical disease detection.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a post-crystallization functionalization method and application of an engineered DNA crystal. Background Art

[0002] DNA self-assembly is one of the most common phenomena in nature: two complementary single-stranded DNA molecules spontaneously hybridize to form a double-stranded DNA structure. This hybridization process is driven by a series of non-covalent interactions, such as hydrogen bonds, van der Waals forces, electrostatic forces, and hydrophobic interactions, and strictly adheres to the Watson-Crick base pairing rules. DNA nanostructured self-assembly can leverage the exceptionally powerful programmed assembly capabilities of DNA molecules to delicately construct ordered DNA supramolecular structures.

[0003] The initial goal of studying DNA self-assembly was to artificially design and construct crystal structures, thereby enabling the ordered three-dimensional assembly and controlled crystallization of protein molecules. The first artificially designed three-dimensional DNA crystals were based on sticky end-guided assembly, which produced DNA crystals with macroscopic dimensions and periodic pore structures. While weak inter-module interactions are often required during the growth of DNA crystals, subsequent characterization and applications (particularly the latter) require stronger interactions to enhance crystal stability.

[0004] DNA ligase is a nuclease that catalyzes the formation of phosphodiester bonds between the 3'-OH and 5'-phosphate termini of adjacent DNA fragments, splicing the two DNA segments together to close the gap between them. T4 DNA ligase (T4Lig), a viral genome-encoded DNA ligase, is currently a widely used DNA ligase because it can complete the ligation reaction under normal conditions. It is primarily isolated from deletion-type bacteriophages. The T4Lig ligation reaction mechanism is based on a nicked double-stranded DNA substrate. Leveraging the known ligation properties of T4Lig, there are three specific methods for ligating DNA fragments: ligating the sticky ends of dsDNA, ligating blunt ends, and ligating artificially synthesized sticky ends. Summary of the Invention

[0005] Based on previous research results from the inventors' research group, a triangular tensegrity motif was designed to grow DNA three-dimensional crystals hundreds of microns in size. These crystals possess a substantial three-dimensional structure and can shrink in DMSO. Adding a target central strand can replace the central strand. The triangular tensegrity motif comprises three different DNA strands: a central strand (L), a side strand (M), and a corner strand (S), for a total of seven strands. The side strands (M) extend in three double-helix directions, while the central strand (L), with three repeats, is located in the center of the motif. Each double-helix structure has two-base sticky ends at each end, interacting to form a highly ordered 3D crystalline network.

[0006] The purpose of the present invention is to provide a method and application for functionalizing engineered DNA crystals after crystallization.

[0007] The method for preparing the engineered three-dimensional DNA crystal provided by the present invention comprises the following specific steps:

[0008] (1) The three DNA single strands constituting the triangular tensegrity structural unit were mixed in a specific buffer according to a stoichiometric ratio (the final concentration of the DNA single strands was 4 μM) and gradually cooled (annealed) from 95°C to 4°C to form the corresponding unit module;

[0009] The three DNA single strands are respectively the central strand L strand, the side strand M strand, and the corner strand S strand, and the two end portions of each S strand are complementary paired with one end portion of the two M strands, respectively, and the number of complementary paired bases is 12 or 11 respectively; and (1) the sticky ends of each M strand and S strand must be two bases in length; (2) each M strand is 42 bases in length; and is divided into three segments from the 5' end to the 3' end, and the number of bases in each segment must be 13, 17, and 12; (3) each L strand is a repetition of three segments of structural motif sequences, and is complementary paired with the middle portions of the three M strands at the center of the triangular tensile structural motif, preferably the length of the complementary pairing between the L strand and each M strand is 17 bases; preferably, the 5' end of the M strand is phosphorylated and the 5' end of the S strand is phosphorylated to facilitate the binding of the sticky ends.

[0010] (2) Crystallization was performed by hanging drop vapor diffusion method. Under the conditions of temperature of 30-35°C, crystallization solution volume of 5 μL, crystallization solution salt concentration of 0.5×TAE / Mg(MgAc2), and crystallization pool salt concentration of 2-5×TAE / Mg(MgAc2), crystals with good crystal shape and high resolution were obtained.

[0011] Furthermore, the method further comprises: (3) preparing a T4 DNA ligase reinforcement solution comprising 10×TAE / Mg(MgAc2), 10 mM ATP, T4Lig and water, adding 5 μL of the reinforcement solution to the grown crystal droplets, and allowing the solution to stand overnight to obtain reinforced engineered DNA crystals;

[0012] Optionally, the method further includes: (4) washing the reinforced crystals with 1×TAE / Mg(MgAc2), washing each crystal droplet 8-10 times to wash away ATP, glycerol and other substances contained in the T4 DNA ligase reinforcement solution;

[0013] In step (1) of the present invention, the single-stranded DNA is preferably purified by hPAGE, more preferably by HPLC.

[0014] In step (1) of the present invention, the solvent for dissolving the single-stranded DNA is preferably double-distilled water.

[0015] In step (1) of the present invention, the annealing step is preferably 95°C, 5 min, 65°C, 30 min, 50°C, 30 min, 37°C, 30 min, 22°C, 30 min, 4°C, 30 min.

[0016] In step (1) of the present invention, the concentration of the DNA single strand after initially being dissolved in double-distilled water is preferably 30-50 μM, more preferably 30-35 μM.

[0017] In step (2) of the present invention, the crystallization plate used in the hanging drop vapor diffusion method is a 24-well crystallization plate produced by Hampton.

[0018] In step (2) of the present invention, the salt concentration of the crystallization liquid pool is preferably 4-5×TAE / Mg(MgAc2), and more preferably the salt concentration of the crystallization liquid pool is 5×TAE / Mg(MgAc2).

[0019] In step (3) of the present invention, the concentration of T4 DNA ligase is preferably 400-1000 U / μL, preferably 600 U / μL.

[0020] In step (3) of the present invention, the final ratio of the T4 DNA ligase reinforcement solution is preferably 5.5 μL of 10×TAE / Mg(MgAc2), 1 μL of 10 mM ATP, 1.34 μL of T4Lig, and 2.16 μL of double-distilled water per 10 μL of reinforcement solution.

[0021] The present invention also provides an engineered DNA crystal, which forms a very ordered 3D crystal network based on a triangular tensile structural unit. The triangular tensile structural unit includes three different DNA component chains: a central chain L chain, an edge chain M chain, and a corner chain S chain. The three edge chains M chains extend in three double helix directions, and each direction is connected by a corner chain to form three double helix structures. The central chain L chain with a three-fold repeat sequence is located in the center of the structural unit. Each double helix structure has a sticky end with two bases at both ends. The sticky ends interact with the triangular tensile structural units to form a very ordered 3D crystal network. Specifically, the engineered DNA crystal is a crystal obtained by the preparation method.

[0022] A method for functionalizing the crystal is further provided, which comprises immersing the crystal in DMSO, first absorbing the DMSO, then washing the crystal 8-10 times with 1×TAE / Mg(MgAc2), adding a fluorescently modified central chain to the crystal droplet, mixing evenly by aspiration with a pipette, and letting it stand overnight.

[0023] Preferably, the concentration of the fluorescently modified central chain is 80-120 μM, preferably 100 μM.

[0024] The present invention also provides the use of the functionalized crystals obtained by the functionalization method in the detection of clinical pathological indicators.

[0025] The crystals obtained by the present invention have application potential. For example, in the prior art, such triangular-designed crystals have been used in the biological field as biocatalysts (Li Z, Liu L, Zheng M, Zhao J, Seeman NC, Mao C. Making Engineered 3D DNA Crystals Robust. J Am Chem Soc. 2019; 141(40): 15850-15855.).

[0026] The DNA crystals prepared by the present invention will undergo irreversible shrinkage in the solvent dimethyl sulfoxide (DMSO) instead of dissolving, collapsing and fragmenting. When the central chain with fluorescent modification is added to the crystal droplet overnight, it is found that the crystal shape has changed back to a three-dimensional structure and the crystal at this time has fluorescence, and the central chain of the crystal has been replaced. The present application can make the structure with a complex central chain that is not easy to grow into a crystal grow from a simple three-dimensional crystal to a complex three-dimensional crystal by replacing the central chain. The crystal structure of the present invention is stable, the target central chain can be added with complex modifications, and it has universality. The crystal has a wide range of uses in medical disease detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the design drawing of the triangular tensile structure.

[0028] Figure 2 Characterized on non-denaturing polyacrylamide gels.

[0029] Figure 3 It is a reinforced three-dimensional DNA crystal.

[0030] Figure 4 This is the principle and flow chart of this patent.

[0031] Figure 5(a) shows the characterization of the functionalization of the crystals of the control group with the central chain of L-CY5 after crystallization.

[0032] Figure 5(b) is the characterization of the functionalization of the crystals of the control group with the central chain of L-CY5 after crystallization (partial magnification).

[0033] Figure 5(c) shows a control group crystal with a central chain L (no fluorescence) that was functionalized after crystallization. TL-BF: bright field; FLUO-CY5: CY5 fluorescence channel.

[0034] Figure 6 Thermal stability test results of reinforced DNA three-dimensional crystals DETAILED DESCRIPTION

[0035] The present invention is further described below with reference to specific examples.

[0036] It is not considered that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be considered to fall within the scope of protection of the present invention.

[0037] Example 1: Crystallization method of DNA three-dimensional crystals

[0038] Specific sequence of the relevant DNA single strand:

[0039] L chain: 5'-CGGTATTCACCACGATGCGGTATTCACCACGATGCGGTATTCACCACGATG-3' (SEQ IDNO: 1);

[0040] M chain: 5'-P-GAAAAACACTGCCTGAATACCGCATCGTGGACTGACTCAAAA-3' (5' end of SEQ ID NO: 2 is phosphorylated);

[0041] S chain: 5'-P-TCTTTTGAGTCAGTGGCAGTGTTTT-3' (the 5' end of SEQ ID NO: 3 is phosphorylated);

[0042] L4 chain: 5'-Py-GAGGAAAAAAAAAAATCCTCCGGTATTCACCACGATGCGGTATTCACCACGATGCGGTATTCACCACGATG-Py-3' (the 5' end of SEQ ID NO: 4 was fluorescently modified with pyrenecarboxamide);

[0043] L-CY5 chain (SEQ ID NO: 5): 5'-CY-5-CGGTATTCACCACGATGCGGTATTCACCACGATGCGGTATTCACCACGATG-3' (the 5' end of SEQ ID NO: 5 is fluorescently modified with CY-5).

[0044] The three DNA single strands L+M+S (with the central strand L) that comprise the triangular tensegrity building block were mixed in a specific buffer at a stoichiometric ratio of 1:1:1 (final DNA concentration of 4 μM). The solution was then gradually cooled (annealed) from 95°C to 4°C. Annealing steps included 95°C for 5 minutes, 65°C for 30 minutes, 50°C for 30 minutes, 37°C for 30 minutes, 22°C for 30 minutes, and 4°C for 30 minutes.

[0045] Use a 24-well plate for hanging drop crystallization. Use a 10-μl pipette to draw 5 μl of crystallization solution and drop it on the center of the glass slide. Then, slowly invert the glass slide into the corresponding well of a 24-well plate coated with vaseline to perform hanging drop vapor diffusion crystallization.

[0046] Among them, the hanging drop vapor diffusion method is used for crystallization, which utilizes the different salt concentrations of the pool liquid and the droplets. After sealing, the water in the droplets will enter the pool liquid according to the difference in the evaporation pressure of water, and the DNA concentration in the droplets will slowly increase from unsaturated, to saturated, to supersaturated, and crystallization will occur in this process.

[0047] The following optimization experiments were also performed:

[0048] Temperature: Crystallization was carried out by hanging drop vapor diffusion method at three temperatures: 4°C, 15°C and 30°C. Crystals could not grow at 4°C, the crystal size was smaller at 15°C, and the crystals had a more ideal size at 30°C; TAE / Mg (MgAc2) concentration: The salt concentration of the pool liquid was set at 2×, 3×, 4×, and 5×TAE / Mg (MgAc2), respectively. It was found that when the salt concentration of the pool liquid was 5×TAE / Mg (MgAc2), the crystal size and quantity were optimal.

[0049] Through optimization and adjustment, crystals with good crystal shape and high resolution were finally obtained under the conditions of temperature of 30°C, crystallization solution volume of 5μL, crystallization solution salt concentration of 0.5×TAE / Mg(MgAc2), and crystallization liquid pool salt concentration of 5×TAE / Mg(MgAc2).

[0050] Characterization of triangular tensile structural elements: 8% native-PAGE was used to verify (e.g. Figure 2 As shown in the figure, it can be seen that the L+M+S chain has clear stripes at the corresponding positions.

[0051] Example 2: Method for Strengthening and Cleaning DNA Three-Dimensional Crystals

[0052] After 7-10 days of crystallization, observe crystal formation using a binocular microscope. Add 5 μL of T4 DNA ligase solution to a droplet with healthy crystal growth. To do this: Select a droplet with healthy crystals, remove the slide from the 24-well plate, and use a 10 μL pipette to pipette 5 μL of T4 DNA ligase solution into the droplet. Pipette the solution thoroughly to mix the solution. Then, place the slide upside down on the 24-well plate and let it sit overnight at room temperature. (Be careful to avoid areas with high concentrations of crystals during aspiration to avoid crystal loss.)

[0053] The final ratio of T4 DNA ligase reinforcement solution is preferably 5.5 μL of 10×TAE / Mg(MgAc2), 1 μL of 10 mM ATP, 1.34 μL of T4 DNA ligase, and 2.16 μL of double-distilled water per 10 μL reinforcement solution. Figure 3 ).

[0054] The crystals that have been strengthened are cleaned using 1×TAE / Mg(MgAc2). First, use a 10-μl pipette to remove the strengthening solution, then aspirate 8-10 μl of 1×TAE / Mg(MgAc2) each time to re-wet the crystals. Use the pipette to repeatedly aspirate 3-5 times, then aspirate the waste liquid generated by this wash, and then repeat this step 8-10 times to complete the cleaning of the crystals to wash away ATP, glycerol, and other substances contained in the T4 DNA ligase strengthening solution. The cleaning process is performed under a binocular stereo microscope. It should be noted that the crystals may be sucked away by the pipette during the cleaning process, so care should be taken to minimize crystal loss.

[0055] Example 3: Post-crystallization functionalization of DNA three-dimensional crystals

[0056] There are two types of replacements in the experiment. One is to use three DNA single chains L+M+S to grow into crystals, then add DMSO, the central chain is removed, and then the target central chain is added to restore the triangular tension structure, and the L chain is replaced by L-CY5 (the result of this replacement is that the crystal changes from no fluorescence to red fluorescence). The second is to use three DNA single chains L-CY5+M+S to grow into crystals, then add DMSO, the central chain is removed, and then the target central chain is added to restore the triangular tension structure, and the L-CY5 is replaced by L4 (the result of this replacement is that the crystal changes from red fluorescence to blue fluorescence). The replacement process is as follows Figure 4 .

[0057] Specific steps: After washing with 1×TAE / Mg(MgAc2), use a pipette to soak the crystals with 8 μl of DMSO. Aspirate 3-5 times to remove the DMSO. Repeat this step 3 times. Finally, aspirate 5 μl of DMSO to soak the crystals. Place the slide upside down on a 24-well plate and let it sit at room temperature overnight. Be careful to avoid areas with a lot of crystals during aspiration to avoid crystal loss.

[0058] After the overnight incubation, the DMSO was aspirated, and the crystals were then washed 8-10 times with 1×TAE / Mg(MgAc2). 1 μL of the fluorescently modified central chain (chain L4 or chain L-CY5) was added to the crystal droplet, and the mixture was mixed by pipetting 3-5 times. The mixture was allowed to stand overnight, and the shape, size, and fluorescence of the crystals were observed using a laser confocal microscope. The results are shown in Figure 5. The fluorescence of the crystals changed from non-fluorescence to red fluorescence (result of replacement one), and from red fluorescence to blue fluorescence (result of replacement two).

[0059] In the horizontal column of Figure 5(a), the control group undergoes no further manipulation after the first wash, leaving the central chain intact, thus providing a control for fluorescence changes. L4 represents observation of the crystal after the central chain has been replaced with L4. In the vertical column of Figure 5(a), the central chain of the control group is L-CY5, resulting in red fluorescence in the CY5 channel and no fluorescence in the Py-Py channel. In the L4 group, central chain replacement has successfully occurred, with the L-CY5 chain removed, resulting in no fluorescence in the CY5 channel; the L4 chain has successfully entered the crystal, resulting in blue fluorescence in the Py-Py channel. A merge plot combines the images of the two channels. Fluorescence changes or occurrences are due to the replacement of the central chain with the corresponding fluorescent modification. The layout and meaning of Figures 5(b) and 5(c) are the same as here.

[0060] Therefore, if Figure 5(a) 、 5(b)As shown in Figure 5(c), crystals with a central chain of L-CY5 in the control group (distinct red fluorescence) were successfully functionalized after crystallization, transforming the central chain into L4, resulting in blue fluorescence. As shown in Figure 5(c), crystals with a central chain of L in the control group (non-fluorescent) were successfully functionalized after crystallization, transforming the central chain into L-CY5, resulting in red fluorescence. This design and operation method can transform structures with complex central chains that are difficult to grow into crystals from simple three-dimensional crystals into complex three-dimensional crystals by replacing the central chain.

[0061] The reinforced crystal prepared in the present invention has good thermal stability. The specific experimental method is as follows: the reinforced crystal droplet is aspirated into a centrifuge tube, placed in a PCR instrument and heated at 50°C for 6 hours, after heating is completed, the crystal droplet is again dropped on a glass slide, and the crystal state is observed under a microscope. The experimental results are as follows: Figure 6 It was observed that the reinforced crystals did not dissolve even when heated to 50°C for 6 hours, and the three-dimensional structure of the crystals was well maintained.

[0062] Therefore, the above experimental results show that the crystal structure of the present invention is stable, and the target central chain can be added with complex modifications, such as modification of nucleic acid aptamer sequences, protein sequences, plasmid sequences, etc., so that the crystal has certain functionalization, and the design and operation method are universal, and the crystal has a wide range of uses in medical disease detection.

Claims

1. A method for functionalizing engineered DNA crystals, characterized in that: Soak the engineered crystal in DMSO, then remove the DMSO by aspiration, wash the crystal with 1×TAE / Mg for 8-10 times, then add the functional central chain to the crystal droplet, mix evenly by pipetting, and let it stand overnight; The central chain is an L4 chain or an L-CY5 chain; The nucleotide sequence of the L4 chain is: 5′- GAGGAAAAAAAAAAATCCTCCGGTATTCACCACGATGCGGTATTCACCACGATGCGGTATTCACCACGATG -3′; L-CY5 chain: 5'-CGGTATTCACCACGATGCGGTATTCACCACGATGCGGTATTCACCACGATG-3'; The method for preparing the engineered DNA crystal comprises the following steps: (1) The three DNA single strands constituting the triangular tensile structural unit are mixed in a stoichiometric ratio and gradually cooled from 95°C to 4°C to form the corresponding unit module; the three DNA single strands are the central chain L chain, the side chain M chain, and the corner chain S chain, and the two ends of each S chain are complementary to one end of the two M chains, and the number of complementary bases is 12 or 11 respectively; and (1) the sticky ends of each M chain and S chain are two bases long; (2) each M chain is 42 bases long; and is divided into three segments from the 5' end to the 3' end, and the number of bases in each segment must be 13, 17, and 12; (3) each L chain is a three-segment repeat sequence, and is complementary to the middle part of the three M chains at the center of the triangular tensile structural unit, and the length of the complementary pairing between the L chain and each M chain is 17 bases; Among them, L chain: 5'-CGGTATTCACCACGATGCGGTATTCACCACGATGCGGTATTCACCACGATG-3'; M chain: 5'-GAAAAACACTGCCTGAATACCGCATCGTGGACTGACTCAAAA-3'; S chain: 5'-TCTTTTGAGTCAGTGGCAGTGTTTT-3'; The final concentration of each DNA single strand is 2 to 6 μM; (2) Crystallization was performed using the hanging drop vapor diffusion method to obtain crystals; crystallization conditions: at 30-35°C, the salt concentration of the crystallization solution was 0.4-0.6×TAE / Mg, and the salt concentration of the crystallization liquid pool was 4-6×TAE / Mg; (3) Adding a reinforcement solution to the crystal solution obtained in step (2) and incubating overnight to obtain reinforced engineered DNA crystals; the reinforcement solution comprises 8-12×TAE / Mg, 8-12 mM ATP, 400-1000 U / μL T4 DNA ligase and water.

2. The functionalization method according to claim 1, characterized in that The 5' end of the M chain is phosphorylated; the 5' end of the S chain is phosphorylated.

3. The functionalization method according to claim 1, characterized in that The final concentration of each DNA single strand is 4 μM; the solvent used to dissolve the DNA single strand is double-distilled water; and the DNA single strand is purified by hPAGE or HPLC.

4. The functionalization method according to claim 1, characterized in that The annealing steps are 95°C, 5 min, 65°C, 30 min, 50°C, 30 min, 37°C, 30 min, 22°C, 30 min, and 4°C, 30 min.

5. The functionalization method according to claim 1, characterized in that The crystallization was carried out at 30°C, with a crystallization solution salt concentration of 0.5×TAE / Mg and a crystallization pool salt concentration of 5×TAE / Mg. A crystallization plate was used in the hanging drop vapor diffusion method.

6. The functionalization method according to any one of claims 1 to 5, characterized in that The following reinforcement steps are also included: The reinforcement solution includes 10×TAE / Mg, 10 mM ATP, 600 U / μL T4 DNA ligase and water.

7. The functionalization method according to claim 1, wherein The following steps are also included: (4) Clean the reinforced crystal 8-10 times using 0.8-1.2×TAE / Mg.

8. The functionalization method according to claim 7, characterized in that The 5-end of the L4 chain is fluorescently modified with pyrenecarboxamide, and the 5-end of the L-CY5 chain is fluorescently modified with CY-5.

9. The functionalization method according to claim 7 or 8, characterized in that The concentration of the functional central chain is 80-120 μM.

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