CN113278607A - Preparation method of circular single-stranded DNA integrated by nucleic acid aptamer and application of circular single-stranded DNA integrated by nucleic acid aptamer in DNA paper folding - Google Patents
Preparation method of circular single-stranded DNA integrated by nucleic acid aptamer and application of circular single-stranded DNA integrated by nucleic acid aptamer in DNA paper folding Download PDFInfo
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Abstract
本发明公开了一种核酸适配体整合的环状单链DNA的制备方法及其在DNA折纸术中的应用。属于DNA纳米技术及生物学应用领域，主要步骤包括：(1.1)、构建含有核酸适配体序列的环状双链重组噬菌粒；(1.2)、将构建的环状双链重组噬菌粒转化大肠杆菌感受态细胞；提取具有核酸适配体整合的环状单链DNA。本发明所述的一种核酸适配体整合的环状单链DNA的制备方法可以设计和生产由用户自定义的骨架链序列，以在用户定义的位置添加各种功能序列，并且突破了传统DNA折纸结构功能化的效率和稳定性低的问题。
The invention discloses a preparation method of a nucleic acid aptamer-integrated circular single-stranded DNA and its application in DNA origami. It belongs to the field of DNA nanotechnology and biological applications, and the main steps include: (1.1), constructing a circular double-stranded recombinant phagemid containing nucleic acid aptamer sequences; (1.2), to construct the circular double-stranded recombinant phagemid Transform E. coli competent cells; extract circular single-stranded DNA with aptamer integration. The method for preparing a circular single-stranded DNA integrated with a nucleic acid aptamer according to the present invention can design and produce a user-defined backbone sequence, so as to add various functional sequences at user-defined positions, and break through the traditional The problem of low efficiency and stability in functionalization of DNA origami structures.
Description
Technical Field
The invention belongs to the field of DNA nanotechnology and biological application, and particularly relates to a preparation method of circular single-stranded DNA integrated by a nucleic acid aptamer and application of the circular single-stranded DNA in DNA paper folding; in particular to preparation of single-stranded DNA (ssDNA) functionalized by different aptamers, and the single-stranded DNA is used as a skeleton chain of DNA origami for constructing a functional DNA origami structure.
Background
DNA is used as a natural biological macromolecule, and has the unique advantage of constructing a functional nano structure by using the unique chemical structure and the unique intermolecular interaction; by utilizing the base complementary pairing effect, DNA molecules can be self-assembled to form an ordered structure with nanometer precision; the DNA nanostructure can also modify DNA chains at specific positions, so that the DNA nanostructure can be used as a scaffold for guiding other molecules or nano materials to carry out controllable self-assembly.
Rothemund proposed a brand-new DNA self-assembly strategy of DNA origami in 2006, and advanced the DNA self-assembly field to a new development stage. The method uses a long single-stranded DNA as a skeleton chain and hundreds of short-stranded DNAs with different sequences as staple chains. Through base complementation, the staple chain folds the backbone chain into various designed shapes. In DNA origami, the DNA sequence of all staple strands is different, making the entire nanostructure spatially fully addressable. Compared with the traditional DNA self-assembly mode, the DNA paper folding technology can not only carry out more accurate assembly and arrangement on the nanoscale to obtain more complex and fine programmable patterns and structures, but also has simpler and more convenient experimental operation and higher assembly efficiency. The structure assembled by the DNA origami can be used as a template to guide the arrangement of other nano materials, drug micromolecules, biological macromolecules and other components, so that nano elements, drug carriers, nano robots and the like with controllable performances of optics, electromagnetics and the like can be obtained, and the DNA origami has very wide application value in the nano field.
Traditional DNA origami uses natural long ssDNA (e.g., M13mp18 phage genome) as the backbone strand and completes assembly by complementary pairing with hundreds of short stapled strands. The range of applications of DNA origami structures is limited to some extent by the size and sequence of the assembled nanostructures. For example, to construct functional DNA origami structures, modified sequence motifs are typically incorporated directly into the stapled chain by chemical synthesis or anchored at specific sites of the DNA nanostructure by complementary hybridization to the extended staple chain. However, the yield of functional DNA origami nanostructures is affected by the hybridization efficiency of such stapled chains. In addition, when the length of the modified functional sequence is too long, the efficiency of synthesis and subsequent incorporation will be greatly affected. In addition, such DNA origami nanostructures with functional dangling strands may be subject to degradation by various nucleases when used in biomedical applications. Over the past few years, many attempts have been made to construct variants of the M13 phage single-stranded genome or alternative long framework strands to assemble various DNA origami nanostructures. For example, the generation of long ssDNA backbone strands is well documented in a strategy based on PCR amplification and recombinant phagemid systems. But still lack a preparation method of biologically functionalized circular single-stranded DNA to break through the problems of low ligation efficiency and stability of traditional DNA origami nanostructure functionalization.
Disclosure of Invention
Aiming at the problems, the invention provides a preparation method of aptamer-integrated circular single-stranded DNA, and the circular single-stranded DNA is constructed into a functional DNA origami structure, so that the problems of low connection efficiency and low stability of DNA origami nanostructure functionalization are solved.
The technical scheme of the invention is as follows: a preparation method of circular single-stranded DNA integrated by nucleic acid aptamers comprises the following specific operation steps:
(1.1) constructing a circular double-stranded recombinant phagemid containing a nucleic acid aptamer sequence;
(1.2) transforming the constructed circular double-chain recombinant phagemid into an escherichia coli competent cell; extracting the circular single-stranded DNA with aptamer integration.
Further, in the step (1.1), the specific operation method for constructing the circular double-stranded recombinant phagemid containing the nucleic acid aptamer sequence is as follows:
first, selected aptamer fragments are obtained by using chemical synthesis, and the aptamer fragments are connected to DNA fragments of selected regions in an M13mp18 RF DNA vector in a manner of overlapping PCR;
then, the DNA fragment of the selected region is inserted into an M13mp18 RF DNA vector by means of Gibson assembly to replace the original DNA fragment of the region, and gene sequencing verification is carried out to obtain the recombinant M13mp18 phagemid with the correct inserted nucleic acid aptamer sequence.
Further, in the step (1.2), the constructed circular double-stranded recombinant phagemid is transformed into escherichia coli competent cells as follows:
firstly, a recombinant M13mp18 phagemid which is correctly inserted into a nucleic acid aptamer sequence and verified by sequencing is transformed into an escherichia coli competent cell and is replicated in an escherichia coli body;
then, by means of blue-white spot screening, single white plaques were picked into tubes containing 2 XYT medium and cultured overnight at 37 ℃ with shaking at 220 rpm; after 12 hours, taking 1mL of cultured cells to transfer to a triangular flask containing 100mL of 2 XYT culture medium, and continuing culturing for 6-8 hours at 37 ℃; thus obtaining a culture solution after the Escherichia coli and the bacteriophage are massively proliferated, and putting the culture solution for standby.
Further, in the step (1.2), a specific operation method for extracting the circular single-stranded DNA integrated with the aptamer is as follows:
firstly, transferring a culture solution after the escherichia coli and the bacteriophage are massively proliferated into a centrifuge tube, and centrifuging for 15min at 10,000 rpm; collecting the supernatant culture solution and placing on ice for later use;
then, adding PEG8000 and NaCl with final concentration of 4.0% and 3.0% into the culture solution, and ice-cooling for more than 30min to precipitate phage; continuously centrifuging at 10,000rpm for 30min at 4 deg.C to collect phage;
finally, adding TE buffer solution to resuspend the phage; extracting single-stranded DNA from the phage suspension by phenol chloroform, and precipitating by absolute ethyl alcohol, wherein the precipitate is the circular single-stranded DNA integrated by the extracted aptamer.
Further, a preparation method of the aptamer-integrated circular single-stranded DNA and application of the aptamer-integrated circular single-stranded DNA prepared by the method in DNA origami.
The invention has the beneficial effects that: (1) the method combining chemical synthesis and Gibson assembly can prepare various functionalized DNA chains, the functionalized positions of the DNA chains can be randomly selected, and the sequence is not limited; (2) the recombinant phagemid constructed by the method can be replicated and amplified in escherichia coli cells in large quantity, and the ssDNA can be efficiently prepared after the infection of the helper phage. The method can also realize large-scale preparation by enlarging a culture system of the Escherichia coli cells; (3) the functional DNA origami constructed by the traditional method modifies a functional sequence to a staple chain in a mode that considers that the break near a cross junction influences the stability of a structure, so that the biggest limitation is to avoid the necessary sacrifice of staple chain cross; the invention breaks through the limitation; (4) the invention can use longer skeleton chain to prepare larger DNA origami carrying more functional units, and provides a new way for constructing more complex and larger-scale functional DNA origami structure; (5) the invention provides a new thought for preparing the functionalized circular ssDNA, and simultaneously provides an effective way for enriching the diversity of the skeleton chain of the functionalized DNA origami and provides meaningful exploration for wider application of the functionalized circular ssDNA.
Drawings
FIG. 1 is a schematic diagram of the preparation of aptamer-integrated ssDNA according to the invention;
FIG. 2 is a schematic diagram of agarose gel electrophoresis analysis of the backbone chain incorporating different aptamers according to the invention. (lane M: DNA marker; lane 1: a skeleton chain into which 3 TBA15 are inserted; lane 2: a skeleton chain into which 2 TBA15 and 1 TBA29 are inserted; lane 3: a skeleton chain into which 3 PDGF aptamers are inserted; lane 4: M13mp18 as a control.)
FIG. 3 is a schematic representation of AFM imaging analysis of the functionalized DNA origami structures incorporating 2 TBA15 and 1 TBA29 in combination with thrombin in accordance with the present invention.
FIG. 4 is a schematic diagram of enzyme digestion stability analysis of TBA functionalized origami structures constructed by different integration modes in the invention.
FIG. 5 is a schematic diagram of the flow analysis of MDA-MB-231 cellular uptake of DNA origami structures incorporating different aptamer amounts in accordance with the present invention.
FIG. 6 is a graph showing the effect of DNA origami structures incorporating different aptamer amounts on MDA-MB-231 and MCF-7 cell proliferation in accordance with the present invention.
Detailed Description
In order to more clearly illustrate the technical solution of the present invention, the following detailed description is made with reference to the accompanying drawings:
as shown in the figure; a preparation method of circular single-stranded DNA integrated by nucleic acid aptamers constructs a functional DNA origami structure by utilizing the circular single-stranded DNA integrated by the nucleic acid aptamers, and the specific operation steps are as follows:
(1.1) constructing a circular double-stranded recombinant phagemid containing a nucleic acid aptamer sequence;
(1.2) transforming the constructed circular double-chain recombinant phagemid into an escherichia coli competent cell; extracting the circular single-stranded DNA with aptamer integration.
Further, in the step (1.1), the specific operation method for constructing the circular double-stranded recombinant phagemid containing the nucleic acid aptamer sequence is as follows:
first, selected aptamer fragments are obtained by using chemical synthesis, and the aptamer fragments are connected to DNA fragments of selected regions in an M13mp18 RF DNA vector in a manner of overlapping PCR; the position of the DNA fragment is determined by the design of step (1.1); purifying the product of the overlapping PCR by agarose gel electrophoresis gel cutting recovery to obtain an M13mp18 RF DNA vector fragment containing a nucleic acid aptamer sequence;
then, inserting the DNA fragment of the selected region into an M13mp18 RF DNA vector in a Gibson assembly mode to replace the original DNA fragment of the region, and performing gene sequencing verification to obtain a recombinant M13mp18 phagemid with a correct inserted nucleic acid aptamer sequence; the recombinant phagemids were further transformed into E.coli DH5 α, plasmids were extracted and recombinant phagemids containing the correct assembly sequence were verified by gene sequencing.
Further, in the step (1.2), the constructed circular double-stranded recombinant phagemid is transformed into escherichia coli competent cells as follows:
firstly, a recombinant M13mp18 phagemid which is correctly inserted into a nucleic acid aptamer sequence and verified by sequencing is transformed into an escherichia coli competent cell and is replicated in an escherichia coli body;
then, by means of blue-white spot screening, single white plaques were picked into tubes containing 2 XYT medium and cultured overnight at 37 ℃ with shaking at 220 rpm; after 12 hours, taking 1mL of cultured cells to transfer to a triangular flask containing 100mL of 2 XYT culture medium, and continuing culturing for 6-8 hours at 37 ℃; thus obtaining a culture solution after the Escherichia coli and the bacteriophage are massively proliferated, and putting the culture solution for standby.
Further, in the step (1.2), a specific operation method for extracting the circular single-stranded DNA integrated with the aptamer is as follows:
firstly, transferring a culture solution after the escherichia coli and the bacteriophage are massively proliferated into a centrifuge tube, and centrifuging for 15min at 10,000 rpm; collecting the supernatant culture solution and placing on ice for later use;
then, adding PEG8000 and NaCl with final concentration of 4.0% and 3.0% into the culture solution, and ice-cooling for more than 30min to precipitate phage; continuously centrifuging at 10,000rpm for 30min at 4 deg.C to collect phage;
finally, adding TE buffer solution to resuspend the phage; extracting single-stranded DNA from the phage suspension by phenol chloroform, and precipitating by absolute ethyl alcohol, wherein the precipitate is the circular single-stranded DNA integrated by the extracted aptamer.
Further, a preparation method of the aptamer-integrated circular single-stranded DNA and application of the aptamer-integrated circular single-stranded DNA prepared by the method in DNA origami.
The first embodiment,
Preparation of human α -thrombin aptamers TBA-15 and TBA-29 ssDNA integrated with PDGF aptamer;
the invention is used for preparing ssDNA integrated by human alpha-thrombin aptamer TBA-15 and TBA-29 and PDGF aptamer, and comprises the following steps:
(1) constructing a circular double-stranded recombinant phagemid containing a nucleic acid aptamer sequence;
obtaining human alpha-thrombin aptamer TBA-15, TBA-29 and PDGF aptamer fragments through chemical synthesis; 2 human alpha-thrombin aptamers TBA-15 and TBA-29 and PDGF aptamer fragments (sequences shown in Table 1) were ligated to DNA fragments of selected regions of M13mp18 RF DNA vector by overlap PCR; the products of the overlapping PCR are purified by agarose gel electrophoresis gel cutting recovery, inserted into an M13mp18 RF DNA vector by a Gibson assembly mode and replace the original DNA fragment of the region, and verified by gene sequencing to obtain the recombinant M13mp18 phagemid with the correct inserted nucleic acid aptamer sequence. In this example, 3 different aptamers (3 TBA-15, 2 TBA-15, 1 TBA-29, and 3 PDGF) were inserted in each of 3 different permutations.
(2) Transforming escherichia coli by using the recombinant phagemid to prepare aptamer-integrated circular single-stranded DNA;
transforming the recombinant phagemid which is correctly inserted with the aptamer sequence and verified by sequencing into an escherichia coli competent cell, picking a single white phagemid into a test tube containing a2 XYT culture medium in a blue-white spot screening mode, and performing shake culture at 220rpm at 37 ℃ for overnight; the next day, 1mL of cultured cells were transferred to a triangular flask containing 100mL of 2 XYT medium and cultured at 37 ℃ for 6-8 h; transferring the cultured escherichia coli into a centrifuge tube, and centrifuging for 15min at 10,000 rpm; collecting the supernatant culture solution and placing on ice for later use; adding PEG8000 and NaCl with final concentration of 4.0% and 3.0% into the culture solution, and ice-cooling for more than 30min to precipitate phage; continuously centrifuging at 10,000rpm for 30min at 4 deg.C to collect phage; finally, the phage was resuspended by adding TE buffer (20mM Tris-Cl, 1mM EDTA, pH 8.0); extracting ssDNA from the phage suspension by phenol chloroform, precipitating by absolute ethyl alcohol, and finally re-dissolving the precipitate in TE buffer solution; the preparation process is shown in figure 1; the resulting ssDNA was characterized by agarose gel electrophoresis as shown in FIG. 2.
TABLE 1 nucleic acid aptamers integrated in the backbone chain
Example two, construction of a functional DNA origami structure of the aptamer integration scaffold strand:
the method for constructing the functional DNA origami structure by using the aptamer-integrated ssDNA prepared by the invention comprises the following specific operations:
mixing the circular ssDNA integrated with different aptamers prepared in the first example with the corresponding staple chains respectively according to a molar ratio of 1: 10; the final concentration of ssDNA was in the range of 1-10nM, as required for subsequent experiments; adding a certain amount of 1 XTAE/Mg2+Buffer solution (Mg)2+Concentration of 12.5mM) to 100 μ L, and placing in a PCR instrument after mixing uniformly, and setting a program gradient annealing from 85 ℃ to 25 ℃ for 16 h; after annealing was complete, excess staple chains were removed by centrifugation using a 100kDa ultrafiltration tube or purified by agarose gel electrophoresis gel recovery.
Example three, in vitro thrombin binding experiments integrating TBA aptamer origami structure:
an in vitro thrombin binding experiment was performed using the origami structure of the integrated TBA aptamer prepared in example two, and the binding efficiency with thrombin was analyzed and compared with the conventional origami structure of the stapler chain extension aptamer by the following specific procedures:
mixing a certain amount of thrombin (about 50nM) with the assembled DNA origami nanostructure (about 10nM) integrated with thrombin aptamer sequence, and incubating at room temperature for 1-2h with shaking to combine the thrombin with the DNA origami structure; after the incubation is finished, adding the DNA origami sample combined with the thrombin into a micro-ultrafiltration tube of 100kDa, centrifuging at 4000rpm for 5min, and then discarding the filtered components; continuously adding a certain amount of 1 XTAE/Mg into the ultrafiltration tube2+The buffer was then centrifuged 2 times to removeUnbound thrombin; finally, 1 μ L of sample was taken and imaged under AFM to observe the binding of thrombin; the same operation as described above was performed on the conventional DNA origami structure of the staple chain extension aptamer, and AFM imaging characterization was performed, with the results shown in FIG. 3.
Example four enzymatic stability analysis of the origami structures of different TBA aptamer integration formats:
and (3) integrating the paper folding structure with the TBA aptamer prepared in the second embodiment to perform enzyme digestion stability experiment, analyzing the enzyme digestion stability of the TBA aptamer, and comparing the enzyme digestion stability with the conventional paper folding structure of the staple chain extension aptamer, wherein the specific operations are as follows:
respectively adding 5 mu L of Exonuclease I and 5 mu L of 10 XExonuclease I Buffer into a paper folding structure containing 40 mu L of different thrombin aptamer integration forms for mixing, so that the final concentrations of Exonuclease and DNA paper folding are 1U and 10nM respectively; incubating the mixed system at 37 ℃ for 1h for enzyme digestion; after incubation, the digested DNA origami sample was transferred to a 100kDa micro-ultrafiltrate tube, centrifuged and washed with 1 XTAE/Mg2+Washing 2 times with buffer to remove Exonaclease I; collecting the sample after ultrafiltration, adjusting the concentration, continuing the in-vitro thrombin combination experiment, and finally observing the thrombin combination by AFM imaging; the same operation as described above was performed on the conventional DNA origami structure of the staple chain extension aptamer, and AFM imaging characterization was performed to analyze the stability of the origami structure, and the results are shown in FIG. 4.
Example five cellular uptake analysis of PDGF aptamer integrated DNA origami structures:
MDA-MB-231 cells were cultured in DMEM medium supplemented with 10% FBS and 1% streptomycin mixed solution (Pen-Strep) at 37 ℃ and 5% CO2Culturing under the conditions of (1); after 2-3 serial passages, 1X 105Transferring the inoculation amount of each hole into a 24-hole plate; after further culturing for 24h, the medium was carefully aspirated, and then DMEM complete medium containing DNA origami structures containing different PDGF aptamer integration formats and DNA origami structures assembled by M13 at a final concentration of 10nM was added, respectively, and a blank control group was set; after 2h incubation, the medium was aspirated and the cells were washed with PBSAfter 2 times, the cells were digested with pancreatin, and a single cell suspension of a certain concentration was prepared and analyzed by flow cytometry, and the results are shown in fig. 5.
Example six, effect of PDGF aptamer integration into DNA origami structure on cell proliferation:
transferring the MDA-MB-231 cells cultured in a culture bottle into a 96-well plate according to the inoculation amount of 1 × 104 per well, culturing for 24h, absorbing the culture medium, then respectively adding complete culture media containing DNA origami structures with different PDGF aptamer integration forms and DNA origami structures assembled by M13 and having a final concentration of 10nM, and setting a blank control group; after the cells are cultured for 12h, 24h and 48h respectively, the proliferation condition of the cells is detected by using a CCK8 kit; the specific detection steps refer to the instruction of the kit. Finally, the cell viability of each experimental group was calculated by absorbance values, and the results are shown in fig. 6.
Finally, it should be understood that the embodiments described herein are merely illustrative of the principles of embodiments of the present invention; other variations are possible within the scope of the invention; thus, by way of example, and not limitation, alternative configurations of embodiments of the invention may be considered consistent with the teachings of the present invention; accordingly, the embodiments of the invention are not limited to the embodiments explicitly described and depicted.
Claims (5)
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