CN117660441A - Method for modifying biotin at two ends of nucleic acid molecule and method for measuring nucleic acid molecule by single molecule force spectrum - Google Patents
Method for modifying biotin at two ends of nucleic acid molecule and method for measuring nucleic acid molecule by single molecule force spectrum Download PDFInfo
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- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 title claims abstract description 102
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 74
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- 235000020958 biotin Nutrition 0.000 title claims abstract description 51
- 239000011616 biotin Substances 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 45
- 238000001228 spectrum Methods 0.000 title claims abstract description 31
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 19
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- 238000012986 modification Methods 0.000 claims description 53
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 43
- 238000012576 optical tweezer Methods 0.000 claims description 19
- 238000003776 cleavage reaction Methods 0.000 claims description 15
- 230000006287 biotinylation Effects 0.000 claims description 14
- 238000007413 biotinylation Methods 0.000 claims description 14
- 230000007017 scission Effects 0.000 claims description 12
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- 108090000790 Enzymes Proteins 0.000 claims description 8
- 102000004190 Enzymes Human genes 0.000 claims description 8
- CIVGYTYIDWRBQU-UFLZEWODSA-N 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid;pyrrole-2,5-dione Chemical compound O=C1NC(=O)C=C1.N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 CIVGYTYIDWRBQU-UFLZEWODSA-N 0.000 claims description 7
- PBVAJRFEEOIAGW-UHFFFAOYSA-N 3-[bis(2-carboxyethyl)phosphanyl]propanoic acid;hydrochloride Chemical compound Cl.OC(=O)CCP(CCC(O)=O)CCC(O)=O PBVAJRFEEOIAGW-UHFFFAOYSA-N 0.000 claims description 6
- YMXHPSHLTSZXKH-RVBZMBCESA-N (2,5-dioxopyrrolidin-1-yl) 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoate Chemical compound C([C@H]1[C@H]2NC(=O)N[C@H]2CS1)CCCC(=O)ON1C(=O)CCC1=O YMXHPSHLTSZXKH-RVBZMBCESA-N 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 230000002194 synthesizing effect Effects 0.000 claims description 5
- 229930024421 Adenine Natural products 0.000 claims description 4
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 4
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 229960000643 adenine Drugs 0.000 claims description 4
- 125000003277 amino group Chemical group 0.000 claims description 4
- 238000011049 filling Methods 0.000 claims description 3
- 238000001298 force spectroscopy Methods 0.000 claims description 3
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- 229920002521 macromolecule Polymers 0.000 abstract description 2
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- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 18
- 229960005156 digoxin Drugs 0.000 description 10
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- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 description 8
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 description 8
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 description 8
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- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the field of mechanical precision measurement of biological macromolecule high-level structures, and discloses a method for modifying biotin at two ends of a nucleic acid molecule and a method for measuring the nucleic acid molecule by using a single-molecule force spectrum. The invention synthesizes the nucleic acid molecule with one end modified by biotin and the other end sulfydryl or amino modified, one end biotin of the nucleic acid molecule is combined with one streptavidin coated microsphere in advance and then seals the streptavidin locus on the nucleic acid molecule, and the other end is biotinylated, so that the nucleic acid molecule can be finally connected with the other streptavidin coated microsphere on the force spectrum, the connection of the two ends of the nucleic acid molecule to the same streptavidin coated microsphere is avoided, so that the manipulation and measurement of the nucleic acid molecule can be performed by using the force spectrum.
Description
Technical Field
The invention relates to the field of mechanical precision measurement of biological macromolecule high-order structures, in particular to a method for modifying biotin at two ends of a nucleic acid molecule and a method for measuring the nucleic acid molecule by using a single-molecule force spectrum.
Background
Single molecule force spectroscopy (optical tweezers, magnetic tweezers, atomic force microscopy) has become a powerful tool for studying the folding and interactions of biomolecules. In order to connect two microspheres at both ends of a molecule to achieve mechanical manipulation of the molecule, conventional molecular connection methods use streptavidin-coated microspheres and digoxin antibody-coated microspheres to bind labels (biotin and digoxin) at both ends of the molecule, respectively.
However, the binding of antigen to antibodies (e.g., digoxin and anti-digoxin antibodies) is weaker than the binding of biotin to streptavidin. The binding of antibodies to antigens is easily dissociated with a small force, e.g. the lifetime of the binding of commonly used digoxin to anti-digoxin antibodies is only 1s at 25pN (Van Patten WJ, walder R, adhikari A, okoniewski SR, ravicchand R, tinberg CE, baker D, perkins TT.2018.improved free-energy landscape Quantification illustrated with a Computationally designed protein-ligand interaction.Chemphysem 19:19-23), resulting in a lower experimental efficiency. If the two ends of the molecule are directly marked as biotin, the two ends of the molecule are connected with the microsphere coated by the same streptavidin, so that the mechanical control can not be performed.
Disclosure of Invention
In view of the above, it is an object of the present invention to provide a method for biotin modification of both ends of a nucleic acid molecule, such that the method is capable of biotinylating both ends of the nucleic acid molecule while avoiding the attachment of both ends of the nucleic acid molecule to the same streptavidin-coated microsphere;
it is a further object of the present invention to provide a method for measuring nucleic acid molecules using single molecule force spectroscopy based on the above-described biotinylation method.
In order to solve or at least partially solve the above technical problems, the present invention provides a method for solving or at least partially solving the above technical problems, as a first aspect of the present invention, a method for biotin modification of both ends of a nucleic acid molecule, comprising:
step 1, synthesizing a nucleic acid molecule with biotin modification at one end and sulfhydryl or amino modification at the other end;
step 2, incubating the microsphere coated by streptavidin with the nucleic acid molecule synthesized in the step 1 to obtain a microsphere-nucleic acid complex;
step 3, blocking the streptavidin binding site on the microsphere-nucleic acid complex; if the other end of the nucleic acid on the microsphere-nucleic acid complex has sulfhydryl modification, reducing the sulfhydryl after blocking the site;
step 4, biotinylating sulfhydryl or amino at the other end of the microsphere-nucleic acid complex.
Optionally, step 1 includes:
introducing biotin modification at the 5 'end of a forward primer of the amplified nucleic acid molecule, introducing sulfhydryl or amino modification at the 5' end of a reverse primer, and then performing PCR amplification to synthesize a nucleic acid molecule with biotin modification at one end and sulfhydryl or amino modification at the other end; or alternatively
Introducing enzyme cutting site into 5 'end of forward primer of amplified nucleic acid molecule, introducing sulfhydryl or amino modification into 5' end of reverse primer, PCR amplifying, enzyme cutting and biotin-dUTP filling to synthesize nucleic acid molecule with biotin modification at one end and sulfhydryl or amino modification at the other end.
Further alternatively, the cleavage site is a cleavage site having an adenine base at the post-cleavage sticky end.
Optionally, the thiol reduction comprises: the mercapto group is reduced with tris- (2-carboxyethyl) phosphine hydrochloride.
Optionally, the biotinylating the thiol group comprises: the reaction was performed with biotin maleimide to biotinylate it.
Optionally, the biotinylating the amino group comprises: biotinylation is carried out by using biotin-N-hydroxysuccinimide.
As a second aspect of the present invention, there is provided a method of single molecule profiling nucleic acid molecules comprising:
step 1, biotinylation modification is carried out on two ends of a nucleic acid molecule according to the biotinylation method;
step 2, capturing a streptavidin-coated microsphere and a biotinylated nucleic acid molecule according to the step 1 by using two single-molecule force spectrums, and then approaching the two single-molecule force spectrums to form stable connection;
and 3, recording parameters of the nucleic acid molecules in different space structure states by carrying out mechanical operation and measurement on the two single-molecule force spectrums.
Optionally, the single-molecule force spectrum includes optical tweezers, magnetic tweezers, and an atomic force microscope.
Optionally, the spatial structural state includes folding, stretching, rotating, and unfolding.
The invention introduces biotin modification or enzyme cutting site at the 5 'end of the forward primer, introduces sulfhydryl or amino modification at the 5' end of the reverse primer, and then digests nucleic acid molecules containing the enzyme cutting site by restriction enzyme and supplements biotin-dUTP to synthesize nucleic acid molecules with biotin modification at one end and sulfhydryl or amino modification at the other end. The biotin at one end of the nucleic acid molecule is combined with a streptavidin-coated microsphere in advance, then the streptavidin locus on the nucleic acid molecule is blocked, and the other end of the nucleic acid molecule is biotinylated, so that the nucleic acid molecule can be finally connected with another streptavidin-coated microsphere on a force spectrum, the connection of the two ends of the nucleic acid molecule to the same streptavidin-coated microsphere is avoided, so that the manipulation and measurement of the nucleic acid molecule can be performed by using the force spectrum.
Description of the drawings:
FIG. 1 is a schematic diagram showing the flow of the biotin modification at both ends of a nucleic acid molecule and optical tweezers for measuring the nucleic acid molecule according to the present invention;
FIG. 2 is a graph showing the results of a DNA stretching experiment using optical tweezers; wherein, (a) an optical tweezers experimental schematic; (b) 9 groups of single-molecule DNA force spectrum data measured by marking one end of the DNA with digoxin and the other end with biotin; (c) 10 groups of single-molecule DNA force spectrum data measured by marking one end of the DNA with DNP and marking the other end with biotin; (d) The two ends of the DNA are marked by biotin to measure 9 groups of single-molecule DNA force spectrum data; (e) The average value and standard deviation of the single-molecule DNA connection breaking force obtained by the data analysis of (b) (c) (d) are 29+/-16 pN (marked by digoxin at one end), 32.2+/-7.1 pN (marked by DNP at one end) and 55+/-20 pN (marked by biotin at both ends); n.s. indicates no significant difference, x indicates significant difference p <0.01.
FIG. 3 shows a single-molecule force spectrum measurement of a DNA hairpin structure using optical tweezers according to the method of the invention; wherein, (a) an optical tweezers experimental schematic; (b) Stretching a force-extension distance curve (sampling frequency is 15 Hz) obtained by molecular record at a constant optical trap moving speed of 50nm/s, wherein a signal indicated by an arrow represents the opening of a DNA hairpin structure; (c) A force-time curve (data sampling frequency is 78125Hz, and the window width is 100 points of smoothing) near the structure opening force in the (b) graph data;
FIG. 4 shows a single-molecule force spectrum measurement of DNA guanine quadruplex structure using optical tweezers in accordance with the method of the present invention; wherein, (a) an optical tweezers experimental schematic; (b) Stretching a force-extension distance curve (sampling frequency is 15 Hz) obtained by molecular record at a constant optical trap moving speed of 50nm/s, wherein a signal indicated by an arrow represents the opening of a DNA guanine tetrad structure; (c) A force-time curve (data sampling frequency is 78125Hz, and the window width is 100 points of smoothing) near the structure opening force in the (b) graph data; (d) The DNA guanine tetrad structure opens the distribution statistical graph of the force, the data represent 24 single molecule force spectrum curves.
The specific embodiment is as follows:
the invention discloses a method for modifying biotin at two ends of a nucleic acid molecule and a method for measuring the nucleic acid molecule by a single molecule force spectrum, and the technical parameters can be properly improved by a person skilled in the art by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the method of the present invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the method described herein without departing from the spirit and scope of the invention.
Aiming at the defects in the current single-molecule force spectrum molecular connection, the invention aims to realize the single-molecule force spectrum connection of which both ends of the nucleic acid molecule are simultaneously modified by biotin so as to enhance the mechanical stability of the nucleic acid molecule connection, thereby improving the efficiency of the single-molecule force spectrum experiment, and avoiding the complex and high-cost scheme of using a plurality of microspheres with different modifications and the scheme of poor connection stability of antigen and antibody.
In a first aspect of the invention, the biotinylation of both ends of the molecule is carried out stepwise, comprising
Step 1, synthesizing a nucleic acid molecule with biotin modification at one end and sulfhydryl or amino modification at the other end;
step 2, incubating the microsphere coated by streptavidin with the nucleic acid molecule synthesized in the step 1 to obtain a microsphere-nucleic acid complex;
step 3, blocking the streptavidin binding site on the microsphere-nucleic acid complex; if the other end of the nucleic acid on the microsphere-nucleic acid complex has sulfhydryl modification, reducing the sulfhydryl after blocking the site;
step 4, biotinylating sulfhydryl or amino at the other end of the microsphere-nucleic acid complex.
Step 1 may use two methods to biotin-modify one end of a nucleic acid molecule, in certain embodiments of the invention, biotin modification is introduced at the 5' end of a forward primer that amplifies the nucleic acid molecule (typically 2-3 kb), followed by PCR amplification, with biotin modification at one end of the PCR product; in still other embodiments of the invention, a cleavage site is introduced at the 5' end of the forward primer of the amplified nucleic acid molecule, followed by PCR amplification, cleavage with restriction enzymes, and finally biotin-dUTP filling with Klenow fragment, with biotin modification at one end of the PCR product. Wherein the number of biotin modifications is 1 or more.
In certain embodiments of the invention, the cleavage site is a cleavage site comprising an adenine base at the post-cleavage sticky end, such as a Bsu36I cleavage site (CCTNAGG, N is any base).
In certain embodiments of the invention, thiol or amino modifications are made to the other end of the nucleic acid molecule, and reverse primers that introduce thiol or amino groups at the 5' end can be used in PCR synthesis of nucleic acid molecules, wherein the number of thiol or amino modifications is 1 or more. The primer containing the thiol modification should be dissolved and stored in an aqueous dithiothreitol solution to prevent disulfide bond formation between the thiols.
In certain embodiments of the invention, biotin may be used to block the streptavidin binding site on the microsphere-nucleic acid complex; in other embodiments of the invention, 10. Mu.g of the microsphere-nucleic acid complex is mixed with 6. Mu.L of streptavidin-coated microsphere (diameter 1.76 μm, concentration 1% w/v) and site blocked by shaking at room temperature for 20 minutes.
In some embodiments of the invention, to facilitate subsequent biotinylation of the thiol group, when thiol modification is selected at the other end of the nucleic acid molecule, the site is further reduced after closure, and tris- (2-carboxyethyl) phosphine hydrochloride may be used to reduce the thiol group. In other embodiments of the invention, to avoid adsorption of the microsphere-nucleic acid complex to the side wall of the vessel after the blocking site and thiol reduction, the invention optionally adds a buffer solution comprising triton, such as 0.1% (w/v) triton in PBS. Finally, the components which are not bound to the microsphere-nucleic acid complex are washed away by centrifuging to discard the supernatant.
In certain embodiments of the invention, the biotinylating the thiol group comprises: the reaction was biotinylated using biotin maleimide (CAS No. 116919-18-7). The biotinylating the amino group comprises: the reaction was performed with biotin-N-hydroxysuccinimide (CAS No. 35013-72-0) to biotinylated it. The final concentration of biotin maleimide and biotin-N-hydroxysuccinimide, which is carried out by shaking reaction at the time of biotinylation, is selected to be 2mM. Finally, washing off the components which are not combined with the microsphere-nucleic acid complex by a method of centrifuging and discarding the supernatant, and finishing biotin modification at two ends.
In a second aspect of the invention there is also provided a method of single molecule profiling a nucleic acid molecule comprising:
step 1, biotinylation modification is carried out on two ends of a nucleic acid molecule according to the biotinylation method;
step 2, capturing a streptavidin-coated microsphere and a biotinylated nucleic acid molecule according to the step 1 by using two single-molecule force spectrums, and then approaching the two single-molecule force spectrums to form stable connection;
and 3, recording parameters of the nucleic acid molecules in different space structure states by carrying out mechanical operation and measurement on the two single-molecule force spectrums.
In certain embodiments of the invention, the spatial structural state includes folding, stretching, rotating, and unfolding.
In certain embodiments of the invention, the single-molecule force profile includes, but is not limited to, optical tweezers, magnetic tweezers, and atomic force microscopy. Taking optical tweezers as an example, referring to the biotin modification method of the first aspect of the present invention and the flow chart of the single-molecule force spectrum measurement using the optical tweezers of the second aspect of the present invention, a schematic diagram is shown in fig. 1.
In other embodiments of the invention, a double optical tweezer system is used to manipulate a nucleic acid single molecule that has been modified with biotin at both ends. And (3) respectively capturing a microsphere coated with streptavidin and the microsphere-nucleic acid compound by an optical trap formed by the two optical tweezers in a microfluidic system, and then approaching the two optical tweezers to form stable connection, so as to finally mechanically control and measure the nucleic acid single molecules. A common measurement scheme is to stretch the nucleic acid molecules at a constant optical trap movement speed, recording a force-extension distance curve.
In the present embodiments, unless specifically stated otherwise, experimental environments and parameter conditions for each set of tests in the examples remain the same, except for the differences explicitly indicated.
The method for modifying biotin at two ends of a nucleic acid molecule and the method for measuring the nucleic acid molecule by using a single molecule force spectrum are further described below.
Example 1:
(1) Synthesizing a DNA molecule with biotin modification at one end and sulfhydryl modification at the other end.
PCR was performed using the following forward and reverse primers and pBR322 plasmid as a template to synthesize a 2.1-kb DNA fragment.
Forward primer: 5' -TCCTAAGGTAAGACACGACTTATCGCCACTG (SEQ ID No. 1);
reverse primer: 5' -Dithio-TAGGAAGCAGCCCAGTAGTAGGTT (SEQ ID No. 2);
wherein the underlined sequence is Bsu36I cleavage site (other cleavage sites comprising adenine bases at the sticky end of the digested DNA can be used). Dithio represents a dimercapto modification. The product was purified using a PCR purification kit. The above product was digested with Bsu 36I. The product was purified using a PCR purification kit. Finally, extension with Klenow enzyme allows biotin-dUTP to be incorporated into the 3' end of the product. The product was purified using a PCR purification kit, and a DNA concentration of 100-200ng/uL was expected.
(2) Streptavidin-coated microspheres were mixed with the above-described synthetic DNA.
10. Mu.g of the DNA molecule solution was mixed with 6. Mu.L of streptavidin-coated microspheres (from Spherech, diameter 1.76 μm, concentration 1% w/v) and shaken at room temperature for 20 minutes.
(3) Blocking the streptavidin binding site on the microsphere and reducing the sulfhydryl group.
To the above solution, an aqueous biotin solution (final concentration of biotin after mixing: 2 mM) was added, and the mixture was stirred at room temperature for 20 minutes. Then, 50mM tris- (2-carboxyethyl) phosphine hydrochloride was added to the solution, and the mixture was shaken at room temperature for 30 minutes. PBS,0.1% (w/v) of triton solution was added to the centrifuge tube where the product was located, and the fraction not bound to the microspheres was washed away by centrifuging (rotation speed 10000rpm, duration 6 min) to discard the supernatant. The microsphere pellet was resuspended in PBS solution.
(4) Thiol-modified biotinylation.
To the above solution was added biotin maleimide (final concentration 2 mM), and the mixture was shaken at room temperature for 2 hours. PBS,0.1% (w/v) of triton solution was added to the centrifuge tube where the product was located, and the fraction not bound to the microspheres was washed away by centrifuging (rotation speed 10000rpm, duration 6 min) to discard the supernatant. The above operation was repeated 1 time. Finally, the microsphere pellet was resuspended in PBS solution.
(5) Single molecule DNA was manipulated and measured using optical tweezers.
And respectively capturing a streptavidin-coated microsphere and the microsphere-DNA compound by using a laser trap in a microfluidic system, then approaching the two microspheres to form stable connection, and finally carrying out mechanical control and measurement on single-molecule DNA. The molecules were stretched at a constant optical trap movement speed and the force-extension distance curve was recorded.
As a comparison with the present invention, the usual method of literature, i.e.DNA labeled with biotin at only one end and labeled with other (digoxin or DNP) at the other end, was used. The preparation method is mainly distinguished by referring to the method in the embodiment, as follows:
1. instead of using a double thiol-modified reverse primer, a double biotin-modified reverse primer was used, with the primer base sequence unchanged.
2. Klenow extension was performed without biotin-dUTP, while Klenow extension was performed with digoxin-dUTP or DNP-dUTP.
By comparing the two methods of the present invention with biotin labeling at only one end, which is digoxin or DNP at the other end, it can be seen that the resulting DNA molecule-microsphere complex of the present invention is capable of undergoing greater stretching and thus a more comprehensive measurement of the mechanical properties of DNA, such as occurs on a 50-60pN DNA overstretch platform (FIG. 2).
Example 2:
(1) Synthesizing a molecule with a DNA handle with one end biotinylated and the other end mercapto-modified, and a DNA hairpin sequence sandwiched between the two handles.
1.1 PCR was performed using the following forward and reverse primers, using the pBR322 plasmid as template, to synthesize 1.5kb handle A.
Forward primer: 5' -TCCTAAGGTAAGACACGACTTATCGCCACTG (SEQ ID No. 1);
reverse primer: 5' -GAATTCTTGAAGACGAAAGGGCCTC (sequence shown as SEQ ID No. 3);
wherein the underlined sequence is Bsu36I cleavage site. The product was purified using a PCR purification kit. The above product was digested with Bsu 36I. The product was purified using a PCR purification kit. Finally, extension with Klenow enzyme allows biotin-dUTP to be incorporated into the 3' end of the product. The product was purified using a PCR purification kit to obtain biotinylated DNA handle a.
1.2 PCR was performed using the following forward and reverse primers, using the pBR322 plasmid as template, to synthesize a 0.6kb handle B.
Forward primer: 5' -AAGCTTTAATGCGGTAGTTTATCACAGT (sequence shown as SEQ ID No. 4);
reverse primer: 5' -Dithio-TAGGAAGCAGCCCAGTAGTAGGTT (SEQ ID No. 2);
wherein Dithio represents a dimercapto modification. The product was purified using a PCR purification kit.
1.3 full-length DNA was synthesized by PCR using the following forward and reverse primers, and the pBR322 plasmid with the DNA hairpin sequence (5' -GAGTCAACGTACTGATCACGCTGGATCCTATTTTTAGGATCCAGCGTGATCAGTACGTTGACTC, sequence shown in SEQ ID No. 5) inserted between EcoRI and HindIII was cloned as a template.
Forward primer: 5' -TCCTAAGGTAAGACACGACTTATCGCCACTG (SEQ ID No. 1);
reverse primer: 5' -TAGGAAGCAGCCCAGTAGTAGGTT (sequence shown as SEQ ID No. 2);
the product was purified using a PCR purification kit.
1.4 annealing. The above-mentioned handle A, handle B and full-length DNA were mixed in a mass ratio of 1.5:0.6:5 in PCR buffer (without Mg2+), incubated at 95℃for 10 minutes, and then naturally cooled to room temperature for 2 hours.
(2) Streptavidin-coated microspheres were mixed with the above synthesized molecules.
Mu.g of the above molecular solution was mixed with 1. Mu.L of streptavidin-coated microspheres (Spherech, diameter 1.76 μm, concentration 1% w/v) and shaken at room temperature for 20 minutes.
(3) Blocking the streptavidin binding site on the microsphere and reducing the sulfhydryl group.
To the above solution, an aqueous biotin solution (final concentration of biotin after mixing: 2 mM) was added, and the mixture was stirred at room temperature for 20 minutes. Then, 50mM tris- (2-carboxyethyl) phosphine hydrochloride was added to the solution, and the mixture was shaken at room temperature for 30 minutes. PBS,0.1% (w/v) of triton solution was added to the centrifuge tube where the product was located, and the fraction not bound to the microspheres was washed away by centrifuging (rotation speed 10000rpm, duration 6 min) to discard the supernatant. The microsphere pellet was resuspended in PBS solution.
(4) Thiol-modified biotinylation.
To the above solution was added biotin maleimide (final concentration 2 mM), and the mixture was shaken at room temperature for 2 hours. PBS,0.1% (w/v) of triton solution was added to the centrifuge tube where the product was located, and the fraction not bound to the microspheres was washed away by centrifuging (rotation speed 10000rpm, duration 6 min) to discard the supernatant. The above operation was repeated 1 time. Finally, the microsphere pellet was resuspended in PBS solution.
(5) The single-molecule DNA hairpin was manipulated and measured using optical tweezers.
And respectively capturing a streptavidin-coated microsphere and the microsphere-DNA compound by using a laser trap in a microfluidic system, then approaching the two microspheres to form stable connection, and finally carrying out mechanical control and measurement on the single-molecule DNA hairpin. The molecules were stretched at a constant optical trap movement speed and the force-extension distance curve was recorded.
The method completely records the force spectrum data of the DNA hairpin to about 52 pN. The opening force of the DNA hairpin was measured to be about 11 pN. The high frequency force versus time curve clearly shows the jump of the hairpin between the open and folded state (fig. 3).
Example 3:
(1) A molecule containing a human telomere DNA guanine quadruplet sequence (TTAGGGTTAGGGTTAGGGTTAGGGTTA, SEQ ID No. 6) was synthesized with one end biotinylated and the other end thiol modified.
1.1 PCR was performed using the following forward and reverse primers, using the pBR322 plasmid as a template, to synthesize the desired molecule.
Forward primer: 5' -Triple biotin-SEQ ID No.6/idSp/SEQ ID No.7;
reverse primer: 5' -Triple SH-TAGGAAGCAGCCCAGTAGTAGGTT (sequence shown as SEQ ID No. 2);
wherein Triple biotin represents three biotin modifications. Triple SH represents three thiol modifications. The sequence of SEQ ID No.6 is a human telomere DNA guanine tetrad sequence. The idSp/represents a base vacancy, and the idSp is connected with the front and rear sequences in a covalent bond mode. SEQ ID No.7 shows the sequence of GGTAAGACACGACTTATCGCCACTG. The product was purified using a PCR purification kit.
(2) Streptavidin-coated microspheres were mixed with the above synthesized molecules.
Mu.g of the above molecular solution was mixed with 1. Mu.L of streptavidin-coated microspheres (Spherech, diameter 1.76 μm, concentration 1% w/v) and shaken at room temperature for 20 minutes.
(3) Blocking the streptavidin binding site on the microsphere and reducing the sulfhydryl group.
To the above solution, an aqueous biotin solution (final concentration of biotin after mixing: 2 mM) was added, and the mixture was stirred at room temperature for 20 minutes. Then, 50mM tris- (2-carboxyethyl) phosphine hydrochloride was added to the solution, and the mixture was shaken at room temperature for 30 minutes. PBS,0.1% (w/v) of triton solution was added to the centrifuge tube where the product was located, and the fraction not bound to the microspheres was washed away by centrifuging (rotation speed 10000rpm, duration 6 min) to discard the supernatant. The microsphere pellet was resuspended in PBS solution.
(4) Thiol-modified biotinylation.
To the above solution was added biotin maleimide (final concentration 2 mM), and the mixture was shaken at room temperature for 2 hours. PBS,0.1% (w/v) of triton solution was added to the centrifuge tube where the product was located, and the fraction not bound to the microspheres was washed away by centrifuging (rotation speed 10000rpm, duration 6 min) to discard the supernatant. The above operation was repeated 1 time. Finally, the microsphere pellet was resuspended in PBS solution.
(5) Single molecule DNA guanine quadruplexes were manipulated and measured using optical tweezers.
And respectively capturing a streptavidin-coated microsphere and the microsphere-DNA complex in a microfluidic system by using a laser trap, then approaching the two microspheres to form stable connection, and finally carrying out mechanical control and measurement on the single-molecule DNA guanine tetrad. The molecules were stretched at a constant optical trap movement speed and the force-extension distance curve was recorded.
The opening force of the DNA guanine quadruplet was 25.1.+ -. 6.2pN (mean.+ -. Standard deviation) as measured by this method. The high frequency force-time curve clearly shows the opening process of the guanine tetrads (fig. 4).
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (9)
1. A method for biotin modification at both ends of a nucleic acid molecule, comprising:
step 1, synthesizing a nucleic acid molecule with biotin modification at one end and sulfhydryl or amino modification at the other end;
step 2, incubating the microsphere coated by streptavidin with the nucleic acid molecule synthesized in the step 1 to obtain a microsphere-nucleic acid complex;
step 3, blocking the streptavidin binding site on the microsphere-nucleic acid complex; if the other end of the nucleic acid on the microsphere-nucleic acid complex has sulfhydryl modification, reducing the sulfhydryl after blocking the site;
step 4, biotinylating sulfhydryl or amino at the other end of the microsphere-nucleic acid complex.
2. The method of claim 1, wherein step 1 comprises:
introducing biotin modification at the 5 'end of a forward primer of the amplified nucleic acid molecule, introducing sulfhydryl or amino modification at the 5' end of a reverse primer, and then performing PCR amplification to synthesize a nucleic acid molecule with biotin modification at one end and sulfhydryl or amino modification at the other end; or alternatively
Introducing enzyme cutting site into 5 'end of forward primer of amplified nucleic acid molecule, introducing sulfhydryl or amino modification into 5' end of reverse primer, PCR amplifying, enzyme cutting and biotin-dUTP filling to synthesize nucleic acid molecule with biotin modification at one end and sulfhydryl or amino modification at the other end.
3. The method of claim 2, wherein the cleavage site is a cleavage site comprising an adenine base at the post-cleavage sticky end.
4. The method of claim 1, wherein reducing the sulfhydryl group comprises: the mercapto group is reduced with tris- (2-carboxyethyl) phosphine hydrochloride.
5. The method of claim 1, wherein biotinylating a thiol group comprises: the reaction was performed with biotin maleimide to biotinylate it.
6. The method of claim 1, wherein biotinylating the amino group comprises: biotinylation is carried out by using biotin-N-hydroxysuccinimide.
7. A method for measuring nucleic acid molecules using single molecule force spectroscopy, comprising:
step 1, biotinylation modification of both ends of a nucleic acid molecule according to the method of any one of claims 1-6;
step 2, capturing a streptavidin-coated microsphere and a biotinylated nucleic acid molecule according to the step 1 by using two single-molecule force spectrums, and then approaching the two single-molecule force spectrums to form stable connection;
and 3, recording parameters of the nucleic acid molecules in different space structure states by carrying out mechanical operation and measurement on the two single-molecule force spectrums.
8. The method of claim 7, wherein the single-molecule force profile comprises optical tweezers, magnetic tweezers, and atomic force microscope.
9. The method of claim 7, wherein the spatial configuration state comprises folding, stretching, rotating, and unfolding.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5484701A (en) * | 1990-01-26 | 1996-01-16 | E. I. Du Pont De Nemours And Company | Method for sequencing DNA using biotin-strepavidin conjugates to facilitate the purification of primer extension products |
| CN104804114A (en) * | 2015-04-28 | 2015-07-29 | 北京中科紫鑫科技有限责任公司 | Preparation method of streptavidin-modified polystyrene microspheres for nucleic acid amplification |
| CN109852667A (en) * | 2018-12-21 | 2019-06-07 | 南开大学 | Method based on single molecule force spectroscopy detection nucleic acid end structure |
| CN114681619A (en) * | 2020-12-30 | 2022-07-01 | 上海交通大学医学院附属仁济医院 | Aptamer compound based on Biotin-SA and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5484701A (en) * | 1990-01-26 | 1996-01-16 | E. I. Du Pont De Nemours And Company | Method for sequencing DNA using biotin-strepavidin conjugates to facilitate the purification of primer extension products |
| CN104804114A (en) * | 2015-04-28 | 2015-07-29 | 北京中科紫鑫科技有限责任公司 | Preparation method of streptavidin-modified polystyrene microspheres for nucleic acid amplification |
| CN109852667A (en) * | 2018-12-21 | 2019-06-07 | 南开大学 | Method based on single molecule force spectroscopy detection nucleic acid end structure |
| CN114681619A (en) * | 2020-12-30 | 2022-07-01 | 上海交通大学医学院附属仁济医院 | Aptamer compound based on Biotin-SA and application thereof |
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