CN111690030A - Single-stranded DNA, G-tetramer and preparation method and application of G-tetramer - Google Patents
Single-stranded DNA, G-tetramer and preparation method and application of G-tetramer Download PDFInfo
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Abstract
The invention discloses a single-stranded DNA, a G-tetramer, a preparation method and application of the G-tetramer. The G-tetramer takes single-stranded DNA as a carrier, cholesterol, carbon dodecalong chain and/or phospholipid molecules are coupled on the single-stranded DNA, and the G-tetramer with a secondary structure is formed through the action of potassium ions. The preparation method comprises the following steps: coupling cholesterol, carbon-dodecalong chain and/or phospholipid molecules on the basic group of the single-stranded DNA in a chemical crosslinking mode to obtain modified single-stranded DNA; the modified single-stranded DNA reacts with potassium ions to form a lipophilic G-tetramer nano structure with a secondary structure. The invention couples lipophilic groups and hydrophobic groups on the basic groups of single-stranded DNA, remarkably enhances the high membrane permeability and hydrophobicity of the channel structure, can be embedded into a phospholipid bilayer and stably exists, carries specific ions or molecules from bottom to top or from top to bottom along concentration gradient, and can be applied to the construction of a bionic nano channel for transporting potassium ions through a membrane.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a single-stranded DNA, a G-tetramer, a preparation method of the G-tetramer and application of the G-tetramer.
Background
The G-tetramer (G-quadruplex) structure is a highly folded stable secondary structure formed by single-stranded DNA rich in guanine G base under the action of specific certain ions or molecules. The G-tetramer structure has a hollow chamber of nanometer order, which allows some specific ions with particle size at micro nanometer level to combine with water molecules, thereby passing through the membrane from bottom to top or from top to bottom.
However, the work of hydrophobic modification of the G-tetramer nano structure to construct a bionic nano channel and control molecular ion transmission on a phospholipid membrane is only reported. Lipophilic cholesterol is modified on the single-stranded DNA rich in G base, and hydrophobic groups such as carbon twelve and the like can enhance the high hydrophobicity and high membrane permeability of the G-tetramer nano-channel structure, namely the opening capability of hydrogen bonds in the channel structure is obviously enhanced. In addition, the lipophilic G-tetramer nano-channel structure is stable at normal temperature, simple to prepare and has excellent selectivity on specific ions or small molecules. Lipophilic G-tetrameric nanostructures are a class of biomimetic nanochannels that are widely and importantly used. The lipophilic G-tetramer nano structure has high membrane permeability and hydrophobicity, so the lipophilic G-tetramer nano structure can be embedded into a phospholipid bilayer and stably exists, carries specific ions or molecules from bottom to top or from top to bottom along a concentration gradient, and can effectively realize the transmission and release of the molecules. The protein channel on the simulated cell membrane regulates the transmembrane transmission of ions, and has considerable application prospect in the fields of clinical treatment, drug delivery, biosensing and the like.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a single-stranded DNA, a G-tetramer and a preparation method and application of the G-tetramer. The single-stranded DNA is used as a carrier of the G-tetramer, so that the biological safety is good; cholesterol, carbon dodecalong chain and/or phospholipid molecules are coupled on the base of the single-stranded DNA in a chemical crosslinking mode, lipophilic cholesterol is modified on the single-stranded DNA rich in G base, hydrophobic groups such as the carbon dodecalong chain can enhance the high hydrophobicity and the high membrane permeability of the G-tetramer nano channel structure, namely the opening capacity of hydrogen bonds in the channel structure is remarkably enhanced. The preparation method is simple, and the method can be applied to the construction of the bionic nanochannel for transporting potassium ions through the membrane.
In order to achieve the aim, the invention provides a single-stranded DNA, and the sequence of the single-stranded DNA is shown as SEQ ID No. 1.
Based on a general technical concept, the present invention provides a G-tetramer having a secondary structure formed by potassium ion action on a single-stranded DNA as a carrier, cholesterol, carbon dodecalong chain and/or phospholipid molecules modified on the single-stranded DNA according to claim 1.
In the G-tetramer, single-stranded DNA is used as a carrier, phospholipid molecules are modified on a phosphate skeleton of the single-stranded DNA, and a G-tetramer with a secondary structure is formed through the action of potassium ions.
The G-tetramer is characterized in that the G-tetramer takes single-stranded DNA as a carrier, and cholesterol is modified at the 3' end of the single-stranded DNA; the carbon dodecalong chain is modified between 5' ends and/or bases of the G-tetramer single-stranded DNA to form a G-tetramer with a secondary structure through the action of potassium ions.
The G-tetramer as described above, further, the cholesterol, carbon dodecalong chain and/or phospholipid molecule is coupled to the single-stranded DNA by a chemical cross-linking reaction; the chemical crosslinking reaction is one or more of chain polymerization reaction, ultraviolet crosslinking method and imide esterification reaction.
Based on a general technical concept, the present invention provides a method for preparing the G-tetramer, comprising the steps of:
s1, modifying cholesterol, carbon-dodecalong chain and/or phospholipid molecules on the single-stranded DNA to obtain modified single-stranded DNA;
s2, allowing the modified single-stranded DNA to react with potassium ions to form a G-tetramer nano-structure with a secondary structure.
In the preparation method, further, the S2 specifically is: centrifuging the modified single-stranded DNA, and dissolving the single-stranded DNA in a solution containing K+In Tris-HCl buffer solution, carrying out thermal denaturation for 10min at the temperature of 90-95 ℃, annealing to slowly reduce the temperature to room temperature, and then incubating for 2-5 h at the temperature of 25-35 ℃ to obtain the lipophilic G-tetramer.
Based on a general technical concept, the invention provides an application of the G-tetramer in constructing a bionic nano channel for transporting potassium ions through a membrane.
The above application, further, the method of the application is:
and incubating the G-tetramer and the liposome at room temperature to enable the G-tetramer to be embedded into the liposome, so that ions are transmitted from outside to inside or from inside to outside.
In the above application, further, the liposome is a phospholipid vesicle or a phospholipid bilayer.
Compared with the prior art, the invention has the advantages that:
(1) the invention provides a single-stranded DNA, which is easy to design, can be synthesized efficiently, has good biological safety, is simple to operate and is convenient to popularize and apply.
(2) The invention provides a G-tetramer, cholesterol, carbon dodecalong chain and/or phospholipid molecules are coupled on a base of single-stranded DNA in a chemical crosslinking mode, lipophilic cholesterol is modified on the single-stranded DNA rich in the G base, hydrophobic groups such as the carbon dodecalong chain can enhance the high hydrophobicity and the high membrane permeability of a G-tetramer nano channel structure, namely the opening capability of hydrogen bonds in the channel structure is obviously enhanced. Therefore, the phospholipid bilayer carrier can be embedded in the phospholipid bilayer and stably exists, and carries specific ions or molecules from bottom to top or from top to bottom along the concentration gradient, so that the transfer and release of the molecules can be effectively realized. The lipophilic G-tetramer nano-channel structure is stable at normal temperature and has excellent selectivity on specific ions or small molecules.
(3) The invention provides a G-tetramer, wherein cholesterol and carbon dodecalong chain are coupled on the base of single-stranded DNA in a chemical crosslinking mode, and the modified single-stranded DNA rich in G base is endowed with strong membrane permeability and membrane hydrophobicity based on strong lipophilicity and hydrophobicity of cholesterol and carbon dodecalong chain, so that the modified single-stranded DNA is easily embedded on a phospholipid membrane and can stably exist.
(4) The invention provides a G-tetramer, and the modified single-stranded DNA forms a highly stable two-dimensional structure G-tetramer with hydrophobic property under the action of potassium ions. The center of the G-tetramer structure is a hollow chamber, the structure is stable, and the G-tetramer structure is a novel transmembrane ion channel based on a DNA tetramer structure and can regulate and control the transmission of ions.
(5) The invention provides a preparation method of a G-tetramer, which is simple and controllable, can intelligently regulate and control configuration reaction conditions of the G-tetramer and the like, and is easy to regulate and control.
(6) The invention provides an application of a G-tetramer in constructing a bionic nano-channel for transporting potassium ions through a membrane, which has a lipophilic G-tetramer bionic nano-channel structure, has the activity of horseradish peroxidase, has a stable structure, can simulate a protein channel on a cell membrane, regulates the transmembrane transmission of ions, and can be embedded into a phospholipid membrane of a bionic cell for transmembrane transmission K from inside to outside or from outside to inside+. Has considerable application prospect in the fields of clinical medicine, drug delivery, biosensing and the like.
Drawings
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.
FIG. 1 shows that a single-stranded DNA having cholesterol modified and carbon dodecalong chain in example 1 of the present invention is present at a certain amount of K+Under the action of the structure, a parallel configuration G-tetramer nano structure graph is formed.
FIG. 2 shows the modified lipophilic single-stranded DNA of example 1 of the present invention at K+Circular dichroism data of G-tetramer formed under the action.
FIG. 3 is a representation of the insertion of G-tetrameric nanochannel structures into the membrane of liposomal phospholipid vesicles in example 2 of the present invention; wherein A is a laser confocal imaging photo of a fluorescence field; b is a bright field laser confocal imaging photo.
Fig. 4 is an experimental result of the application of the bionic potassium ion nanochannel in the embodiment 3 of the present invention to the transmission of potassium ions. The principle of application of the G-tetramer is A. B is HPTS transmission experiment of 0-5 mu M ionophore; c is the EC50 and n value fitted by the Hill function (EC50 refers to the activity at an effective concentration of 50%, and n value refers to the number of DNA single strands required to form a molecular vector).
Detailed Description
The invention is further described below with reference to specific preferred embodiments, without thereby limiting the scope of protection of the invention.
Examples
The materials and equipment used in the following examples are commercially available.
Example 1:
the G-tetramer takes single-stranded DNA rich in guanine G base as a carrier, the 5 'end and the middle of the single-stranded DNA are modified with 3 hydrophobic carbon dodeca long chains, the 3' end is modified with a cholesterol molecule, and the sequence of the single-stranded DNA rich in guanine G base is shown in SEQ ID NO.1, and specifically comprises the following steps:
PS2.M：5’-GTGGGTAGGGCGGGTAGGGTTGG-3’。
the modified single-stranded DNA is:
lipoPS2.M：5’-/SpacerC12/GTGGGT/SpacerC12/AGGGCGGGT/SpacerC12/AGGGTTGGChol-3’。
spacer C12 in the sequence is a carbon twelve long chain; chol is cholesterol.
The single-stranded DNA forms a highly stable two-dimensional structure G-tetramer with hydrophobic property under the action of potassium ions. The G-tetramer structure center is a hollow chamber which can be used as a potassium ion specific recognition channel for controllable ion transmission. The amphiphilic G-tetramer structure is incubated with the liposome phospholipid vesicle, so that transmembrane transmission of ions can be regulated.
A method for preparing the G-tetramer of example 1, comprising the steps of:
(1) the single-stranded DNA is synthesized by a solid-phase phosphite triester method, and in the synthesis process, cholesterol (Chol) and carbon dodecalong chain (C12) are directly coupled on the base of the single-stranded DNA in a chemical crosslinking mode. Wherein, cholesterol is modified at the 3 'end, three C12 are respectively modified at the 5' end and the middle part, and the middle C12 is directly connected between two bases to obtain the lipophilic single-stranded DNA. The ratio of single stranded DNA to cholesterol and carbon twelve long strands was 1: 3 during the modification.
(2) And forming a secondary structure G-tetramer nano-structure by the lipophilic single-stranded DNA under the action of potassium ions. The method comprises the following specific steps: 1OD unopened lipophilic single-stranded DNA was centrifuged at 4000rpm at 4 ℃ for 10min, and then gently removed from the sample and dissolved in 33.4. mu.L of 20mM Tris-HCl buffer (containing 100mM K)+) Performing thermal denaturation at 95 ℃ for 10min, annealing to slowly cool the mixture to room temperature, and then incubating for 3h at 30 ℃; this gave 100. mu.M of lipophilic G-tetramer in parallel configuration.
FIG. 1 shows the modification of cholesterol and a carbon-dodecyl long-chain strand of a single-stranded DNA rich in G base in potassium ion (K)+) Under the action of the nano-structure diagram, a lipophilic G-tetramer is formed. As can be seen from the figure: the single-stranded DNA is amphiphilic after being modified by lipophilic cholesterol and hydrophobic carbon-dodecalong chain, and forms a G-tetramer with a hollow cavity and a parallel configuration under the action of potassium ions with certain concentration.
FIG. 2 shows that the single-stranded DNAs of example 1 of the present invention are each at 100mM K+10mM of K+0mM K+Circular dichroism data of G-tetramer formed under the action. This figure shows that single stranded DNA is at K of 100mM+10mM of K+Under the action of the light, a positive characteristic absorption peak is formed at the position of 265nm, and K is not formed+And the action, no characteristic absorption peak exists at the wavelength of 265 nm. The modified single-stranded DNA is shown to form a G-tetramer under the action of potassium ions and is a G-tetramer in a parallel configuration, and the G-tetramer has concentration gradient dependence on the potassium ions.
Example 2:
the G-tetramer takes single-stranded DNA rich in guanine G base as a carrier, 2 hydrophobic carbon dodeca long chains are modified in the middle of the single-stranded DNA, a cholesterol molecule is modified at the 3 'end, and the FAM fluorescent dye modifies the 5' end of the single-stranded DNA. The sequence of the single-stranded DNA rich in guanine G base is shown as SEQ ID NO.1, and specifically comprises the following steps:
PS2.M：5’-GTGGGTAGGGCGGGTAGGGTTGG-3’。
the modified single-stranded DNA is:
lipoPS2.M:5’-FAMGTGGGT/SpacerC12/AGGGCGGGT/SpacerC12/AGGGTTGG Chol-3’。
spacer C12 in the sequence is a carbon twelve long chain; chol is cholesterol.
A method for preparing a biomimetic potassium ion nanochannel according to embodiment 1, comprising the following steps:
(1) the single-stranded DNA is synthesized by a solid-phase phosphite triester method, and in the synthesis process, FAM dye, cholesterol (Chol) and carbon dodecalong chain (C12) are directly coupled on the base of the single-stranded DNA in a chemical crosslinking mode. Wherein, cholesterol is modified at the 3 'end, two C12 are directly connected between two bases, and FAM dye is modified on the G base at the 5' end to obtain the hydrophobic single-stranded DNA modified with the FAM dye. During modification, the ratio of the concentration of single-stranded DNA to FAM dye, cholesterol and carbon twelve long chains was 1: 2.
(2) And forming a G-tetramer nano structure with a secondary structure by using the hydrophobic single-stranded DNA modified with the FAM dye under the action of potassium ions. The method comprises the following specific steps: 1OD modified FAM dye hydrophobic single-stranded DNA was centrifuged at 4000rpm at 4 ℃ for 10min, and then gently removed and dissolved in 33.4. mu.L of 20mM Tris-HCl buffer (containing 100mM K)+) Annealing at 95 deg.C for 10min, slowly cooling to room temperature, and allowing to stand at 30 deg.C for 3 hr; this gave a lipophilic G-tetramer stock solution in parallel configuration at a concentration of 100. mu.M.
(3) After being uniformly mixed, the phospholipid solution is spin-dried on a rotary evaporator under the conditions of 60 ℃ and 200rpm, so that a thin phospholipid membrane is attached to the wall of the round-bottom flask. 1mL of 10mM Tris-HCl buffer (containing 100mM K)+) The liquid was poured slowly along a certain point of the round bottom flask mouth to allow the liquid to go over the phospholipid membrane on the wall of the flask. Slowly adding the G-tetramer stock solution obtained in the step (2) into a bottle, and placing the bottle in a chamberStanding at room temperature in the dark for 5h to obtain GUV (large vesicle) solution with G-tetramer embedded in phospholipid membrane for use.
FIG. 3 is a representation of the insertion of the G-tetramer structure into the membrane of the liposome vesicle in example 2 of the present invention; wherein (A): fluorescence field (B): bright field confocal laser imaging. The fluorescence on the membrane indicates that the single-stranded DNA containing the FAM fluorescent dye forms G-tetramer and is embedded into the phospholipid membrane.
Example 3:
the application of the G-tetramer in the embodiment 1 of the invention as a bionic potassium ion nano channel to transmit potassium ions comprises the following steps:
(1) LUV vesicles were synthesized using lipid membrane hydration:
1.1, uniformly mixing phospholipid solution containing 30 μ M DPPC and 5.1 μ M cholesterol, and spin-drying on a rotary evaporator at 60 ℃ and 200rpm to attach a thin phospholipid membrane on the wall of the round-bottomed flask.
1.2 after placing the round-bottomed flask containing the phospholipid membrane under vacuum at 0 ℃ for 5 hours, 1mL of 10mM Tris-HClbuffer (containing 0.5mM HPTS and 100mM K) was added+) Slowly injecting liquid over the phospholipid membrane on the bottle wall along a certain branch point of the bottle mouth of the round-bottom flask, suspending and stirring for 1h, performing ultrasonic treatment in water bath for 15min, and repeatedly performing cyclic freeze thawing with liquid nitrogen for 8 times. Rotating at room temperature for 4 hr, and refrigerating at 4 deg.C to obtain LUV (large vesicle) solution containing HPTS.
(2) In turn, 10 EP tubes of 200. mu.L were used to prepare 180. mu.L of each fluorescence reaction system containing 2.5mM LUV (macrovesicle) solution, 0 to 5.0. mu.M of the G-tetramer stock solution of example 1, and 10mM Tris-HCl buffer (containing 0.5mM HPTS and 100mM K)+). After mixing well, the fluorescence change of HPTS was measured on a fluorescence spectrophotometer F-7000.
FIG. 4-A is a mechanism of potassium ion transport on phospholipid membranes by the resulting parallel G-tetramer having lipophilicity.
FIG. 4-B is an HPTS ion selectivity experiment, and the data in the figure shows that the potassium ion transport efficiency of lipophilic G-tetramer is in positive correlation with the concentration of the carrier G-tetramer, namely at a certain G-tetramer concentration, the potassium ion transport speed of the lipophilic G-tetramer is increased along with the increase of the G-tetramer concentration.
FIG. 4-C is a plot of the HPTS experimental data of FIG. 4-B, using a nonlinear fit using origine 9.0 software, using the values of EC50 and n fitted by a Hill function. HPTS: the trisodium salt of 8-hydroxypyrene-1, 3, 6-trisulfonate has weak fluorescence and is a fluorescent dye which is very sensitive to pH. The data from the figure show that: 56% activity with an effective concentration of G-tetramer of up to 50%; the number of DNA single strands required for one molecular vector was 0.93.
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make many possible variations and modifications to the disclosed embodiments, or equivalent modifications, without departing from the spirit and scope of the invention, using the methods and techniques disclosed above. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention.
Sequence listing
<110> university of Hunan
<120> single-stranded DNA, G-tetramer, and preparation method and application of G-tetramer
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>23
<212>DNA
<213> Artificial sequence (artifical sequence)
<220>
<221>misc_feature
<222>(1)..(23)
<223> designed according to the experimental requirements, as a vector.
<400>1
gtgggtaggg cgggtagggt tgg 23
Claims (10)
1. A single-stranded DNA, characterized in that the sequence of the single-stranded DNA is shown in SEQ ID NO. 1.
2. A G-tetramer, wherein the single-stranded DNA of claim 1 is used as a carrier, and cholesterol, carbon dodecalong chain and/or phospholipid molecules are modified on the single-stranded DNA to form a G-tetramer with a secondary structure by potassium ion.
3. The G-tetramer according to claim 2, wherein the G-tetramer is supported by single-stranded DNA, and phospholipid molecules are modified on the phosphate backbone of the single-stranded DNA to form a G-tetramer with a secondary structure by potassium ion.
4. The G-tetramer according to claim 2, wherein the G-tetramer is carried by a single-stranded DNA, and cholesterol is modified at the 3' -end of the single-stranded DNA; the carbon-dodecalong chain modification forms a G-tetramer with a secondary structure between the 5' end and/or the base of the single-stranded DNA through the action of potassium ions.
5. The G-tetramer of any one of claims 2 to 4, wherein the cholesterol, carbon dodecalong chain and/or phospholipid molecules are coupled to the single-stranded DNA by a chemical cross-linking reaction; the chemical crosslinking reaction is one or more of chain polymerization reaction, ultraviolet crosslinking method and imide esterification reaction.
6. A process for preparing the G-tetramer of any one of claims 2 to 5, comprising the steps of:
s1, modifying cholesterol, carbon-dodecalong chain and/or phospholipid molecules on the single-stranded DNA to obtain modified single-stranded DNA;
s2, allowing the modified single-stranded DNA to react with potassium ions to form a G-tetramer nano-structure with a secondary structure.
7. The preparation method according to claim 6, wherein the S2 is specifically: centrifuging the modified single-stranded DNA, and dissolving the single-stranded DNA in a solution containing K+In Tris-HCl buffer solution, carrying out thermal denaturation for 10min at the temperature of 90-95 ℃, annealing to slowly reduce the temperature to room temperature, and then incubating for 2-5 h at the temperature of 25-35 ℃ to obtain the lipophilic G-tetramer.
8. Use of the G-tetramer of any one of claims 2 to 5 for constructing a biomimetic nanochannel for transporting potassium ions across a membrane.
9. The application according to claim 8, wherein the method of application is:
and (3) incubating the G-tetramer and the liposome at room temperature to enable the G-tetramer to be embedded on the liposome, so that ions are transmitted from outside to inside or from inside to outside.
10. The use of claim 9, wherein the liposome is a phospholipid vesicle or a phospholipid bilayer.
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Citations (2)
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| CN110172438A (en) * | 2019-06-11 | 2019-08-27 | 湖南大学 | A kind of functional modification method of cell membrane |
| CN110257053A (en) * | 2019-07-02 | 2019-09-20 | 上海交通大学 | A kind of customized frame nucleic acid nano illuminator of shape and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110172438A (en) * | 2019-06-11 | 2019-08-27 | 湖南大学 | A kind of functional modification method of cell membrane |
| CN110257053A (en) * | 2019-07-02 | 2019-09-20 | 上海交通大学 | A kind of customized frame nucleic acid nano illuminator of shape and its preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| CHUNYING LI，等: "Single-stranded DNA designed lipophilic G-quadruplexes as transmembrane channels for switchable potassium transport", 《CHEMCOMM》 * |
| FRANK W. KOTCH，等: "A Lead-Filled G-Quadruplex: Insight into the G-Quartet’s Selectivity for Pb2+ over K+", 《ORG. LETT.》 * |
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