CN114392357A - Cell membrane anchored nucleic acid medicine, preparation method and application thereof - Google Patents
Cell membrane anchored nucleic acid medicine, preparation method and application thereof Download PDFInfo
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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Abstract
The invention discloses a nucleic acid medicament anchored by cell membranes, a preparation method and application thereof. The nucleic acid drug comprises a first anchor nucleic acid sequence and a regulatory nucleic acid sequence; the regulatory nucleic acid sequence comprises an aptamer sequence and a second anchor nucleic acid sequence; the 5' end of the aptamer sequence is connected with a second anchoring nucleic acid sequence, the second anchoring nucleic acid sequence and the first anchoring nucleic acid sequence are completely complementary and hybridized to form a double-stranded structural cell membrane anchoring sequence, and the cell membrane anchoring sequence modifies one or two cell membrane anchoring groups. The preparation method of the aptamer comprises the following steps: s1, synthesizing a first anchoring nucleic acid sequence; s2, synthesizing a regulating nucleic acid sequence; s3, mixing the two nucleic acid sequences according to the molar concentration of 1:1, heating at 95 ℃ for 5 minutes, and cooling. The nucleic acid medicament can improve the combination stability of the nucleic acid aptamer medicament and target receptor protein, prolong the action time on cell membranes and improve the regulation and control effect of the nucleic acid aptamer medicament on the cell membrane receptor protein.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a nucleic acid drug anchored by a cell membrane, a preparation method and application thereof.
Background
The receptor protein on the surface of the cell membrane is an important signal protein for maintaining the normal physiological function of the cell. Abnormal expression or dysfunction of these receptor proteins is closely associated with many disease processes, including cancer, diabetes and neurodegenerative diseases. In recent years, many studies have been conducted to inhibit protein activity by using cell membrane receptor proteins as drug targets and developing various monoclonal antibodies, small molecule inhibitors, and the like. However, further applications are limited due to the time-consuming and labor-intensive and expensive production of these formulations, the difficult quality control and the susceptibility to immune reactions. In recent years, due to the advantages of easy synthesis and modification, controllable quality, easy storage and low immunogenicity, aptamer drugs are gradually exposed in research of nucleic acid drugs with disease treatment functions. Some specific aptamers can be directly used as antitumor drugs and show application potential of inhibiting the activity of target proteins by preventing the interaction of the target proteins and other signal proteins, such as the binding of receptors and ligands. Although aptamer drugs can play a therapeutic role, there are some limitations. In a first aspect, aptamer drugs typically use self-steric hindrance upon target binding to inhibit the pro-receptor-ligand interaction-mediated protein activation process. In a complex cellular microenvironment, free aptamer drugs are subject to dissociation after binding to target molecules due to their small size, resulting in the need for large doses of aptamer drugs to perform inhibitory functions. In the second aspect, due to the endocytosis of cells, the aptamer drug taking the cell membrane receptor protein as the target cannot continuously play an inhibition function after being endocytosed by tumor cells, so that the inhibition time of the aptamer drug is short. Therefore, the stability of the combination of the nucleic acid aptamer medicaments is improved, the residence time of the nucleic acid aptamer medicaments on cell membranes is prolonged, the inhibition effect of the nucleic acid aptamer medicaments can be improved, and the application prospect of the nucleic acid aptamer medicaments in preparing the nucleic acid medicaments for treating diseases is wider.
In order to solve the above technical problems, there is a need to develop a stable and efficient cell membrane anchored nucleic acid drug, a preparation method and applications thereof.
The invention content is as follows:
the invention aims to provide a cell membrane anchored nucleic acid medicament, a preparation method and application thereof, and aims to solve the technical problems of the invention, including but not limited to any one of the following technical problems: in a first aspect, how to improve the binding stability of aptamer drugs to target receptor proteins; in the second aspect, how to realize the high-efficiency inhibition of the activity of cell membrane receptor protein by the aptamer drug and reduce the dosage of the aptamer drug; in a third aspect, the long-acting inhibition of the activity of a cell membrane receptor protein of an aptamer drug is realized.
In order to solve any one or more of the technical problems, the invention adopts the following technical scheme:
providing a cell membrane anchored nucleic acid drug formed by assembling a first anchoring nucleic acid sequence and a regulatory nucleic acid sequence comprising two functional sequences: an aptamer sequence and a second anchor nucleic acid sequence; the second anchoring nucleic acid sequence is connected to the 5' end of the nucleic acid aptamer sequence, the nucleic acid aptamer sequence specifically targets a cell membrane receptor protein, the nucleic acid aptamer sequence is shown in SEQ ID NO.1, the second anchoring nucleic acid sequence and the first anchoring nucleic acid sequence are in complete complementary hybridization to form a cell membrane anchoring sequence with a double-chain structure, and one or two cell membrane anchoring groups are modified on the cell membrane anchoring sequence. In this embodiment, a cell membrane anchoring group is modified in the second anchor nucleic acid sequence or the first anchor nucleic acid sequence at the end or other position; two cell membrane anchoring groups are modified at the ends or other positions of the second anchor nucleic acid sequence and the first anchor nucleic acid sequence, respectively.
On the basis of the above scheme, in another improved technical scheme, 1-5 random base sequences are arranged between the nucleic acid aptamer sequence and the second anchoring nucleic acid sequence. By arranging 1-5 random base sequences between the nucleic acid aptamer sequence and other sequences, the aim is to separate two functional sequences, maintain the configuration of the nucleic acid aptamer sequence and not influence the function of the nucleic acid aptamer sequence.
On the basis of the above scheme, in another improved technical scheme, the cell membrane anchoring group is one of hydrophobic molecules, and the hydrophobic molecules comprise cholesterol molecules, tocopherol molecules and diacyl liposome.
On the basis of the above-mentioned scheme, in another improved technical scheme, the first anchor nucleic acid sequence and the second anchor nucleic acid sequence complementary hybridization form a length of 18-24bp double-stranded structure.
In a further development of the invention, both of the cell membrane anchoring groups are modified in the middle between the first and the second anchoring nucleic acid sequence; the intermediate position is: the first anchor nucleic acid sequence and the second anchor nucleic acid sequence is n bases, when n is an even number, two of the cell membrane anchoring groups are modified between the (n/2) th and (n/2) +1 th bases of the first anchor nucleic acid sequence (orientation 5 '-3') and the second anchor nucleic acid sequence (orientation 3 '-5'), respectively; when n is an odd number, two of the cell membrane anchoring groups are modified between the (n/2) -0.5 and (n/2) +0.5 bases of the first (5 '-3') and second (3 '-5') anchoring nucleic acid sequences, respectively. This arrangement can further improve the stability of the cell membrane anchoring sequence anchored to the cell membrane.
On the basis of the scheme, in another improved technical scheme, the cell membrane receptor protein is a mesenchymal epidermal transformation factor (c-Met receptor protein) which can be activated through receptor dimerization mediated by a Hepatocyte Growth Factor (HGF) serving as a ligand of the mesenchymal epidermal transformation factor.
On the basis of the above scheme, in another improved technical scheme, the binding site of the aptamer and the cell membrane receptor protein and the binding site of the ligand and the cell membrane receptor protein are partially overlapped or identical.
The invention also provides a preparation method of the cell membrane anchored nucleic acid drug, which comprises the following steps,
s1, synthesizing a first anchoring nucleic acid sequence;
s2, synthesizing a regulating nucleic acid sequence;
s3, mixing the two nucleic acid sequences in the steps S1 to S2 according to the molar concentration of 1:1, heating at 95 ℃ for 5 minutes after mixing, annealing and mutually hybridizing, and slowly cooling to room temperature.
The invention also provides application of the cell membrane anchored nucleic acid medicament in nucleic acid aptamer medicaments.
The invention also provides application of the cell membrane anchored nucleic acid drug in research on regulation of c-Met receptor protein activity and correlation of cell functions.
The technical scheme of the invention at least has the following beneficial effects: the invention provides a nucleic acid medicament anchored by cell membranes, a preparation method and application thereof. The nucleic acid medicament anchored by the cell membrane can obviously improve the stability of the combination of the nucleic acid aptamer medicament and the target protein. The nucleic acid drug anchored by the cell membrane can be applied to the regulation of the activity of the cell membrane receptor protein and the cell function.
(1) Some nucleic acid aptamers can be directly used as anti-tumor drugs, and the interaction between target proteins and other signal proteins is prevented by utilizing the self-steric hindrance after the binding of targets, such as the binding of receptors and ligands, so that the application potential of inhibiting the activity of the target proteins is shown. However, in a complex cellular microenvironment, free aptamers are likely to dissociate after binding to a target molecule due to their small size, and thus a large dose of aptamer is usually required to exert inhibitory function. Compared with free aptamer, the nucleic acid medicament enriches more aptamer to the surface of a cell membrane through the cell membrane anchoring sequence with a double-chain structure, and simultaneously increases the binding stability, so that the c-Met receptor protein activity can be obviously inhibited at lower concentration, and the inhibiting effect of the nucleic acid aptamer medicament is enhanced.
(2) Due to the endocytosis of cells, the aptamer drug taking the cell membrane receptor protein as a target cannot continuously play an inhibition function after being endocytosed by tumor cells, so that the inhibition time of the aptamer drug is short. The nucleic acid medicament of the invention utilizes the cell membrane anchoring sequence to slow down the endocytosis process of the aptamer sequence, and aims at the targets on the cell membrane such as c-Met receptor protein, thereby prolonging the inhibiting time. That is, the invention can effectively prolong the action time of the aptamer on the surface of the cell membrane and reduce the drug effect loss caused by endocytosis of the cell. Therefore, the nucleic acid medicament anchored by the cell membrane provides a new platform for developing high-efficiency nucleic acid medicaments, and further expands the application prospect of the nucleic acid medicaments in biomedicine.
Drawings
FIG. 1 is a schematic representation of the enhanced inhibition of target receptor protein activity by cell membrane anchored nucleic acid drugs of examples 1 and 2 of the present invention;
FIG. 2 is confocal images of HeLa cells treated with different cell membrane anchoring sequences in a medium containing 10% FBS in examples 1 and 2 of the present invention (A) - (E), (F) quantitative analysis of fluorescence intensity of each group of cells; a scale: 20 μm;
FIG. 3 is a gel electrophoresis image of the assembly of the first anchor nucleic acid sequence and the regulatory nucleic acid sequence in examples 1 and 2 of the present invention;
FIG. 4 is a confocal image of 200nM 2CH-ab: b or 2CH-ad: d cells incubated with HeLa cells for 10 min at room temperature in examples 1 and 2 of the present invention; a scale: 20 μm;
FIG. 5 is a confocal image showing the anchoring orientation of 2CH-ab b on the cell membrane in examples 1 and 2 of the present invention; a scale: 20 μm;
FIG. 6 is a confocal image comparing the binding stability of 2CH-ab b and free a to the c-Met receptor protein in example 1 and example 2 of the present invention. A scale: 20 μm;
FIG. 7 shows the effect of immunoblot analysis on the activity of c-Met receptor protein in comparison 2CH-ab: b and ab: b in example 1 and comparative example 1;
FIG. 8 shows the effect of immunoblotting analysis of free a on the activity of c-Met receptor protein in comparative example 1 and comparative example 2;
FIG. 9 immunoblot analysis comparing the modulating effects of 2CH-ad: d and ad: d on the activity of c-Met receptor protein in example 2 of the present invention and comparative example 2;
FIG. 10 is a graph of the migration ability of DU145 cells analyzed in the cell scratch test in examples 1 and 2 of the present invention under different conditions, on a scale: 100 μm;
FIG. 11 shows the effect of immunoblot analysis comparing 2CH-ab: b and ab: b on the regulation of c-Met receptor protein activity after 24 hours incubation with DU145 cells in examples 1 and 2 of the present invention.
Detailed Description
Hereinafter, specific examples of the present invention will be described in more detail using examples and comparative examples, and the technical scope of the present invention is not limited to the following examples.
Referring to the schematic of FIG. 1, the cell membrane anchored nucleic acid drug of the present invention is formed by assembling a first anchoring nucleic acid sequence and a regulatory nucleic acid sequence, which includes two functional sequences: an aptamer sequence and a second anchor nucleic acid sequence; the aptamer targets a cell membrane receptor protein, the sequence of the aptamer is shown in SEQ ID NO.1, the second anchoring nucleic acid sequence and the first anchoring nucleic acid sequence are fully complementary and hybridized to form a cell membrane anchoring sequence with a double-chain structure, and cell membrane anchoring groups are modified on the second anchoring nucleic acid sequence and the first anchoring nucleic acid sequence.
In this example, 1 random base sequence is placed between the aptamer sequence and the second anchor nucleic acid sequence. This does not affect the ligation of the aptamer sequence to the second anchor nucleic acid sequence, while separating the two and not affecting the conformation of the aptamer sequence. In other embodiments, an integer number of 5 or less random base sequences may be provided. However, the number of the random base sequences in the scheme is not suitable to be too large, the length of the aptamer sequence is 50 bases, the random base sequences are used for ensuring that the configuration of the aptamer sequence is not influenced, if too many random base sequences are arranged, DNA is wasted on one hand, and on the other hand, the configuration of the aptamer sequence can be influenced by overlong random base sequences.
On the basis of the above embodiment, in another improved technical embodiment, the cell membrane anchoring group is one of hydrophobic molecules, the hydrophobic molecule is a cholesterol molecule, the cholesterol molecule is modified on the phosphate group of the DNA skeleton, and an ester bond is formed by the reaction of the hydroxyl group and the phosphate group after deprotection on the cholesterol molecule. In other embodiments, the hydrophobic molecule is a tocopherol molecule or a diacyl liposome. Because the cholesterol molecule, the tocopherol molecule and the diacyl liposome are all common cell membrane anchoring groups and can be modified in the DNA sequence, the three molecules can anchor the DNA sequence on the cell membrane. In the experimental part of the present specification, cholesterol molecules are used as cell membrane anchoring groups, and it is known from the common knowledge in the art that the corresponding experimental results can be achieved by using tocopherol molecules or diacyl liposomes instead of cholesterol molecules.
Based on the above example, in another improved technical example, the first anchor nucleic acid sequence and the second anchor nucleic acid sequence are complementary hybridized to form a double-stranded structure with a length of 18-24 bp. The second anchor nucleic acid sequence and the first anchor nucleic acid sequence are completely complementary and hybridized to form a double-stranded structure of the cell membrane anchor sequence, if the length of the cell membrane anchor sequence is too long, the difficulty of modifying the hydrophobic molecule is increased, DNA raw materials are wasted, and the cost is increased.
In a further improved technical embodiment, on the basis of the above embodiment, the cell membrane anchoring groups are each modified in a position intermediate to the first anchoring nucleic acid sequence and the second anchoring nucleic acid sequence; the intermediate position is: the first anchor nucleic acid sequence and the second anchor nucleic acid sequence is n bases, when n is an even number, two cell membrane anchoring groups are respectively modified between the (n/2) th and (n/2) +1 th bases of the first anchor nucleic acid sequence (direction is 5 '-3') and the second anchor nucleic acid sequence (direction is 3 '-5'); when n is an odd number, the two cell membrane anchoring groups are modified between the (n/2) -0.5 and (n/2) +0.5 bases of the first anchor nucleic acid sequence (oriented 5 '-3') and the second anchor nucleic acid sequence (oriented 3 '-5'), respectively. This arrangement can further improve the stability of the cell membrane anchoring sequence anchored to the cell membrane.
In a further improved technical embodiment, based on the above embodiment, the cell membrane receptor protein is a mesenchymal epidermal transformation factor (c-Met receptor protein) which is activated by receptor dimerization mediated by its ligand Hepatocyte Growth Factor (HGF). The aptamer sequence of this example forms a competitive relationship with the c-Met receptor protein at the position where it binds to the c-Met receptor protein, partially overlapping the position where ligand HGF binds to the c-Met receptor protein. That is, after the aptamer sequence binds to the c-Met receptor protein first, HGF is not easily bound to the c-Met protein. After the free aptamer sequence is combined with the c-Met protein, dissociation and endocytosis of cells are easy to occur, so that partial HGF can be combined with the c-Met receptor protein to activate the c-Met receptor protein. After the cell membrane anchoring sequence with the double-chain structure is arranged on the nucleic acid aptamer sequence, more nucleic acid aptamers are enriched on the surface of the cell membrane, the anchoring effect increases the combination stability, and the inhibition effect of the nucleic acid aptamer sequence becomes better. Therefore, the activity of the c-Met receptor protein can be obviously inhibited at a lower concentration, and the inhibiting effect of the aptamer drug is enhanced; meanwhile, the process that the aptamer sequence is endocytosed by cells is slowed down by utilizing the cell membrane anchoring sequence, the action time of the aptamer sequence on the surface of the cell membrane is effectively prolonged aiming at targets on the cell membrane such as c-Met receptor protein, and the drug effect loss caused by endocytosis of the cells is reduced.
In a further improved technical embodiment, based on the above embodiments, the binding site of the aptamer to the cell membrane receptor protein and the binding site of the ligand to the cell membrane receptor protein are partially overlapped or identical.
The invention also provides a preparation method of the nucleic acid medicament, which comprises the following steps,
s1, synthesizing a first anchoring nucleic acid sequence;
s2, synthesizing a regulating nucleic acid sequence;
s3, mixing the two DNA sequences in the steps S1 to S2 according to the molar concentration of 1:1, heating at 95 ℃ for 5 minutes after mixing, annealing and mutually hybridizing, and slowly cooling to room temperature.
The invention also provides application of the nucleic acid medicament in nucleic acid aptamer medicaments.
The invention also provides application of the nucleic acid medicament in research on regulation of c-Met receptor protein activity and correlation of cell functions.
According to the invention, the hydrophobic cell membrane anchoring groups are modified at the middle positions of the first anchoring nucleic acid sequence and the second anchoring nucleic acid sequence, so that the nucleic acid aptamer formed after assembly can be stably anchored on the surface of a cell membrane. When the cell membrane-anchored nucleic acid drug of the present invention is used as an aptamer drug, the protein activity can be significantly inhibited at a lower concentration and the inhibition time of the aptamer drug can be prolonged, as compared with a free aptamer drug. Therefore, the aptamer drug is anchored on the surface of the cell membrane by using the cell membrane anchoring sequence, so that the dosage of the aptamer drug can be reduced, and the inhibition effect of the aptamer drug is enhanced.
The main instruments used in this embodiment are:
Milli-Q Integarl pure water/ultrapure water integrated system (Millipore Corp., USA); cary Eclipse fluorescence spectrometer (Agilent Technologies, USA); a small vertical electrophoresis cell (Bio-Rad, USA); ChemiDocTM Touch gel imaging system (Bio-Rad, USA); SH-1000UV-Vis Spectrophotometer (Corona Electric Co., Japan); a1 confocal laser scanning microscope (nikon, japan); Ti-S inverted microscope (Nikon, Japan); CHB 202 constant temperature metal bath (hangzhou bord technologies, china).
The main reagents used in this embodiment are:
the DNA sequences (Table 1) used in this experiment were synthesized by Shanghai Bioengineering technology, Inc., China and purified by HPLC. Recombinant human HGF was purchased from PeproTech, usa. anti-c-Met antibody, anti-p-Met antibody, anti-Akt antibody, anti-p-Akt antibody, anti-ERK 1/2 antibody, anti-p-ERK 1/2 antibody, and HRP-labeled secondary antibody were purchased from Cell Signaling Technology, Inc., USA. MEM medium, cell culture double antibody and Fetal Bovine Serum (FBS) were purchased from Gibco, usa. RIPA cell lysate, protein concentration quantification kit, protease inhibitor and phosphatase inhibitor were purchased from shanghai bi yunnan biotechnology limited, china. DNase I was purchased from Thermo, USA. Phosphate Buffered Saline (PBS) was purchased from Gibco, usa.
Example 1
The cell membrane anchored nucleic acid drug in this example is assembled from a first anchoring nucleic acid sequence and a regulatory nucleic acid sequence, which includes two functional sequences: an aptamer sequence and a second anchor nucleic acid sequence; the sequence of the nucleic acid aptamer is shown as SEQ ID NO.1, the second anchoring nucleic acid sequence and the first anchoring nucleic acid sequence are completely complementary and hybridized to form a cell membrane anchoring sequence with a double-chain structure, and cell membrane anchoring groups are modified on the second anchoring nucleic acid sequence and the first anchoring nucleic acid sequence. The second anchor nucleic acid sequence forms a cell membrane anchor sequence upon annealing complementary hybridization with the first anchor nucleic acid sequence.
Referring to the oligonucleotide sequences shown in table 1 below, in this example 1, a cell membrane receptor protein, i.e., a mesenchymal epidermal transformation factor (c-Met receptor protein), which plays an important role in tumor cell activities, was selected as a target receptor protein, and a double-stranded complementary nucleic acid sequence of 18 bases in length was selected as a cell membrane anchoring sequence. CH-b is a first anchor nucleic acid sequence modified with a cell membrane anchoring group (in this example, a hydrophobic molecule, cholesterol molecule), and the sequence of b is shown in SEQ ID NO. 2. a is a nucleic acid aptamer sequence specifically targeting c-Met receptor protein, CH-b is a second anchor nucleic acid sequence modified with cholesterol molecules, the second anchor nucleic acid sequence CH-b is added at the 5' end of the nucleic acid aptamer sequence a to form CH-ab, and in order to ensure that the configuration of the nucleic acid aptamer sequence is not affected, a random base sequence T is arranged between the nucleic acid aptamer sequence a and the second anchor nucleic acid sequence b in the embodiment 1, wherein the sequence of b is shown in the following table 1, and the sequence of ab is shown in SEQ ID NO. 3. B in the second anchor nucleic acid sequence CH-b is completely complementary to b in the first anchor nucleic acid sequence CH-b. And (2) mixing the CH-b and the CH-ab according to the molar concentration of 1:1, heating at 95 ℃ for 5 minutes to carry out annealing and mutual hybridization, forming a double-chain structure by CH-ab and CH-b, and slowly cooling to room temperature to form the nucleic acid drug 2CH-ab: b anchored by cell membranes. In another embodiment, one random base sequence disposed between the aptamer sequence a and the second anchor nucleic acid sequence b can be a or C or G; the number of other random base sequences may be no more than 5 bases, as long as the configuration of the aptamer sequence is not affected.
Comparative example 1
The conditions were the same except that the aptamer a of the c-Met receptor protein and ab: b of the unmodified cell membrane anchoring group were used instead of 2CH-ab: b in example 1.
TABLE 1 oligonucleotide sequences used in example 1
The "-CH-" in Table 1 above represents a cholesterol molecule.
Example 2
In this example 2, a mesenchymal epidermal transformation factor (c-Met receptor protein) which plays an important role in tumor cell activities was selected as a target receptor protein, and a double-stranded complementary nucleic acid sequence of 24 bases in length was selected as a cell membrane anchoring sequence. CH-d is a first anchor nucleic acid sequence modified with a cell membrane anchoring group (in this example, a hydrophobic molecule, cholesterol molecule), and the sequence of d is shown in SEQ ID NO. 4; a is an aptamer specifically targeting c-Met receptor protein, CH-d is a second anchor nucleic acid sequence modified with cholesterol molecules, the second anchor nucleic acid sequence CH-d is added at the tail end of the aptamer a of the c-Met receptor protein to form CH-ad, and in order to ensure that the configuration of the aptamer sequence is not affected, a random base sequence T is arranged between the aptamer sequence a and the second anchor nucleic acid sequence d in the embodiment 2, wherein the sequence of the ad is shown as SEQ ID No.5, and the sequences of d and d are completely complementary. The corresponding nucleic acid sequences were prepared according to the same procedure as in example 1, and the sequences of CH-ad and CH-d in example 2 are shown in Table 2.
Comparative example 2
The same experiment was carried out except that 2CH-ad: d in example 2 was replaced by ad: d of an unmodified cell membrane anchoring group.
TABLE 2 oligonucleotide sequences used in example 2 and comparative example 2
The "-CH-" in Table 2 above represents a cholesterol molecule.
(one) related experimental procedures of example 1, comparative example 1, example 2, comparative example 2
The nucleic acid probes in example 1, comparative example 1, example 2, and comparative example 2 were subjected to the following experiments:
A. anchorage stability optimization of cell membrane anchoring sequences
Optimizing the number and the positions of cholesterol molecule modifications: after incubation of 200nM of different cell membrane anchoring sequences (2CH-b: b, 2CH-c: c, CH-b: b, CH-b and CH-c) with HeLa cells for 10 min, excess probe was washed off with PBS and confocal fluorescence imaging was performed. The cells were then incubated with medium containing 10% fetal bovine serum for 30 minutes, washed 3 times with PBS, and again confocal fluorescence imaged.
B. Anchoring effect and anchoring directivity verification
200nM 2CH-ab: b was incubated with HeLa cells for 10 min, washed 3 times with PBS and then confocal fluorescence imaged to verify the anchoring effect. To verify their anchoring directionality, DNase I (0.5U/. mu.l) was then added and incubated with the cells for 5 min, washed 3 times with PBS and again confocal fluorescence imaged.
C. Aptamer competition assay
Mixing 10 μ M CH-ab with 10 μ M CH-b, annealing at 95 deg.C for 5 min, cooling to room temperature for more than 2 hr, and storing at 4 deg.C. To characterize the stability of aptamer binding, HeLa cells were first incubated with 400nM a labeled with the Cy3 fluorophore (Cy3-Free a) or 2CH-ab: b labeled with the Cy3 fluorophore (Cy3-2CH-ab: b) for 10 min, washed 3 times with PBS, and subjected to a first confocal fluorescence imaging. Then, cells were incubated with 400nM a labeled Cy5 fluorophore a (Cy5-Free a) for 15 minutes, washed 3 times with PBS, and imaged a second time.
D. Immunoblotting experiments
DU145 cells were seeded in 6-well plates (3X 10 per well)5Cells) were cultured for 24 hours, and then the culture was starved for 24 hours by replacing the culture with MEM medium containing 0.5% BSA, and an experiment was performed:
(1) performance study of the effect of cell membrane anchored nucleic acid drugs in enhancing the activity of profilins: DU145 cells were incubated with nucleic acid probes (2CH-ab: b, or a) at different concentrations for 15 minutes, 20ng/mL HGF was added and the cells were incubated for 30 minutes, the cells were washed 3 times with cold PBS, the cells were lysed with RIPA lysis buffer to which protease inhibitors (1X) and phosphatase inhibitors (1X) were added, proteins were extracted and the concentrations were determined, and then immunoblotting experiments were performed.
To verify the versatility of this strategy, 30nM different nucleic acid probes (2CH-ad: d or 2CH-d: d) were incubated for 15 min, 20ng/mL HGF was added and incubated with cells for 30 min, after washing the cells 3 times with cold PBS, the cells were lysed with RIPA lysis buffer supplemented with protease inhibitors (1X) and phosphatase inhibitors (1X), proteins were extracted and the concentration was determined followed by immunoblotting experiments.
(2) Performance study of cell membrane anchored nucleic acid drugs to prolong inhibition time: after addition of 250nM2CH-ab: b or ab: b to DU145 cells, incubation was performed for 24 hours in a cell culture incubator. And adding 20ng/mL HGF to incubate with the cells for 30 minutes, washing the cells for 3 times by using cold PBS, cracking the cells to extract protein, and performing an immunoblotting experiment after determining the protein concentration. Cells incubated with 20ng/mL HGF alone for 30 minutes were used as a positive control.
E. Cell migration ability analysis
DU145 cells were seeded in 12-well plates to fill the entire plate. The experiment was performed after replacing the complete culture with MEM medium containing 0.5% BSA starving the cells for 24 hours. Scratches were made in the center of the well plate using a sterile 200 μ L tip and the width of the scratch was recorded under an inverted microscope. Cell migration experiments were divided into four groups. A first group: blank control group; second group: only 20ng/mL HGF was added as a positive control; third group: 30nM ab: b; and a fourth group: 30nM 2CH-ab: b. After the third and fourth groups were incubated for 15 minutes with the nucleic acid probe, 20ng/mL HGF was added for incubation. After each set of cells was placed in an incubator for an additional 24 hours, the scratch width was recorded under an inverted microscope. Migration distance of cells within 24 hours was quantified using Image J software.
(II) results of the correlation experiment
A. Anchorage stability optimization of cell membrane anchoring sequences
First, in order to achieve the best anchoring effect, we optimized the number and position of cholesterol molecules modified on the anchoring nucleic acid sequence, and constructed 5 kinds of cell membrane anchoring sequences, including 2CH-b: b, 2CH-c: c, CH-b: b, CH-b and CH-c, and examined their respective anchoring stability on cell membrane. Wherein, 2CH-b is double-stranded cell membrane anchoring sequence, and one cholesterol molecule is modified in the middle position of two anchoring nucleic acid sequences (a first anchoring nucleic acid sequence and a second anchoring nucleic acid sequence); c is double-stranded cell membrane anchoring sequence, and the 3 'end of one anchoring nucleic acid sequence and the 5' end of the other anchoring nucleic acid sequence are respectively modified with one cholesterol molecule; b is double-chain cell membrane anchoring sequence, and a cholesterol molecule is modified in the middle of one anchoring nucleic acid sequence; CH-b is a single-stranded anchor nucleic acid sequence, and a cholesterol molecule is modified in the middle of the sequence; CH-c is a single-stranded anchor nucleic acid sequence, and a cholesterol molecule is modified at the 5' end of the sequence. We labeled Cy3 fluorescence on both of these anchor nucleic acid sequences. As shown in the confocal images of FIGS. 2A-2E, these cell membrane anchoring nucleic acid sequences were successfully anchored to the cell membrane surface. The double-stranded nucleic acid sequence (i.e., 2CH-b: b) modified by two cholesterol molecules at the middle position of the cell membrane anchoring nucleic acid sequence in the presence of 10% fetal bovine serum shows more stable cell membrane anchoring performance. By increasing the number of membrane anchoring molecules (i.e. membrane anchoring groups) better anchoring stability can be obtained compared to CH-b: b. Compared with 2CH-c, when the cell membrane anchoring molecule is modified in the middle position of the double-stranded anchoring nucleic acid sequence, the cell membrane anchoring nucleic acid sequence with the double-stranded structure can be inserted into a deeper position of a cell membrane, so that the anchoring stability is improved. Therefore, 2CH-b: b was chosen as a cell membrane anchoring sequence for subsequent experiments.
From the above experimental results, it can be seen that: (1) two cholesterol molecules are respectively modified at the middle positions of the first anchor nucleic acid sequence and the second anchor nucleic acid sequence, and the cholesterol molecules are positioned at the symmetrical positions of double chains after the first anchor nucleic acid sequence and the second anchor nucleic acid sequence are hybridized; (3) cholesterol is one of the hydrophobic molecules that is inserted into the phospholipid layer of the cell membrane by its hydrophobicity, and so on to other hydrophobic molecules such as tocopherol molecules and diacyl liposomes, and is also modified to be more effective at the middle of the double-stranded anchor nucleic acid sequence.
B. Construction and characterization of cell membrane anchored nucleic acid drugs
In example 1, comparative example 1, example 2 and comparative example 2, the c-Met receptor protein was selected as the target receptor protein model, and an aptamer targeting the c-Met receptor protein, namely a, was selected to verify our design. The c-Met receptor protein is an important drug target, and the over-activation of the c-Met receptor protein leads to the migration and invasion of tumor cells. Extending the second anchor nucleic acid sequence, i.e., CH-ab, at the terminus of aptamer a and hybridizing it to the first anchor nucleic acid sequence CH-b to form 2 CH-ab. We first verified the hybridization of the nucleic acid drug in PBS buffer. As shown in the attached figure 3, from left to right, the lane 1 is a CH-ab band, the lane 2 is a CH-b band, and due to the hybridization of CH-ab and CH-b, the lane 3 shows a new band at the higher position, thereby proving the successful assembly of 2CH-ab: b, and proving that the modification of cholesterol molecules does not influence the hybridization of CH-ab and CH-b. Next, as shown in FIG. 4A, the 3 '-end of the probe CH-ab was labeled with Cy3 fluorescence, and the 5' -end of the probe CH-b was labeled with Cy5 fluorescence. When the assembled 2CH-ab: b was incubated with HeLa cells for ten minutes, fluorescence of Cy3 and Cy5 was observed simultaneously on the cell membrane, demonstrating that 2CH-ab: b was successfully anchored on the cell membrane surface of HeLa cells.
In example 2, to verify the versatility of the method, we designed a first anchor nucleic acid sequence and a second anchor nucleic acid sequence of 24 bases in length, and designed the nucleic acid drug 2CH-ad: d. As shown in fig. 4B, 2CH-ad: d was successfully anchored to the cell membrane surface of HeLa cells.
The recognition and binding ability of aptamer drugs to target proteins is critical for their inhibitory efficacy, thus requiring aptamers to be located outside the cell membrane. To examine the directionality of the anchoring of 2CH-ab b on the cell membrane, we labeled Cy3 fluorescence at the 3 'end of probe CH-ab and Cy5 fluorescence at the 3' end of probe CH-b. As shown in fig. 5, anchoring the assembled probe 2CH-ab: b to HeLa cell membrane enabled simultaneous observation of a bright cycle of Cy3 and Cy5 fluorescence. DNase I is an endonuclease capable of hydrolysing single-or double-stranded DNA. Since DNase I cannot cross the cell membrane, we used it to investigate the directionality of the 2CH-ab: b anchor. The fluorescence of Cy3 on the cell membrane after DNase I treatment was significantly reduced, whereas the fluorescence of Cy5 remained essentially unchanged, indicating that aptamer a was located outside the cell membrane. Next, we investigated the stability of 2CH-ab: b binding to the target c-Met receptor protein by aptamer competition experiments. As shown in FIG. 6A, almost no Cy5 fluorescence signal was observed on Cy3-2CH-ab: b anchored cell membranes. However, a ring of significant Cy5 fluorescence signal was observed on the cell membrane incubated with Cy3-Free a, indicating Cy5-Free a binding (FIG. 6B). It was thus demonstrated that cell membrane anchored 2CH-ab b binds more stably to the target c-Met receptor protein than free aptamer a.
C. Cell membrane anchored nucleic acid drugs enhance arrestin activity and cellular function
In example 1, comparative example 1, example 2 and comparative example 2, the c-Met receptor protein was selected as a model of the target receptor protein, and it was examined whether the cell membrane-anchored nucleic acid drug could enhance the inhibition of c-Met receptor phosphorylation by HGF. We also compared the effect of probes 2CH-ab: b, ab: b with free a on inhibiting the activity of the c-Met receptor. As shown in the attached figures 7A and 7B, the inhibition ability of 2CH-ab: B is obviously better than that of ab: B, and the protein expression of phosphorylated c-Met (p-Met) can be effectively inhibited at lower concentration. Meanwhile, ab: B shows similar inhibitory ability to free a (fig. 7B and fig. 8), further verifying that 2CH-ab: B capable of anchoring on cell membranes significantly enhances the inhibitory efficacy of aptamer drugs and can reduce the dosage of aptamer drugs.
In example 2, to verify the versatility of the method, we designed a first anchor nucleic acid sequence and a second anchor nucleic acid sequence of 24 bases in length as another set of cell membrane anchor sequences, and then designed the nucleic acid drug 2CH-ad: d. The cells treated with 30nM 2CH-ad d showed almost no detectable protein expression of p-Met, whereas the cells treated with the same concentration of free ad d showed similar expression level of p-Met as the positive control treated with HGF only, failing to play a significant inhibitory effect. Meanwhile, the cell membrane anchoring sequence (2CH-d: d) does not affect the activity of the c-Met receptor protein. The experimental results demonstrate that the cell membrane anchored nucleic acid drug 2CH-ad: d is able to inhibit c-Met receptor protein activity more efficiently (see the results in fig. 9), demonstrating the versatility of this strategy.
From the comparison of example 1 and example 2, it can be seen that the first anchor nucleic acid sequence and the second anchor nucleic acid sequence are not limited to the specific sequences listed in the above-mentioned examples 1 and 2, as long as the first anchor nucleic acid sequence and the second anchor nucleic acid sequence satisfy the cell membrane anchoring sequence forming a double-stranded structure of stable hybridization of 18 to 24bp, and both the first anchor nucleic acid sequence and the second anchor nucleic acid sequence are modified with a cell membrane anchoring group. Because the length of the aptamer sequence in the scheme is 50 bases, the length of the cell membrane anchoring sequence is not too long, and if the length is too long, the difficulty of modifying cholesterol molecules is increased, DNA raw materials are wasted, and the cost of synthesizing DNA is increased; on the other hand, too long a cell membrane anchoring sequence may affect the function of the aptamer sequence. The experimental results of example 1 and example 2 show that when the aptamer sequence is provided with a cell membrane anchoring sequence having a double-stranded structure of an appropriate length (18-24bp) and the cell membrane anchoring group is provided on the cell membrane anchoring sequence, the cell membrane anchoring nucleic acid sequence having the double-stranded structure binds to the cell membrane, so that the aptamer can be enriched on the surface of the cell membrane, the binding stability with the cell membrane receptor protein is increased, the activity of the c-Met receptor protein can be significantly inhibited at a lower concentration, and the inhibition effect of the aptamer drug is enhanced.
Activation of the c-Met receptor protein is closely related to cell migration behavior. Therefore, we examined the effect of 2CH-ab b on the regulation of cell behavior using a cell-scratch experiment. Consistent with the expected effect, as shown in fig. 10, the application of 2CH-ab: b incubated cells healed the scratches more slowly, indicating a better ability to inhibit cell migration behavior.
D. Cell membrane anchored nucleic acid drugs prolong inhibition time
Although probes 2CH-ab: b and ab: b showed good inhibition at 250nM for a shorter time (15 min) (FIG. 7). As shown in fig. 11, only 2CH-ab b was able to resist HGF stimulation-mediated activation of c-Met receptor after 24 hours of incubation with cells, and still maintained good inhibitory effect. Ab b has essentially no inhibitory effect after 24 hours of incubation with cells, and the expression level of p-Met protein has been comparable to that of the positive control group treated with HGF alone. The experimental results indicate the feasibility of 2CH-ab: b in long-acting inhibition of protein activity.
The analysis of the above experimental results shows that the cell membrane anchoring sequence with double-chain structure constructed by the invention can enrich more aptamer drugs on the cell membrane surface, and increase the binding stability (see the result in figure 6), so that better inhibition effect can be realized only by smaller dose (see the results in figures 7 to 10), meanwhile, the cell membrane anchoring sequence can slow down the endocytosis process of the aptamer drugs by cells, and the inhibition time is prolonged for the target on the cell membrane, namely the c-Met receptor protein (see the result in figure 11)
The nucleic acid drug anchored by the cell membrane constructed by the invention can obviously improve the combination stability of the aptamer drug and the target protein, can obviously inhibit the activity of the c-Met receptor protein at a lower concentration, and greatly enhances the inhibition effect of the aptamer drug. In addition, the strategy can effectively prolong the action time of the aptamer drug on the surface of a cell membrane and reduce the drug effect loss caused by endocytosis of the cell. The designed nucleic acid drug anchored by the cell membrane is simple and controllable, provides a new platform for developing high-efficiency nucleic acid drugs, and further expands the application prospect of the nucleic acid drug in biomedicine.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present application and not for limiting the protection scope thereof, and although the present application is described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that: numerous variations, modifications, and equivalents will occur to those skilled in the art upon reading the present application and are within the scope of the claims as issued or as granted.
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Claims (10)
1. A cell membrane anchored nucleic acid drug formed by the assembly of a first anchoring nucleic acid sequence and a regulatory nucleic acid sequence comprising two functional sequences: an aptamer sequence and a second anchor nucleic acid sequence; the second anchoring nucleic acid sequence is connected to the 5' end of the nucleic acid aptamer sequence, the nucleic acid aptamer sequence specifically targets a cell membrane receptor protein, the nucleic acid aptamer sequence is shown in SEQ ID NO.1, the second anchoring nucleic acid sequence and the first anchoring nucleic acid sequence are in complete complementary hybridization to form a cell membrane anchoring sequence with a double-chain structure, and one or two cell membrane anchoring groups are modified on the cell membrane anchoring sequence.
2. The nucleic acid drug of claim 1, wherein between 1 and 5 random base sequences are disposed between the aptamer sequence and the second anchor nucleic acid sequence.
3. The nucleic acid drug of claim 2, wherein the cell membrane anchoring group is one of hydrophobic molecules, the hydrophobic molecules including cholesterol molecules, tocopherol molecules, and diacyl liposomes.
4. The nucleic acid drug of claim 3, wherein the first anchor nucleic acid sequence and the second anchor nucleic acid sequence hybridize complementarily to form a double-stranded structure having a length of 18-24 bp.
5. The nucleic acid drug of claim 4, wherein both of said cell membrane anchoring groups are modified at a position intermediate to said first anchoring nucleic acid sequence and said second anchoring nucleic acid sequence; the intermediate position is: the first anchor nucleic acid sequence and the second anchor nucleic acid sequence are n bases, when n is an even number, two of the cell membrane anchoring groups are modified on the DNA backbone between the (n/2) th and (n/2) +1 th bases of the first anchor nucleic acid sequence and the second anchor nucleic acid sequence, respectively; when n is an odd number, two of the cell membrane anchoring groups are modified on the DNA backbone intermediate the (n/2) -0.5 and (n/2) +0.5 bases of the first and second anchor nucleic acid sequences, respectively.
6. The nucleic acid drug of any one of claims 1-5, wherein the cell membrane receptor protein is a mesenteric epidermal transformation factor that can be activated by receptor dimerization mediated by its ligand hepatocyte growth factor.
7. The nucleic acid drug of claim 6, wherein the aptamer sequence overlaps or is identical to the binding site of the cell membrane receptor protein and the ligand partially overlaps or is identical to the binding site of the cell membrane receptor protein.
8. A method for preparing a nucleic acid drug according to any one of claims 2 to 7, comprising the steps of:
s1, synthesizing a first anchoring nucleic acid sequence;
s2, synthesizing a regulating nucleic acid sequence;
s3, mixing the two nucleic acid sequences in the steps S1 to S2 according to the molar concentration of 1:1, heating at 95 ℃ for 5 minutes after mixing, annealing and mutually hybridizing, and slowly cooling to room temperature.
9. The nucleic acid drug according to any one of claims 1 to 7 for use in a nucleic acid aptamer drug.
10. Use of the nucleic acid medicament according to any one of claims 1 to 7 for the regulation of c-Met receptor protein activity and cell function-related studies.
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