Nucleic acid aptamer AS1411 modified DNA tetrahedron and preparation method thereof

Abstract

This invention discloses a nucleic acid aptamer AS1411 modified DNA tetrahedron. The preparation method includes the steps of (1) binding an AS1411 sequence to the 5 terminal of any DNA single-strand in a DNA tetrahedron, synthesizing the DNA, dissolving obtained DNA powder with ddH.sub.2O; (2) measuring an absorbance of the DNA and then calculating a total volume of the four single strands; (3) pipetting the DNA obtained in step (1) according to the calculation results in step (2), mixing the DNA with a TM buffer, mixing the mixture uniformly with vortex vibration, and performing a PCR progress. The preparation method is simple. The produced product can effectively solve the problem that the unmodified DNA tetrahedron cannot enter the nucleus and the problem that the AS1411 cannot carry drugs directly.

Claims

1. A method for preparing a modified DNA tetrahedron modified by a nucleic acid aptamer AS1411, wherein the nucleic acid aptamer AS1411 is a DNA single-strand having a sequence of SEQ ID NO: 5, the method comprises the following steps: (1) binding the nucleic acid aptamer AS1411 to any one of four DNA single-strands of a DNA tetrahedron at the 5 terminal of the DNA, synthesizing the DNA, dissolving the obtained DNA powder with ddH.sub.2O, storing at a condition of 4 C.; wherein the four DNA single strands include a first DNA single-strand having a sequence of SEQ ID NO: 1, a second DNA single-strand having a sequence of SEQ ID NO: 2, a third DNA single-strand having a sequence of SEQ ID NO: 3 and a fourth DNA single-strand having a sequence of SEQ ID NO: 4; (2) measuring an absorbance of the DNA at wavelengths of 260 nm and 330 nm by ultraviolet quantitation method, then calculating a volume of each single strand in a 100 L, 1 M system according to the following formula:
V=100[(A.sub.260A.sub.330)10.sup.5/(15.2a number of adenine in a single strand+7.4a number of cytosine in the single strand+11.4a number of guanine in the single strand+8.3a number of thymine in the single strand)], calculating a total volume of the four single strands according to the above calculated results of each single strand; (3) pipetting the DNA obtained in step (1) according to the calculated results in step (2), mixing the DNA with a TM buffer to obtain a mixture, mixing the mixture uniformly with vortex vibration, and putting the mixture into a PCR apparatus; raising the temperature to 95 C. and maintaining for 10 min; and then reducing to 4 C. and maintaining for 20 min, and finally storing at 20 C. to obtain the modified DNA tetrahedron modified by the nucleic acid aptamer AS1411.

2. The method of claim 1, wherein in step (1), the DNA powder is dissolved in 10 L ddH.sub.2O.

3. The method of claim 1, wherein the TM buffer in step (3) having a pH value of 8.0 includes 5-10 mM Tris-HCl and 5-50 mM MgCl.sub.2.

4. The method of claim 1, wherein the TM buffer in step (3) having a pH value of 8.0 includes 10 mM Tris-HCl and 50 mM MgCl.sub.2.

5. A modified DNA tetrahedron modified by a nucleic acid aptamer AS1411, wherein the nucleic acid aptamer AS1411 is a DNA single-strand having a sequence of SEQ ID NO: 5, and the modified DNA tetrahedron is prepared through a method including the following steps: (1) binding the nucleic acid aptamer AS1411 to any one of the four DNA single-strands of a DNA tetrahedron at the 5 terminal of the DNA, synthesizing the DNA, dissolving the obtained DNA powder with ddH.sub.2O, storing at a condition of 4 C.: wherein the four DNA single strands include a first DNA single-strand having a sequence of SEQ ID NO: 1, a second DNA single-strand having a sequence of SEQ ID NO: 2, a third DNA single-strand having a sequence of SEQ ID NO: 3 and a fourth DNA single-strand having a sequence of SEQ ID NO: 4; (2) measuring an absorbance of the DNA at wavelengths of 260 nm and 330 nm by ultraviolet quantitation method, then calculating a volume of each single strand in a 100 L, 1 M system according to the following formula:
V=100/[(A.sub.260A.sub.330)10.sup.5/(15.2a number of adenine in a single strand+7.4a number of cytosine in the single strand+11.4a number of guanine in the single strand+8.3a number of thymine in the single strand)], calculating a total volume of the four single strands according to the above calculated results of each single strand; (3) pipetting the DNA obtained in step (1) according to the calculated results in step (2), mixing the DNA with a TM buffer to obtain a mixture, mixing the mixture uniformly with vortex vibration, and putting the mixture into a PCR apparatus; raising the temperature to 95 C. and maintaining for 10 min; and then reducing to 4 C. and maintaining for 20 min, and finally storing at 20 C. to obtain the modified DNA tetrahedron modified by the nucleic acid aptamer AS1411.

6. The modified DNA tetrahedron of claim 5, wherein in step (1), the DNA powder is dissolved in 10 L ddH.sub.2O.

7. The modified DNA tetrahedron of claim 5, wherein the TM buffer in step (3) having a pH value of 8.0 includes 5-10 mM Tris-HCl and 5-50 mM MgCl.sub.2.

8. The modified DNA tetrahedron of claim 5, wherein the TM buffer in step (3) having a pH value of 8.0 includes 10 mM Tris-HCl and 50 mM MgCl.sub.2.

Description

DETAILED DESCRIPTION OF THE INVENTION

Embodiment 1

(1) A method for preparing a nucleic acid aptamer AS1411 modified DNA tetrahedron includes the following steps: (1) binding an AS1411 sequence to S1 of the DNA tetrahedron at the 5 terminal, synthesizing the DNA using an automatic DNA synthesizer to obtain a DNA powder, centrifuging the obtained DNA powder at a high speed to ensure that the DNA powder aggregates at a bottom of the tube, dissolving the obtained DNA powder with ddH.sub.2O (1 nmol DNA is dissolved in 10 L ddH.sub.2O), storing at the condition of 4 C.; wherein the AS1411 sequence is shown in SEQ ID NO: 5, and S1 has a sequence of SEQ ID NO: 1; (2) measuring an absorbance of the DNA at wavelengths of 260 nm and 330 nm by ultraviolet quantitation method, then calculating the volume of each single strand in a 100 L, 1 M system according to the following formula:
V=100/[(A.sub.260A.sub.330)10.sup.5/(15.2a number of adenine in a single strand+7.4a number of cytosine in the single strand+11.4a number of guanine in the single strand+8.3a number of thymine in the single strand)], calculating a total volume of the four single strands according to the above calculation results;

(2) (3) pipetting the DNA obtained in step (1) according to the calculated total volume in step (2), mixing the DNA with a TM buffer (10 mM Tris-HCl, 50 mM MgCl.sub.2, pH 8.0) to obtain a mixture, mixing the mixture uniformly with vortex vibration, and putting the mixture into a PCR apparatus; raising the temperature to 95 quickly and maintaining for 10 min; and then cooling down to 4 C. and maintaining for 20 min, and finally storing at 20 C. to obtain the nucleic acid aptamer AS1411 modified DNA tetrahedron.

Embodiment 2

(3) A method for preparing a nucleic acid aptamer AS1411 modified DNA tetrahedron includes the following steps:

(4) (1) binding an AS1411 sequence to 52 of the DNA tetrahedron at the 5 terminal, synthesizing the DNA using an automatic DNA synthesizer to obtain a DNA powder, centrifuging the obtained DNA powder at a high speed to ensure that the DNA powder aggregates at a bottom of the tube, dissolving the obtained DNA powder with ddH.sub.2O (1 nmol DNA is dissolved in 10 L ddH.sub.2O), storing at the condition of 4 C.; wherein the AS1411 sequence is shown in SEQ ID NO: 5, and S2 has a sequence of SEQ ID NO: 2;

(5) (2) measuring an absorbance of the DNA at wavelengths of 260 nm and 330 nm by ultraviolet quantitation method, then calculating the volume of each single strand in a 100 L, 1 M system according to the following formula:
V=100/[(A.sub.260A.sub.330)10.sup.5/(15.2a number of adenine in a single strand+7.4a number of cytosine in the single strand+11.4a number of guanine in the single strand+8.3a number of thymine in the single strand)], calculating a total volume of the four single strands according to the above calculation results;

(6) (3) pipetting the DNA obtained in step (1) according to the calculated total volume in step (2), mixing the DNA with a TM buffer (10 mM Tris-HCl, 50 mM MgCl.sub.2, pH 8.0) to obtain a mixture, mixing the mixture uniformly with vortex vibration, and putting the mixture into a PCR apparatus; raising the temperature to 95 C. quickly and maintaining for 10 min; and then cooling down to 4 C. and maintaining for 20 min, and finally storing at 20 C. to obtain the nucleic acid aptamer AS1411 modified DNA tetrahedron.

Embodiment 3

(7) A method for preparing a nucleic acid aptamer AS1411 modified DNA tetrahedron includes the following steps:

(8) (1) binding an AS1411 sequence to S3 of the DNA tetrahedron at the 5 terminal, synthesizing the DNA using an automatic DNA synthesizer to obtain a DNA powder, centrifuging the obtained DNA powder at a high speed to ensure that the DNA powder aggregates at a bottom of the tube, dissolving the obtained DNA powder with ddH.sub.2O (1 nmol DNA is dissolved in 10 L ddH.sub.2O), storing at the condition of 41; wherein the AS1411 sequence is shown in SEQ ID NO: 5, and S3 has a sequence of SEQ ID NO: 3;

(9) (2) measuring an absorbance of the DNA at wavelengths of 260 nm and 330 nm by ultraviolet quantitation method, then calculating the volume of each single strand in a 100 L, 1 M system according to the following formula:
V=100/[(A.sub.260A.sub.330)10.sup.5/(15.2a number of adenine in a single strand+7.4a number of cytosine in the single strand+11.4a number of guanine in the single strand+8.3a number of thymine in the single strand)], calculating a total volume of the four single strands according to the above calculation results;

(10) (3) pipetting the DNA obtained in step (1) according to the calculated total volume in step (2), mixing the DNA with a TM buffer (10 mM Tris-HCl, 50 mM MgCl.sub.2, pH 8.0) to obtain a mixture, mixing the mixture uniformly with vortex vibration, and putting the mixture into a PCR apparatus; raising the temperature to 95 C. quickly and maintaining for 10 min; and then cooling down to 4 C. and maintaining for 20 min, and finally storing at 20 C. to obtain the nucleic acid aptamer AS1411 modified DNA tetrahedron.

Embodiment 4

(11) A method for preparing a nucleic acid aptamer AS1411 modified DNA tetrahedron includes the following steps:

(12) (1) binding an AS1411 sequence to S4 of the DNA tetrahedron at the 5 terminal, synthesizing the DNA using an automatic DNA synthesizer to obtain a DNA powder, centrifuging the obtained DNA powder at a high speed to ensure that the DNA powder aggregates at a bottom of the tube, dissolving the obtained DNA powder with ddH.sub.2O (1 nmol DNA is dissolved in 10 L ddH.sub.2O), storing at the condition of 4 C.; wherein the AS1411 sequence is shown in SEQ ID NO: 5, and S4 has a sequence of SEQ ID NO: 4;

(13) (2) measuring an absorbance of the DNA at wavelengths of 260 nm and 330 nm by ultraviolet quantitation method, then calculating the volume of each single strand in a 100 L, 1 M system according to the following formula:
V=100/[(A.sub.260A.sub.330)10.sup.5/(15.2a number of adenine in a single strand+7.4a number of cytosine in the single strand+11.4a number of guanine in the single strand+8.3a number of thymine in the single strand)], calculating a total volume of the four single strands according to the above calculated results; (3) pipetting the DNA obtained in step (1) according to the calculated total volume in step (2), mixing the DNA with a TM buffer (10 mM Tris-HCl, 50 mM MgCl.sub.2, pH 8.0) to obtain a mixture, mixing the mixture uniformly with vortex vibration, and putting the mixture into a PCR apparatus; raising the temperature to 95 C. quickly and maintaining for 10 min; and then cooling down to 4 C. and maintaining for 20 min, and finally storing at 20 C. to obtain the nucleic acid aptamer AS1411 modified DNA tetrahedron.

(14) Verification of the nucleic acid aptamer AS1411 modified DNA tetrahedrons obtained by the above methods: a polyacrylamide gel electrophoresis (PAGE) method is used. That is, diluting each of the unmodified single strand of the DNA tetrahedron by 100-fold, taking 5 L of the dilution to mix with 1 L of 6loading buffer, taking 5 L of the modified DNA tetrahedron to mix with 1 L of 6loading buffer, adding the solutions into a prepared gel (the ingredients of the gel: ultrapure water 4.2 mL, 40% acrylamide 1.2 mL, 10TAE 0.6 mL, 10% APS 60 L, and TEMED 6 L) respectively, adding a 20 bp marker in a side of the gel as a control, conducting electrophoresis in a 1TAE buffer at a constant voltage of 100V for 80 minutes. Adding 50 ml ddH.sub.2O and 5 L Gel-red to a box without light, mixing uniformly, putting the gel into the box, shaking in a shaker for 15-20 minutes, and taking a picture via a gel imaging system. The electrophoretogram is shown in FIGURE.

Embodiment 5

(15) 20 mM pH 7.0 sodium phosphate buffer, 150 mM sodium chloride buffer, and 0.5 mM EDTA buffer are taken to prepare the following reaction system: 201M of the nucleic acid aptamer AS1411 modified DNA tetrahedron obtained in Embodiment 4, 4001 M of Adriamycin, 0.37% vol formaldehyde. The above reaction system is kept at 10 C. to react for 12 h. The obtained product using HPLC, molecular sieve, or ethanol precipitation method (the nucleic acid aptamer AS1411 modified DNA tetrahedron obtained in the present invention binding with drugs is referred as a sample, and the nucleic acid aptamer AS1411 modified DNA tetrahedron obtained without binding drugs is referred as a control) is purified.

(16) The specificity and affinity of the samples against tumor cells are studied by flow cytometry. That is, the samples and the control (200 nM respectively) with tumor cells (A549) are incubated on the ice for half an hour respectively. The substance nonspecifically adsorbed on the cells is washed out with a DPBS buffer. The obtained cells are resuspended in a DPBS (including 5 mM Mg.sup.2+), and are analyzed using flow cytometry. The result shows that compared with the control, the samples have a specific recognition ability and higher affinity with respect to tumor cells.

Embodiment 6

(17) Plating the cultured tumor cells A549 in 96-well cell culture plates (100 L, 510.sup.4 cell/well), adding pure Adriamycin or samples with predetermined concentrations (0, 0.1 M, 0.2 M, 0.4 M, 0.8 M, 1.6 M, 3.2 M) into the wells respectively, culturing in a cell incubator for 1.5 h, centrifuging, removing the cell culture medium in the supernatant, adding fresh culture medium to continue culturing for 48 h, then testing the proliferation rate of the cells using a MTS reagent.

(18) The testing result shows that, as the concentration increases, the proliferation rate of tumor cells decreases gradually, indicating that the inhibitory effect of the Adriamycin and samples against tumor cells increases gradually. Compared with the pure Adriamycin, the samples have a stronger inhibitory effect.