WO2024093150A1

WO2024093150A1 - Method for purifying dye-modified nucleic acid probe

Info

Publication number: WO2024093150A1
Application number: PCT/CN2023/088237
Authority: WO
Inventors: 蔡晶晶
Original assignee: 百力格生物科技(上海)股份有限公司
Priority date: 2022-11-03
Filing date: 2023-04-14
Publication date: 2024-05-10
Also published as: CN115414703A; CN115414703B

Abstract

A method for purifying a dye-modified nucleic acid probe. By means of the acting force between an adsorbent and the non-polar functional groups of a free modification dye and a dye-modified nucleic acid probe, the excess free modification dye and the dye-modified nucleic acid probe are retained on an adsorbent, then a column containing the adsorbent is eluted with an aqueous solution containing an organic solvent, and the dye-modified nucleic acid probe is selectively eluted from the adsorbent using the polarity difference between the free modification dye and the dye-modified nucleic acid probe. This method does not require the pre-cooling of a reagent and has a simple process flow; and the operation time for removing the free modification dye is shortened from 4-15 h to 10-20 min, which greatly shortens the operation cycle.

Description

Method for purifying nucleic acid probes modified with dyes

Technical Field

The invention relates to the field of nucleic acid synthesis, and in particular to a method for purifying a nucleic acid probe modified with a dye.

Background technique

Single-stranded DNA and RNA are composed of a certain number of deoxynucleotides or nucleotides. By labeling the structure of DNA or RNA with some modified dyes, DNA modified probes and RNA modified probes are formed. DNA/RNA modified probes are widely used in molecular diagnosis QPCR (real-time fluorescence quantitative), STR (short tandem repeat sequence), and FISH (fluorescence in situ hybridization) and other fields.

Generally, there are two common methods for labeling single-stranded DNA and RNA nucleic acids with modified dyes. One is the solid phase phosphoramidite triester method, and the other is the liquid phase amino activated ester addition method. The traditional liquid phase amino activated ester addition method is to dissolve the DNA/RNA nucleic acid probe containing amino active groups in an alkaline buffer, and then add 3 times the molar amount of the activated ester modified dye dissolved in an organic solvent, and react at room temperature for 4-12 hours. In order to remove the excess free modified dye in the reaction, the DNA/RNA modified probe needs to be precipitated by ethanol precipitation after the reaction is completed, and the precipitate is washed with 70% frozen ethanol to obtain a precipitated solid.

In the prior art, after the reaction is completed, the system contains excess free modification dyes and buffer salts. For specific application fields, these unreacted free modification dyes and buffer salts need to be removed in advance in subsequent production. Therefore, in the post-reaction treatment, a large amount of organic solvents are required to be repeatedly precipitated and cleaned, which produces a lot of harmful waste liquid. At the same time, for large-scale DNA/RNA modified probes, the processing time is as long as 4-15 hours, which is a great waste of time. The ethanol reagent used in the precipitation and washing process needs to be pre-cooled at -20°C, which has high process requirements and cumbersome processes.

At the same time, the ethanol precipitation method cannot completely precipitate the DNA/RNA modified probes, and the process loss is large, with a general precipitation loss rate of about 3-10%. In addition, a large amount of organic solvents are required to repeatedly precipitate and clean during the treatment process, and the material cost is too high. For large-scale DNA/RNA nucleic acid probes, because the free modification dyes are wrapped around the nucleic acid macromolecular structure, even after multiple precipitation and cleaning, a small amount of free modification dyes will still remain in the product after purification by high-performance liquid chromatography. For probes such as STR, a small amount or trace amount of residual free modification dyes will affect the interpretation of the results.

Summary of the invention

In order to solve the above technical problems, a method for purifying a dye-modified nucleic acid probe is invented. The method retains excess free modifying dye and the dye-modified nucleic acid probe on the adsorbent through the interaction between the adsorbent and the non-polar functional groups of the free modifying dye and the dye-modified nucleic acid probe, and then elutes the column containing the adsorbent with an aqueous solution containing an organic solvent. The polarity difference between the free modifying dye and the dye-modified nucleic acid probe is utilized to selectively elute the dye-modified nucleic acid probe from the adsorbent, while the free modifying dye is retained on the adsorbent, so as to purify the dye-modified nucleic acid probe; the adsorbent is a C18 adsorption column, and the volume of acetonitrile in the aqueous solution containing an organic solvent is 5-15%.

The modified dye of the nucleic acid probe in the present invention is a dye used in a fluorescent probe, such as CY7 SE (cyanine 7 succinimide activated ester), CY5 SE (cyanine 5 succinimide activated ester), CY5.5 SE (cyanine 5.5 succinimide activated ester), CY3 SE (cyanine 3 succinimide activated ester), TAMRA SE (abbreviated as TAM) (hydroxytetramethylrhodamine succinimide activated ester), ROX SE (6-hydroxy-X-rhodamine succinimide activated ester), TexasRed SE (abbreviated as TXR) (Texas Red succinimide activated ester).

In one embodiment, the volume of acetonitrile in the aqueous solution containing an organic solvent is 10%.

In one embodiment, the aqueous solution containing the organic solvent is used for elution twice.

In one embodiment, the dye is ROX, TXR, TAM, CY3, CY5, CY5.5 or CY7.

In one embodiment, the nucleic acid probe is a DNA nucleic acid probe or an RNA nucleic acid probe. The present invention provides a method for quickly removing free modified dyes in a nucleic acid probe after dye modification, the method does not require pre-cooling reagents, and the process flow is simple; the operation process time of the method is shortened from 4-15 hours to 10-20 minutes, which greatly shortens the operation cycle; the method has high retention of DNA/RNA nucleic acid probes, and the process loss is very small, and the general loss rate is about 1-4%; the method solves the problem of free modified dyes remaining in DNA/RNA nucleic acid probes after purification by high performance liquid chromatography, and improves the purity of the product. The method only needs to add a small amount of organic solvent for elution, which greatly reduces the generation of harmful waste liquid.

Detailed ways

In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative work should belong to the scope of protection of this application. In the following embodiments, unless otherwise specified, they are all conventional methods in this field.

Example 1 Selection experiment of different adsorbents

In this experiment, the types of adsorbents were screened, and two adsorbents, octadecylsilane bonded silica gel filler (C8) and octadecylsilane bonded silica gel filler (C18), were selected and respectively loaded in plastic column tubes with sieve plates to make C8 adsorption columns and C18 adsorption columns. These two adsorption columns were used to compare the adsorption effects of dye-modified nucleic acid probes and free modified dyes under the same experimental conditions. Because the maximum absorption wavelength of the free modified dye is consistent with the maximum absorption wavelength of the dye modified nucleic acid probe, the maximum absorption wavelength of the modified dye was selected as the detection wavelength on the high performance liquid chromatography, and the peak area ratio of the free modified dye in the original solution and the recovered solution was compared to characterize the adsorption degree of the free modified dye. The absorbance value of the nucleic acid probe in the solution was measured with an enzyme marker and then converted into a nmol value to calculate the nucleic acid probe content.

The specific experimental plan is as follows:

1. Dissolve 400 nmol of DNA/RNA probe containing amino structure in 400 ul of water at pH 8.5.

0.5M Na2CO3/NaHCO3 buffer, shake well to mix;

2. Use 150ul DMF solvent to dissolve 1200nmol activated ester modified dye solid, add it to the above buffer, and shake thoroughly to mix;

3. Place the mixed solution in a shaker and shake for 4-12 hours;

4. Take the crude product volume corresponding to 4 nmol, analyze it on HPLC, and calculate the percentage of free dye peak area at the wavelength of maximum absorption of the dye;

5. Add 10 ml of methanol solution to the C8 adsorption column and C18 adsorption column respectively, let it stand for 5 minutes, then slowly drip it and discard the waste liquid

6. Add 10 ml of ultrapure water to the C8 adsorption column and C18 adsorption column respectively, drip slowly, and discard the waste liquid;

7. Place 10ml centrifuge tubes at the lower ports of the C8 adsorption column and the C18 adsorption column respectively;

8. Divide the solution in step (3) into two equal parts, pour them into C8 and C18 adsorption columns respectively, drip them slowly, and collect the recovered solution;

9. Measure the absorbance of the recovery solution using an ELISA instrument and calculate the nmol content of the modified probe in the recovery solution.

10. Take the volume of the recovered solution corresponding to 4 nmol, analyze it on a high performance liquid chromatograph, and calculate the percentage of the peak area of the free modified dye at the maximum absorption wavelength of the modified dye;

11. The base numbers in the table refer to the number of deoxynucleotides or nucleotides in DNA/RNA. The adsorption rate data of nucleic acid probes on different adsorption columns are shown in Table 1, the adsorption amount data of free dyes on different adsorption columns are shown in Table 2, and the nucleic acid sequences of each sample are shown in Table 3. The calculation of the data

The formula is as follows:

Nucleic acid probe adsorption rate = (nucleic acid probe feed amount - nucleic acid probe content in the recovery solution) / nucleic acid probe feed amount.

Table 1

Table 2

table 3

Conclusion: From Table 1, it can be seen that the adsorption effect of C8 adsorbent on short-chain nucleic acid probes is acceptable, but the adsorption effect on long-chain nucleic acid probes is poor; the adsorption effect of C18 adsorbent on both short-chain and long-chain nucleic acid probes is better than that of C8 adsorbent, and the adsorption rate of modified probes is above 95%. From Table 2, it can be seen that when using C8 adsorbent and C18 adsorbent for adsorption, the peak area ratio of free modified dye in the recovered liquid has little deviation, and the ratio is less than 5%, which meets the expected requirements; and the corresponding C18 has a significantly smaller peak area ratio of free dye in the C8 solution. Considering the comprehensive data, C18 adsorbent is the best option.

Example 2 Selection experiment of different eluents

In this embodiment, the type of eluent is screened. Considering the solubility of DNA/RNA nucleic acid probes in organic solvents, the eluents used in the experiment are all 80% organic solvent + 20% ultrapure water (volume ratio). Under the same experimental conditions, the elution effects of the nucleic acid probe modified with the dye and the free modified dye are compared. In high performance liquid chromatography, the maximum absorption wavelength of the modified dye is still selected as the detection wavelength, and the peak area ratio of the free modified dye in the original solution and the recovered solution is compared to characterize the residual degree of the free modified dye. The absorbance value of the nucleic acid probe in the solution is measured with an enzyme marker and then converted into a nmol value to calculate the nucleic acid probe content.

The specific operations are as follows:

1. Dissolve 800 nmol of DNA/RNA nucleic acid probe containing amino structure in 800 ul of 0.5 M Na2CO3/NaHCO3 buffer with pH 8.5, and shake thoroughly to mix;

2. Use 300ul DMF solvent to dissolve 2400nmol activated ester modified dye solid, add it to the above buffer, and shake thoroughly to mix;

3. Place the mixed solution in a shaker and shake for 4-12 hours;

5. Add 10 ml of methanol solution to the C18 adsorption column, let it stand for 5 minutes, then slowly drip it and discard the waste liquid;

6. Add 10 ml of ultrapure water to the C18 adsorption column, drip slowly, and discard the waste liquid;

7. Divide the solution in step (3) into 4 equal parts, pour them into 4 C18 adsorption columns respectively, drip them slowly, and discard the waste liquid;

8. Place 10ml centrifuge tubes at the lower ports of the four C18 adsorption columns;

9. Add 8 ml of 80% methanol, 80% acetonitrile, 80% ethanol, and 80% propanol solution to each of the four C18 adsorption columns, drip slowly, and collect the recovered solution.

10. Measure the absorbance of the recovery solution and calculate the nmol content of the modified probe in the recovery solution.

11. Take the volume of the recovered solution corresponding to 4 nmol, analyze it on a high performance liquid chromatograph, and calculate the percentage of the peak area of the free modified dye at the maximum absorption wavelength of the modified dye;

12. The number of bases in the table refers to the number of deoxynucleotides or nucleotides in DNA/RNA. The recovery rate data of nucleic acid probes are shown in Table 4, the peak area percentage data of free dyes are shown in Table 5, and the nucleic acid sequences of each sample are shown in Table 6. The data calculation formula is as follows:

Nucleic acid probe recovery rate = nucleic acid probe content in the recovery solution / nucleic acid probe feed amount

Table 4

table 5

Table 6

Conclusion: From the data in Table 4, the recovery rate of modified nucleic acid probes using 80% methanol elution was significantly lower than that using 80% acetonitrile, 80% ethanol and 80% propanol elution; at the same time, from the data in Table 5, when eluted with the above four solvents, the residual free modification dye in the recovery liquid was relatively high, exceeding the expected standard, but when eluted with 80% methanol and 80% acetonitrile, the residual free modification dye in the recovery liquid was significantly lower than that using 80% ethanol and 80% propanol elution. Comprehensive consideration was given to both increasing the recovery rate of dye-modified nucleic acid probes as much as possible and reducing the residual free modification dye in the recovery liquid as much as possible. Therefore, elution with 80% acetonitrile solution was the best solution for this experiment.

Example 3 Experiment of elution with acetonitrile-water solution of different volume concentrations For the present invention, after elution with 80% acetonitrile, the residual free modifying dye in the recovered solution is still very high. In order to verify whether the content of organic solvent in the elution solvent has an effect on the residual free modifying dye in the recovered solution, this example selects five aqueous solutions containing acetonitrile in different proportions, and conducts elution experiments under the same conditions. In the experiment, the maximum absorption wavelength of the modifying dye is still selected as the detection wavelength, and the peak area ratio of the free modifying dye in the original solution and the recovered solution is compared to characterize the residual degree of the free modifying dye. The absorbance value of the nucleic acid probe in the solution is measured with an enzyme marker and then converted into a nmol value to calculate the nucleic acid probe content.

The specific operations are as follows;

1. Dissolve 1000 nmol of DNA/RNA nucleic acid probe containing amino structure in 1000 ul of 0.5 M Na2CO3/NaHCO3 buffer with pH 8.5, and shake thoroughly to mix;

2. Use 375ul DMF solvent to dissolve 3000nmol activated ester modified dye solid, add it to the above buffer, and shake thoroughly to mix;

3. Place the mixed solution in a shaker and shake for 4-12 hours;

7. Divide the solution in step (3) into 5 equal parts, pour them into 5 C18 adsorption columns respectively, drip them slowly, and discard the waste liquid;

8. Place 10ml centrifuge tubes at the lower ports of the five C18 adsorption columns;

9. Add 8 ml of 1% acetonitrile, 10% acetonitrile, 30% acetonitrile, 50% acetonitrile, and 70% acetonitrile solution (volume ratio) to 5 C18 adsorption columns respectively, drip slowly, and collect the recovered solution 1;

10. Replace the 10ml centrifuge tubes at the lower ports of 5 C18 adsorption columns;

11. Add 8 ml of 1% acetonitrile, 10% acetonitrile, 30% acetonitrile, 50% acetonitrile, and 70% acetonitrile solution (volume ratio) to 5 C18 adsorption columns respectively, drip slowly, and collect the recovered solution 2;

12. Replace the 10ml centrifuge tubes at the lower ports of 5 C18 adsorption columns;

13. Add 8 ml of 1% acetonitrile, 10% acetonitrile, 30% acetonitrile, 50% acetonitrile, and 70% acetonitrile solution (volume ratio) to 5 C18 adsorption columns respectively, drip slowly, and collect the recovered solution 3;

14. Measure the absorbance values of recovery solution 1, recovery solution 2, and recovery solution 3 for the five samples, and calculate the nmol content of the modified probe in the recovery solution;

15. Combine recovery solution 1, recovery solution 2, and recovery solution 3, take a combined liquid volume corresponding to 4 nmol, analyze on a high performance liquid chromatograph, and calculate the percentage of the free modification dye peak area at the maximum absorption wavelength of the modification dye;

16. The base numbers in the table refer to the number of deoxynucleotides or nucleotides in DNA/RNA. The recovery rate data of nucleic acid probes are shown in Table 7, the peak area percentage data of free dyes are shown in Table 8, and the nucleic acid sequences of each sample are shown in Table 9. The data calculation formula is as follows:

Table 7

Table 8

Table 9

Conclusion: From the data in Table 7, the total recovery rate of modified probes is low when eluted with 1% acetonitrile solution, which cannot meet the requirements. The total recovery rate of probes can reach more than 97% when eluted with 10% acetonitrile, 30% acetonitrile, 50% acetonitrile and 70% acetonitrile solutions. However, from the content of modified probes in recovery solution 1, recovery solution 2 and recovery solution 3, the content of modified probes in recovery solution 3 is extremely low, accounting for a very small proportion of the total recovery amount; from the data in Table 8, the content of acetonitrile in the eluent has a great influence on the residual free modified dye. The higher the proportion of acetonitrile, the greater the residual free modified dye. From the data value of recovery solution 1, the residual proportion of free modified dyes has exceeded the expected standard of no more than 5% when eluted with 30% acetonitrile, 50% acetonitrile and 70% acetonitrile solutions. Considering the high recovery rate of modified probes and the low residual free modified dyes, the best solution for this experiment is to use 10% acetonitrile and elute and recover twice. In order to further optimize the concentration of the eluent, a comparative test was conducted again using 5%, 10%, 15%, 20% and 25% acetonitrile aqueous solutions. Referring to the above test process, two elution experiments were conducted for the above five concentrations. The two elution recovery solutions were combined to calculate the total recovery rate of the nucleic acid probe. The data are shown in Table 10, and the peak area percentage data of the free dye are shown in Table 11:

Table 10

Table 11

The test results show that the total recovery rate of the nucleic acid probe eluted twice with 5%, 10%, 15%, 20% and 25% acetonitrile solutions can reach more than 70%, but when eluted with 20% and 25% acetonitrile solutions, the peak area proportion of the free modification dye in the recovery solution has exceeded 5%, which exceeds the expected standard range. Considering the recovery rate of the nucleic acid probe and the residual proportion of the free modification dye, 5-15% acetonitrile solution as the eluent can meet the purification purpose of the present invention, and 10% acetonitrile solution as the eluent has the best effect.

Example 4 Comparative test of different types of activated esters In order to expand the general adaptability of the present invention, experiments were carried out on nucleic acid probes modified with different dyes under the same experimental conditions, and the recovery rate of the nucleic acid modified probes and the peak area ratio of the free modified dye were compared. Because the maximum absorption wavelength of the free modified dye is consistent with the maximum absorption wavelength of the dye modified nucleic acid probe, the maximum absorption wavelength of the modified dye is selected as the detection wavelength on the high performance liquid chromatography, and the peak area ratio of the free modified dye in the original solution and the recovered solution is compared to characterize the degree of removal of the free modified dye. The absorbance value of the nucleic acid probe in the solution is measured by an enzyme marker and then converted into a nmol value to calculate the nucleic acid probe content.

The specific experimental plan is as follows:

1. Dissolve 200 nmol of DNA/RNA probe containing amino structure in 200 ul of 0.5 M Na2CO3/NaHCO3 buffer at pH 8.5, and shake thoroughly to mix;

2. Use 75ul DMF solvent to dissolve 600nmol activated ester modified dye solid, add it to the above buffer, and shake thoroughly to mix;

3. Place the mixed solution in a shaker and shake for 4-12 hours;

7. Pour the solution in step (3) into the C18 adsorption column, drip slowly, and discard the waste liquid;

8. Place a 10ml centrifuge tube at the lower port of the C18 adsorption column;

9. Add 8 ml of 10% acetonitrile to the C18 adsorption column, drip slowly, and collect the recovered solution 1

10. Replace the 10ml centrifuge tube at the lower port of the C18 adsorption column

11. Add 8 ml of 10% acetonitrile to the C18 adsorption column, drip slowly, and collect the recovered solution 2

12. Combine recovery solution 1 and recovery solution 2, measure the absorbance, and calculate the nmol content of the modified probe in the recovery solution;

13. Take the combined solution volume corresponding to 4 nmol, analyze it on HPLC, and calculate the percentage of free modified dye peak area at the maximum absorption wavelength of the modified dye

14. The base numbers in the table refer to the number of deoxynucleotides or nucleotides in DNA/RNA. The recovery rate data of nucleic acid probes are shown in Table 12, the peak area percentage data of free dyes are shown in Table 13, and the nucleic acid sequences of each sample are shown in Table 14. The data calculation formula is as follows:

Table 12

Table 13

Table 14

Conclusion: From the experimental data, it can be seen that the present invention has a good effect on the removal of different types of free modified dye residues in DNA/RNA nucleic acid probes, especially for ROX, TXR,

The residual removal effect of TAM was better than that of CY3, CY5, CY5.5 and CY7.

It should be understood that the invention disclosed is not limited to the specific methods, protocols, and materials described, as these may vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments.

The embodiments are intended, but not intended to limit the scope of the invention, which is limited only by the appended claims.

Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

Claims

A method for purifying a dye-modified nucleic acid probe, characterized in that the method retains excess free modifying dye and the dye-modified nucleic acid probe on an adsorbent through the interaction between the adsorbent and the non-polar functional groups of the free modifying dye and the dye-modified nucleic acid probe, and then elutes the column containing the adsorbent with an aqueous solution containing an organic solvent, and utilizes the polarity difference between the free modifying dye and the dye-modified nucleic acid probe to selectively elute the dye-modified nucleic acid probe from the adsorbent, while the free modifying dye is retained on the adsorbent, so as to purify the dye-modified nucleic acid probe; the adsorbent is a C18 adsorption column, and the volume of acetonitrile in the aqueous solution containing an organic solvent is 5-15%.
The nucleic acid probe purification method according to claim 1 is characterized in that the volume of acetonitrile in the aqueous solution containing an organic solvent is 10%.
The method for purifying a nucleic acid probe according to claim 1 is characterized in that the aqueous solution containing the organic solvent is used for elution twice.
The nucleic acid probe purification method according to claim 1 is characterized in that the modified dye is ROX, TXR, TAM, CY3, CY5, CY5.5 or CY7.
The nucleic acid probe purification method according to claim 1 is characterized in that the nucleic acid probe is a DNA nucleic acid probe or an RNA nucleic acid probe.