CN110511935B - Aptamer truncation optimization method based on S1 enzyme cleavage - Google Patents
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Abstract
An aptamer truncation optimization method based on S1 nuclease cleavage belongs to the field of medical diagnosis. The method comprises magnetic bead and protein coupling (M & P), aptamer and M & P incubation (M & P & A), S1 nuclease truncation optimization and mass spectrometry. The aptamers achieve truncation optimization of sequence length by this method. Based on steric hindrance, nuclease cuts aptamer and target to combine with low efficiency of compact segment sequence, and the segment is preserved in a large amount and presents larger ion abundance in mass spectrometric detection. The KD value of the truncated sequence and the target PD-L1 protein is reduced by 54.67 percent compared with the original sequence, thereby verifying the feasibility of the method. The present invention can provide an experiment-based method for truncation optimization of aptamers and can be used to determine the binding region of an aptamer to a target.
Description
Technical Field
The invention belongs to the field of medical diagnosis, and particularly relates to a method for optimizing a screened aptamer by utilizing nuclease digestion. Currently, truncation optimization of aptamers is mainly software computational simulation, and direct truncation optimization based on experiments is rarely involved. The method can provide an experimental method for truncation optimization of the aptamer and provides a basis for clarifying an interaction mechanism of the aptamer and the target.
Background
With the rapid development of socioeconomic and the accelerated aging of population in China, cancer has become a high-mortality disease second to cardiovascular diseases. Cancer is a chronic disease, and early diagnosis and early treatment have great significance for improving the survival rate of patients. Tumor marker detection is an important means for early detection, identification and prognosis of cancer, and no matter the content of the tumor marker in a patient or a healthy person is trace, a highly sensitive detection method is required. At present, the detection method of tumor markers mainly utilizes specific interaction between proteins for detection. Programmed cell death ligand 1(PD-L1) is an important tumor marker, a transmembrane protein, which is overexpressed on a variety of cancer cells and is the primary ligand for programmed cell death receptor 1 (PD-1). The PD-L1 protein plays an important role in regulating the immunity of an anti-tumor host, enables tumor cells to escape the immune system of the host and promotes the metastasis of the cancer cells.
The expression and purification cost of the antibody protein is high, the antibody protein is volatile, the batch-to-batch difference is large, and the adverse effect is generated on the detection result of the tumor marker. Aptamers are single-stranded oligonucleotide sequences screened from DNA or RNA libraries that bind with high specificity to a target substance. The aptamer binds specifically to the target substance through base pair stacking, hydrogen bonding, electrostatic and hydrophobic interactions, and the like. Compared with protein, the aptamer has the advantages of high affinity, strong specificity, small molecular weight, easy synthesis and modification, short screening period and the like, and is a good substitute for protein antibodies.
The aptamer obtained by directly screening from the library can not be directly applied to a system to be detected with complex components, and further truncation optimization is needed. As the length of nucleic acids increases, they have a greater chance of being locked into a metastable inactive conformation due to accidental interactions. Nucleotide base stacking forces allow separation of the inactive and active conformations, indicating that shorter aptamers have a greater tendency to change to the active conformation. Aptamer truncation is mainly performed by molecular docking simulation through software, and sequence truncation is performed according to simulated aptamer-target action sites. The invention takes PD-L1 as a target molecule and S1 nuclease as a truncated enzyme, and based on that the enzyme digestion efficiency is adversely affected by the binding region of an aptamer and the target, the binding region fragment presents larger ion abundance in mass spectrometric detection, and the sequence with higher affinity is optimized by truncation.
Disclosure of Invention
The invention aims to overcome the defects of truncation optimization of the existing aptamer, provides a novel truncation optimization method of the aptamer, has more practicability compared with molecular dynamics simulation based on software, and provides a basis for clarifying an interaction mechanism of the aptamer and a target. The invention takes PD-L1 as a target molecule and S1 nuclease as a truncating enzyme, and truncates a known aptamer to optimize a sequence with higher affinity.
The above purpose is realized by the following technical scheme:
1. synthesizing a PD-L1 aptamer represented by the following sequence:
5’-GCT GTG TGA CTC CTG CAA GAC GGA CCA GCC TTG CCG CAA GAC GGA CCA GGG ATT CAA ACG AGC AGC TGT ATC TTG TCT CC-3’。
2. optimized aptamers using nuclease truncation
The enzyme cutting and cutting optimization process uses the following reagents: 2 × protein binding solution concentration composition: 100mM sodium phosphate, 600mM NaCl, 0.02% Tween-20, pH8.0(1 × protein binding solution is 2 × protein binding solution diluted 1 times).
SELEX binding solution: 150mM NaCl, 5mM KCl, 1mM MgCl2，1mM CaCl240mM 4-hydroxyethylpiperazine ethanesulfonic acid, pH 7.4.
Eluent: 20mM Tris, 500mM imidazole, pH 7.5.
5 × reaction solution: 200mM sodium acetate, 1.5M NaCl, 10mM ZnSO4, pH 4.5.
Human PD-L1/B7-H1 protein, His Tag (HPLC-verified) was purchased from Acrobiosystems (Bethesda, Md.).
2.1 reconstitution of PD-L1 protein
Lyophilized PD-L1 was reconstituted to a 400. mu.g/mL stock solution with 250. mu.L of sterile deionized water and solubilized at room temperature for 30-60 minutes. The mixture was divided into 10 portions of 25. mu.L each, and placed in a refrigerator at-80 ℃.
2.2 connection of PD-L1 protein to magnetic beads
2.2.1 Add 5. mu.L of PD-L1 protein solution to 700. mu.L of 1 Xprotein binding solution.
2.2.2 mix the beads in the vial (vortex > 30 seconds or tip, spin 5 minutes).
2.2.3 transfer 50. mu.L of magnetic beads to a microcentrifuge tube and place on a magnetic separation rack for 2 minutes, aspirate and remove supernatant. The sample (prepared in 1 × protein binding solution) was added to the magnetic beads and mixed well.
2.2.4 incubation on roller at 37 ℃ for 5 min, after completion of incubation, place on magnetic separation rack for 2 min, and discard supernatant.
2.2.5 washing the magnetic bead-protein complex 3 times (300. mu.L each) with 1 Xprotein binding solution, magnetic separation for 2 minutes and discarding the supernatant.
2.3 aptamer incubation
2.3.1 aptamer was dissolved in sterile water, heat denatured at 95 ℃ for 5 minutes, ice-bathed for 10 minutes, and mixed with equal volume of SELEX binding solution.
2.3.2 adding into the magnetic bead-protein complex, mixing evenly, and incubating for 30 minutes at room temperature.
2.3.3 at the end of the incubation the supernatant was magnetically separated off and washed 2 times with PBS-T buffer (200. mu.L each).
2.4S 1 nuclease assay
2.4.1 the magnetic bead complex obtained in 2.3.3 and an appropriate amount of S1 nuclease were added to a 1 Xreaction solution system, reacted at 37 ℃ for 30 minutes, magnetically separated, washed 2 times with PBS-T (500. mu.L each), and the supernatant was removed.
2.4.2 adding 200 μ L of eluent, mixing, boiling water bath at 100 deg.C for 10 min (winding centrifugal tube with adhesive tape during boiling, inserting into foam or sponge, and placing into boiling water), magnetically separating, and collecting supernatant.
3. Mass spectrometric analysis
3.1 analysis of the supernatant obtained in 2.4.2 using a Saimer FeilLTQXL type linear ion trap mass spectrometer, the results are shown in FIG. 1.
3.2 comparing the mass spectrogram with the truncated subsequence to obtain a truncated optimized subsequence as follows: 5'-AAGACGGACCAGCCTTGCCGCAAGACGGACCAGGGATT-3' are provided. FIG. 2 is a secondary structure diagram of the position of a truncated aptamer in a pro-aptamer.
4 affinity analysis:
4.1 original PD-L1 aptamer with fluorescent group marked at 5' end and truncated PD-L1 aptamer are prepared into a series of concentrations by SELEX binding solution.
4.2 mu.L of each concentration gradient was taken, and the mixture was incubated in a water bath at 95 ℃ for 5 minutes and in an ice bath for 10 minutes, and then added to the PD-L1-His tag magnetic bead complex and incubated at 37 ℃ for 30 minutes.
4.3 the 4.2 complexes of magnetic bead-PD-L1-aptamer were washed 2 times with 1 Xprotein binding solution (200. mu.L each) and finally resuspended in 200. mu.L and the fluorescence intensity was measured using a microplate reader.
4.4 with the aptamer concentration as abscissa and the fluorescence intensity as ordinate, drawing a binding saturation curve, and obtaining the Kd value of the original PD-L1 aptamer to be 115.15nM and the Kd value of the truncated PD-L1 aptamer to be 52.20 nM. As shown in fig. 3, after truncation, the aptamer Kd value decreased by 54.67%, indicating that the aptamer was optimized for effective truncation.
The invention has the advantages that:
(1) compared with the traditional truncation optimization technology (software prediction truncation), the method has the advantages of direct truncation optimization based on experiments and strong operability.
(2) The invention provides a factual basis for clarifying the interaction mechanism of the aptamer and the target.
(3) The aptamer obtained by truncation optimization of the invention can bind to PD-L1, and has higher affinity than the original aptamer.
(4) The aptamer is used for modifying different reporter molecules, so that various biosensors can be constructed, and the biosensors are used for detecting exosomes carrying PD-L1 in human plasma and can also be used for separating and detecting cancer cells of high-surface PD-L1.
Drawings
FIG. 1: diagram of mass spectrum result after digestion of aptamer
FIG. 2: secondary structure diagram of the position of a truncated aptamer in a pro-aptamer
FIG. 3: non-linear fitting of kd values of aptamers before and after truncation optimization (A. original sequence; B. truncated sequence)
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1: synthesis of PD-L1 aptamer represented by the following sequence
PD-L1 aptamer:
5’-GCT GTG TGA CTC CTG CAA GAC GGA CCA GCC TTG CCG CAA GAC GGA CCA GGG ATT CAA ACG AGC AGC TGT ATC TTG TCT CC-3’。
example 2: optimized aptamers using nuclease truncation
2. Optimization of enzyme digestion truncation
The enzyme cutting and cutting optimization process uses the following reagents: the final concentration of 2 × protein binding solution was: 100mM sodium phosphate, 600mM NaCl, 0.02% Tween-20, pH8.0(2 × protein binding solution is 2 × protein binding solution diluted 1 times).
SELEX binding solution: 150mM NaCl, 5mM KCl, 1mM MgCl2，1mM CaCl240mM 4-hydroxyethylpiperazine ethanesulfonic acid, pH 7.4.
Eluent: 20mM Tris, 500mM imidazole, pH 7.5.
Human PD-L1/B7-H1 protein, His Tag (HPLC-verified) was purchased from Acrobiosystems (Bethesda, Md.).
2 × Reaction Buffer: 200mM sodium acetate, 1.5M NaCl, 10mM ZnSO4, pH 4.5.
2.1 reconstitution of PD-L1 protein
Lyophilized PD-L1 was reconstituted to a 400. mu.g/mL stock solution with 250. mu.L of sterile deionized water and solubilized at room temperature for 30-60 minutes. The mixture was divided into 10 portions of 25. mu.L each, and placed in a refrigerator at-80 ℃.
2.2 connection of PD-L1 protein to magnetic beads
2.2.1 Add 5. mu.L of PD-L1 protein solution to 700. mu.L of 1 Xprotein binding solution.
2.2.2 mix the beads in the vial (vortex > 30 seconds or tip, spin 5 minutes).
2.2.3 transfer 50. mu.L of magnetic beads to a microcentrifuge tube and place on a magnetic separation rack for 2 minutes, aspirate and remove supernatant. The sample (prepared in 1 × protein binding buffer) was added to the beads and mixed well.
2.2.4 incubation on roller at 37 ℃ for 5 min, after completion of incubation, place on magnetic separation rack for 2 min, and discard supernatant.
2.2.5 washing the magnetic bead-protein complex 3 times (300. mu.L each) with 1 Xprotein binding solution, magnetic separation for 2 minutes and discarding the supernatant.
2.3 aptamer incubation
2.3.1 aptamer was dissolved in sterile water, heat denatured at 95 ℃ for 5 minutes, ice-bathed for 10 minutes, and mixed with equal volume of SELEX binding solution.
2.3.2 adding into the magnetic bead-protein complex, mixing evenly, and incubating for 30 minutes at room temperature.
2.3.3 at the end of the incubation the supernatant was magnetically separated off and washed 2 times with PBS-T buffer (200. mu.L each).
2.4S 1 nuclease assay
2.4.1 the magnetic bead complex obtained in 2.3.3 and an appropriate amount of S1 nuclease were added to a 1 Xreaction solution system, reacted at 37 ℃ for 30 minutes, magnetically separated, washed 2 times with PBS-T (500. mu.L each), and the supernatant was removed.
2.4.2 adding 200 μ L of eluent, mixing, boiling water bath at 100 deg.C for 10 min (winding centrifugal tube with adhesive tape during boiling, inserting into foam or sponge, and placing into boiling water), magnetically separating, and collecting supernatant.
Example 3 Mass Spectrometry
3.1 analysis of the supernatant obtained in 2.4.2 using a Saimer FeilLTQXL type linear ion trap mass spectrometer, the results are shown in FIG. 1.
3.2 comparing the mass spectrogram with the truncated subsequence to obtain a truncated optimized subsequence as follows: 5'-AAG ACG GAC CAG CCT TGC CGC AAG ACG GAC CAG GGA TT-3' are provided. FIG. 2 is a secondary structure diagram of the position of a truncated aptamer in a pro-aptamer.
Example 4 affinity assay:
4.1 original PD-L1 aptamer with fluorescent group marked at 5' end and truncated PD-L1 aptamer are prepared into a series of concentrations by SELEX binding solution.
4.2 mu.L of each concentration gradient was taken, and the mixture was incubated in a water bath at 95 ℃ for 5 minutes and in an ice bath for 10 minutes, and then added to the PD-L1-His tag magnetic bead complex and incubated at 37 ℃ for 30 minutes.
4.3 the 4.2 complexes of magnetic bead-PD-L1-aptamer were washed 2 times with 1 Xprotein binding solution (200. mu.L each) and finally resuspended in 200. mu.L and the fluorescence intensity was measured using a microplate reader.
4.4 with the aptamer concentration as abscissa and the fluorescence intensity as ordinate, drawing a binding saturation curve, and obtaining the Kd value of the original PD-L1 aptamer to be 115.15nM and the Kd value of the truncated PD-L1 aptamer to be 52.20 nM. As shown in fig. 3, after truncation, the aptamer Kd value decreased by 54.67%, indicating that the aptamer was optimized for effective truncation.
Sequence listing
<110> Beijing university of chemical industry
<120> aptamer truncation optimization method based on S1 enzyme cleavage
<141> 2019-01-28
<160> 2
<170> SIPOSequenceListing 1.0
<210> 2
<211> 80
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
gctgtgtgac tcctgcaaga cggaccagcc ttgccgcaag acggaccagg gattcaaacg 60
agcagctgta tcttgtctcc 80
<210> 2
<211> 38
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
aagacggacc agccttgccg caagacggac cagggatt 38
Claims (2)
1. A method for optimizing aptamer truncation based on S1 nuclease cleavage,
PD-L1 is used as a target molecule, S1 nuclease is used as a truncated enzyme, and the method is realized by the following technical scheme:
1) synthesizing a PD-L1 aptamer represented by the following sequence:
5’-GCT GTG TGA CTC CTG CAA GAC GGA CCA GCC TTG CCG CAA GAC GGA CCA GGG ATT CAA ACG AGC AGC TGT ATC TTG TCT CC-3’；
2) the truncated optimized aptamer is cut by nuclease, and the method comprises the following steps:
the enzyme cutting and cutting optimization process uses the following reagents: 2 × protein binding solution concentration composition: 100mM sodium phosphate, 600mM NaCl, 0.02% Tween-20, pH 8.0; 1 × protein binding solution is to dilute 2 × protein binding solution by 1 time;
SELEX binding solution: 150mM NaCl, 5mM KCl, 1mM MgCl2，1 mM CaCl240mM 4-hydroxyethylpiperazine ethanesulfonic acid, pH 7.4;
eluent: 20mM Tris, 500mM imidazole, pH 7.5;
5 × reaction solution: 200mM sodium acetate, 1.5M NaCl, 10mM ZnSO4, pH 4.5;
2.1 reconstitution of PD-L1 protein
2.2 connection of PD-L1 protein to magnetic beads
2.2.1 adding 5. mu.L of PD-L1 protein solution into 700. mu.L of 1 Xprotein binding solution;
2.2.2 mixing of magnetic beads in vials
2.2.3 transfer 50. mu.L of magnetic beads to a microcentrifuge tube, place on a magnetic separation rack for 2 minutes, aspirate and remove supernatant; adding a sample into the magnetic beads, and uniformly mixing;
2.2.4 incubation on roller at 37 ℃ for 5 minutes, placing on magnetic separation rack for 2 minutes after incubation is finished, and discarding supernatant;
2.2.5 washing the magnetic bead-protein complex 3 times with 1 Xprotein binding solution, magnetically separating for 2 minutes and discarding the supernatant;
2.3 aptamer incubation
2.3.1 dissolving the aptamer in sterile water, performing thermal denaturation at 95 ℃ for 5 minutes, performing ice bath for 10 minutes, and uniformly mixing with the SELEX binding solution with the same volume;
2.3.2 adding the mixture into the magnetic bead-protein complex, uniformly mixing, and incubating for 30 minutes at room temperature;
2.3.3 magnetic separation to remove supernatant after incubation, washing for 2 times with PBS-T buffer solution;
2.4 nuclease cleavage assay of S1
2.4.1 adding the magnetic bead complex obtained in 2.3.3 and S1 nuclease into a 1 Xreaction solution system, reacting for 30 minutes at 37 ℃, carrying out magnetic separation, washing for 2 times by PBS-T, and removing supernatant;
2.4.2 adding 200 μ L of eluent, mixing well, boiling water bath at 100 deg.C for 10 min, magnetically separating, and collecting supernatant;
3) mass spectrometry analysis
3.1 analyzing the supernatant obtained by the 2.4.2 by using a Saimer FeilLTQXL type linear ion trap mass spectrometer;
3.2 comparing the mass spectrogram with the truncated subsequence to obtain a truncated optimized subsequence as follows: 5'-AAGACGGACCAGCCTTGCCGCAAGACGGACCAGGGATT-3' are provided.
2. A truncated optimized sequence having the sequence: 5'-AAGACGGACCAGCCTTGCCGCAAGACGGACCAGGGATT-3' are provided.
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| CN104630230A (en) * | 2015-01-06 | 2015-05-20 | 江南大学 | Group of nucleic acid aptamers for specifically recognizing okadaic acid |
| CN104894135A (en) * | 2015-04-28 | 2015-09-09 | 中国人民解放军第二军医大学 | High affinity adapter body capable of specifically binding with saxitoxin acetate and application thereof |
| CN108977448A (en) * | 2018-08-03 | 2018-12-11 | 湖北师范大学 | For detecting aptamer and its screening technique and the application of clenobuterol hydrochloride |
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| CN104630230A (en) * | 2015-01-06 | 2015-05-20 | 江南大学 | Group of nucleic acid aptamers for specifically recognizing okadaic acid |
| CN104894135A (en) * | 2015-04-28 | 2015-09-09 | 中国人民解放军第二军医大学 | High affinity adapter body capable of specifically binding with saxitoxin acetate and application thereof |
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