A method for rapid screening of aptamer based on exonuclease degradation rate

Abstract

The application provides a method for rapidly screening aptamers based on exonuclease degradation rate, a reaction system with added aptamers and targets, and a control system with only added aptamers, after adding exonuclease, part of samples are taken out every certain time and added into a quenching solution containing a fluorescent dye, the fluorescent intensity is detected and the relative fluorescent intensity of the reaction system and the control system is compared, and the binding capacity of the aptamers and the targets is indirectly represented. The application can qualitatively detect the affinity of multiple groups of aptamers in a high-throughput and large-batch manner, greatly shortens the screening period, reduces the cost, and is beneficial to subsequent quantitative detection.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and immune screening, and more specifically to a method for rapidly screening aptamers based on the degradation rate of exonucleases. Background Technology

[0002] Nucleic acid aptamers are RNA or single-stranded DNA molecules that can form a specific spatial structure and bind specifically to a target substance. The range of target substances is broad, including proteins, small molecules, metal ions, and even whole cells. Nucleic acid aptamers are obtained through in vitro screening. By repeatedly screening and enriching random libraries, specific aptamers with high affinity for the target can be identified. Compared to antibodies, aptamers are more stable, less susceptible to environmental influences, and can be chemically synthesized, ensuring sequence accuracy, reducing batch-to-batch variability, and at a lower synthesis cost. Therefore, using aptamers as probes for small molecule detection is receiving increasing attention in clinical testing, treatment, and environmental monitoring.

[0003] To meet practical applications, aptamers must possess sufficient affinity and specificity for their corresponding targets. With the development of DNA sequencing technology, enriched sequences obtained through in vitro screening can be sequenced and analyzed to yield tens of thousands of candidate sequences. The affinity and specificity of these sequences for the target need to be tested one by one.

[0004] Existing methods for detecting nucleic acid aptamer affinity mainly rely on specialized instruments and equipment, such as isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and microscale thermophoresis (MST). These specialized instruments can obtain complete binding parameters of the aptamer-target interaction. However, these methods can only detect the interaction between one aptamer and the target at a time, resulting in low throughput, long processing times, high instrument costs, high detection costs, and the need for skilled personnel, making them unsuitable for large-scale aptamer screening. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a method for evaluating the affinity between aptamers and targets based on changes in the degradation rate of exonuclease-coupled target aptamers.

[0006] Technical Solution: To achieve the above-mentioned objective, the present invention provides a method for rapidly screening aptamers based on the degradation rate of exonucleases, comprising: preparing a reaction system containing aptamers and targets, and a control system containing only aptamers; after incubation of the reaction system and the control system, adding exonucleases to each and mixing well; starting from t=0, taking a portion of the sample at regular intervals and adding it to a quenching solution containing fluorescent dye, detecting the fluorescence intensity, and obtaining a reaction system dataset and a control system dataset based on relative fluorescence intensity; comparing the reaction system dataset and the control system dataset.

[0007] Because exonucleases exhibit degradative activity towards aptamers, the degradation rate decreases as the exonuclease degrades the aptamer to the target-binding region after it binds to its target. The stronger the binding affinity between the aptamer and the target, the slower the degradation rate. Based on this, this invention utilizes specific fluorescent dyes to monitor aptamer degradation, indirectly characterizing the binding affinity between the aptamer and the target.

[0008] It should be noted that the reaction system is not limited to a single system, but can be a collection of multiple reaction systems formed by aptamers with different types, concentrations, or coupling environments. Therefore, large-scale aptamer screening can be performed on multiple aptamers, multiple targets, multiple coupling environments, and multiple control systems to identify aptamers with high affinity for the target, providing a data foundation for subsequent quantitative analysis. The reaction system and control system prioritize aptamer binding to the target; this system may not be optimal for enzyme activity, and DMSO, BSA, and MgCl2 may be added as necessary.

[0009] The aptamers described in this invention are selected from, but are not limited to, single-stranded DNA, single-stranded RNA, double-stranded DNA, and double-stranded RNA, with a preferred aptamer length of 15-90 bp. These aptamers can form stable secondary structures such as stem-loop structures and hairpin structures in their corresponding buffer systems.

[0010] The target described in this invention refers to a small molecule that can specifically bind to a given aptamer. The aptamer can be screened using SELEX technology and can bind to the aptamer through hydrogen bonding, intermolecular forces, electrostatic adsorption, or base stacking forces.

[0011] To achieve the purpose of the invention, the fluorescent dye is selected from those that exhibit a significant increase in fluorescence intensity upon binding to single-stranded or double-stranded nucleotides, but no increase in fluorescence intensity upon binding to single nucleotides. If the fluorescent dye has a stronger binding affinity to single nucleotides, it will result in a high background value, failing to reveal differences in degradation rates.

[0012] Specifically, the fluorescent dye is selected from, but is not limited to, any one of purpurin-based nucleic acid staining agents or EB. More preferably, the fluorescent dye is selected from, but is not limited to, SYBR from Invitrogen. TM SYBR series dyes TM Gold, SYBR TM Green I, SYBR TM Green II, SYBR TM Safe), GelRed series dyes from Biotium; GoldView series dyes, EB, etc.

[0013] The exonucleases described in this invention are selected from, but are not limited to, any one or two of T5 exonuclease, exonuclease I (E. coli), exonuclease III (E. coli), and exonuclease T. These exonucleases can specifically degrade single-stranded or double-stranded nucleic acid sequences from one or both ends; particularly when nucleic acids are bound to other molecules, the degradation rate of the enzyme slows down. For example, T5 exonuclease degrades DNA along the 5'→3' direction; it can degrade double-stranded DNA, single-stranded DNA, and nicked plasmid DNA. It can initiate DNA degradation from the 5' end, as well as from nicks or notches in linear or circular double-stranded DNA, but it cannot degrade supercoiled double-stranded DNA. Exonuclease I (E. coli) is a single-strand-specific 3'→5' exonuclease that breaks down ssDNA from the 3' end to generate 5'-mononucleotides; this enzyme has very high specificity for single-stranded DNA and does not degrade double-stranded DNA or RNA. Exonuclease III (E. coli) possesses 3'→5' exonuclease activity, acting on double-stranded DNA. It progressively cleaves single nucleotides from the 3' end, removing only a few nucleotides with each substrate binding, resulting in a progressive deletion within the DNA molecule. The substrate is double-stranded DNA with blunt ends or 5' overhangs, but it can also act on nicks in double-stranded DNA, degrading the DNA molecule from the 3' end and releasing 5' single nucleotides. It almost completely does not cleave 3' overhangs, especially those longer than 4 nt, and it also cannot cleave thiophosphoryl bonds. Different exonucleases have different optimal conditions and sites of action; therefore, the type and concentration of enzyme must be selected based on aptamer length and its predicted secondary structure, buffer system, and enzymatic digestion temperature.

[0014] Furthermore, the aptamer undergoes pretreatment before being added to the reaction system or control system. The pretreatment step involves purification, dissolution in ultrapure water, and then heating to 90-95°C for denaturation for 10-20 minutes, followed by transfer to ice water or cooling at room temperature for later use. More preferably, denaturation is performed at approximately 95°C for 10 minutes, followed by transfer to ice water for later use.

[0015] Furthermore, the final concentration of the exonuclease after being added to the reaction system or control system is 0.01-2 U / μl, preferably 0.15-0.5 U / μl.

[0016] The quenching solution serves two purposes: firstly, it inactivates the exonuclease, stopping the enzymatic digestion reaction; secondly, it dissociates the double-stranded nucleic acid aptamer into a single-stranded state, promoting binding to the fluorescent dye. Specifically, the quenching solution comprises: 10-50 mM Tris-HCl (pH 7.4), 10%-80% v / v deionized formamide, and 2-100 mM EDTA. The fluorescent dye is then diluted with this quenching buffer, resulting in a final concentration of 1-10 times for the detection system.

[0017] As a further optimization of the present invention, the quenching buffer comprises:

[0018] Tris-HCl 10–50 mM, pH 7.4

[0019] Deionized formamide 10-80 v / v%.

[0020] EDTA 2-100mM.

[0021] More preferably, the quenching buffer solution is:

[0022] Tris-HCl 10-20 mM, pH 7.4

[0023] Deionized formamide 10-50 v / v%.

[0024] EDTA 2-50mM

[0025] Dilute the fluorescent dye 1-2 times.

[0026] The relative fluorescence intensity R = F of this invention t / F0×100%, where F t Let t be the fluorescence intensity of the reaction system or control system at time t, and F0 be the initial fluorescence intensity of the reaction system or control system. Based on this, a dataset of the reaction system and a dataset of the control system based on relative fluorescence intensity are obtained, and the two are compared. The preferred comparison method is based on time and relative fluorescence intensity; specifically, it is based on comparing the area under the fluorescence signal curves of the reaction system and the control system over time.

[0027] AUC = (AUC Target -AUC ck ) / AUC ck

[0028] Among them, AUC Target The AUC is the area under the fluorescence curve of the time-dependent target system. ck The area under the fluorescence curve of the control system without the target is represented; the greater the difference between the two, the stronger the affinity.

[0029] The following provides supplementary explanations of the technical terms involved in this invention:

[0030] The incubation of the reaction system and control system described in this invention refers to the binding of the purified aptamer to the target at a specific temperature. Conventional purification methods are selected from either HPLC or PAGE purification. The incubation temperature is determined based on enzyme activity and the optimal temperature for aptamer-target binding, generally between 25-37°C.

[0031] Quenching refers to the process of adding reagents such as EDTA to a buffer system to inhibit the enzymatic hydrolysis of aptamers by exonucleases, thereby terminating the reaction.

[0032] Exonuclease: Among nucleic acid hydrolases, exonucleases are enzymes that sequentially hydrolyze phosphodiester bonds from the ends or specific positions of the molecular chain to generate mononucleotides.

[0033] The method of this invention can simultaneously and rapidly screen the affinity of multiple aptamers for a target. Combined with automated pipetting equipment, it enables automated, large-scale screening of aptamer-target affinity, significantly improving screening efficiency and reducing labor costs. Furthermore, the instruments, equipment, and reagents used in this screening method are readily available and easy to operate, which helps reduce screening costs. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the technical principle of the present invention;

[0035] Figure 2 The variation trend of relative fluorescence intensity over time in different reaction systems of Example 2 is shown;

[0036] Figure 3 The curves showing the change of relative fluorescence intensity over time under different enzyme concentrations in Example 3 are shown. The corresponding exonucleases are Exo III (0.5 U / μl) + Exo I (1.5 U / μl).

[0037] Figure 4 The curves showing the change of relative fluorescence intensity over time under different enzyme concentrations in Example 3 are shown. The corresponding exonucleases are Exo III (1.0 U / μl) + Exo I (3.0 U / μl).

[0038] Figure 5 Example 4 compares the relative fluorescence intensity trends of aptamers with different aldosterone affinities;

[0039] Figure 6 for Figure 5 The slope curve after the curve has been transformed;

[0040] Figure 7 for Figure 5 The curve obtained after polynomial fitting.

[0041] Figure 8 The ITC caloric curve of the affinity aldosterone aptamer in Example 5 is shown.

[0042] Figure 9 for Figure 8 The fitted curve;

[0043] Figure 10 The ITC caloric curve of the affinity-free aldosterone aptamer in Example 5 is shown.

[0044] Figure 11 for Figure 10 The scatter distribution shows that it cannot fit the curve.

[0045] Figure 12 The relative fluorescence intensity curves for affinity verification of known aptamers in Example 6 are shown below; where (a) and (b) represent the relative fluorescence intensity changes of the thrombin aptamer in systems containing and without thrombin, respectively; and (c) and (d) represent the relative fluorescence intensity changes of the APT aptamer in systems containing and without ATP, respectively. Detailed Implementation

[0046] Example 1

[0047] This embodiment provides a method for rapid screening of aptamers based on the degradation rate of exonucleases, and prepares a reaction system with aptamers and targets added and a control system with only aptamers added.

[0048] The aptamer is selected from any one of single-stranded DNA, single-stranded RNA, double-stranded DNA, and double-stranded RNA, and the aptamer length is preferably 15-90 bp; it is denatured and cooled before use.

[0049] After incubating the reaction system and control system at 25°C for 1 hour, exonuclease was added to each and the mixture was stirred. Starting from t=0, a portion of the sample was taken at regular intervals and added to a quenching solution containing fluorescent dye (final concentration: 10mM Tris-HCl, 20% deionized formamide, 10mM EDTA, 1×SYBR Gold), and the relative fluorescence intensity was measured.

[0050] Relative fluorescence intensity R = F t / F0×100%, where F t Ft represents the fluorescence intensity of the reaction system or control system at time point t, and F0 represents the initial fluorescence intensity of the reaction system or control system. The relative fluorescence intensities obtained at different time points for each system are calculated and compared to determine the affinity of the aptamer for the target. The reaction mechanism of this invention can be found in [reference needed]. Figure 1 As shown, the top route indicates that the aptamer has an affinity for the target, which inhibits enzymatic digestion. After the reaction, adding a fluorescent dye will produce a fluorescent signal. The bottom route indicates that the aptamer has no affinity for the target, and the exonuclease can degrade the aptamer. Finally, adding a fluorescent dye will not produce a signal.

[0051] Table 1 lists several preferred targets and their corresponding aptamer sequences. The reaction system contains 5 μl of aptamer, 40 μl of buffer system with or without the target, and 5 μl of exonuclease. The buffer system can be PBS or Tris-HCl. The exonuclease is selected from any one or two of T5 Exo, Exo I, Exo III, and Exo T, and is adaptively adjusted according to the affinity between the target and the aptamer and different aptamer sequences.

[0052] Table 1 Examples of targets and corresponding aptamers

[0053]

[0054] The above method can be used for rapid initial screening of aptamers, thereby improving the efficiency of obtaining high-affinity aptamers. The corresponding aptamer nucleotide sequences are only preferred embodiments, and those skilled in the art can make adaptive modifications as needed. All such modifications should be understood to be within the scope of protection of this invention without departing from the principles of this invention.

[0055] Example 2: Analysis of the degradation ability of exonucleases on ssDNA in different reaction systems

[0056] Prepare the DPBS buffer system: 1 mM CaCl2, 2.7 mM KCl, 1.5 mM KH2PO4, 0.5 mM MgCl2, 137 mM NaCl, 8 mM Na2HPO4; then prepare a buffer system containing 0.1 mg / ml BSA and a buffer system with increased magnesium ion concentration.

[0057] Prepare a Tris-HCl buffer system: 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2.

[0058] Two reaction systems were prepared based on the above buffer system. ssDNA (SEQ NO.1) was added to the reaction systems to a final concentration of 1 μM. The specific preparation details of the reaction systems are shown in Table 2.

[0059] Table 2 Preparation of different reaction systems

[0060]

[0061] Every 30 or 60 minutes, 5 μl of the reaction solution was added to a quenching solution containing 195 μl of fluorescent dye (final concentration: 10 mM Tris-HCl, 20% (v / v) deionized formamide, 10 mM EDTA, 1×SYBR Gold). Fluorescence intensity was detected using a fluorescence spectrophotometer at an excitation wavelength of 495 nm and an emission wavelength of 545 nm. Data sets of the reaction system and control system based on relative fluorescence intensity were obtained.

[0062] Relative fluorescence intensity R = F t / F0×100%, where F t Ft represents the fluorescence intensity of the reaction system at time t, and F0 represents the initial fluorescence intensity of the reaction system.

[0063] The relative fluorescence intensities of the different reaction systems and exonuclease reactions were statistically analyzed, and the results are as follows: Figure 2 As shown.

[0064] The above experimental results show that:

[0065] 1) The enzyme activity and degradation rate of aptamers vary under different buffer system conditions. Enzyme activity needs to be verified according to the buffer system. The addition of BSA and MgCl2 is beneficial to improve enzyme activity. The two should be optimized according to the buffer system used.

[0066] 2) The sequences provided in this experiment can form a neck-loop structure with complementary terminal pairings through software simulation. Combined with the activity characteristics of the two enzymes, the binding effect is better. In practical applications, it is necessary to predict the aptamer structure to select the most suitable enzyme.

[0067] Example 3: Effect of exonuclease concentration on degradation ability

[0068] The use of enzymes is a crucial factor affecting the accuracy of the detection results in this invention. Excessively high enzyme concentrations result in rapid aptamer cleavage, potentially missing the opportunity for differential detection. To further improve the accuracy of the evaluation, it is necessary to configure appropriate exonucleases and their dosages based on the method described in this invention, corresponding to different targets / aptamers and reaction systems.

[0069] The experimental group used reaction system R6 from Example 2, employing a combination of Exo III (0.5 U / μl) and Exo I (1.5 U / μl). A control group was also set up, with the enzyme concentration doubled. Changes in fluorescence intensity were compared between the target-containing and target-free systems. The aptamer nucleotide sequence used in this example is shown in SEQ ID NO.2, corresponding to aldosterone. Results are as follows: Figure 3 As shown, the fluorescence intensity of the target-containing and target-free systems changed over time. Within 4 hours, the difference between the two gradually increased, indicating that the target binds to the aptamer and hinders the degradation of the aptamer by the exonuclease. Figure 4 Based on the same reaction system, when the enzyme concentration is doubled, it can be seen that the binding of the target to the aptamer exhibits an inhibitory effect within half an hour, after which the enzyme can degrade it.

[0070] Example 4: Comparison of the affinity of different aptamer sequences based on changes in relative fluorescence intensity

[0071] Different sequences exhibit varying fluorescence intensities, making direct comparison impossible. A unified standard is needed to compare their ability to inhibit enzymatic digestion. This example compares the relative fluorescence intensity changes of each system at different times, providing a direct comparison of system fluorescence retention rates and illustrating the affinity between the aptamer and the target.

[0072] This embodiment further uses aldosterone as the target. The nucleotide sequence of the affinity aptamer is shown in SEQ ID NO.2, and the nucleotide sequence of the incompatible aptamer is shown in SEQ ID NO.3. Based on reaction system R6 in Example 2, the changes in relative fluorescence intensity in different reactions were statistically analyzed, and the results are as follows: Figure 5 As shown.

[0073] Furthermore, to visually represent the enzymatic hydrolysis of the two aptamers over time, their degradation curves can be fitted using software. A linear fit can be used, and their slopes can be compared (e.g., ...). Figure 6 (As shown) or by fitting the fluorescence retention curve with a polynomial function and comparing the area under the curve (e.g.) Figure 7 (As shown).

[0074] Example 5: ITC Validation Test

[0075] Isothermal titration calorimetry (ITC) is a thermodynamic technique for monitoring any chemical reaction initiated by the addition of binding components, and it has become the preferred method for identifying interactions between biomolecules. In ITC, a reactant is first placed in a temperature-controlled sample cell and coupled to a reference cell via a thermocouple circuit. Both the sample and reference cells are under the same external environment. A specific titrant (selected according to experimental needs) is quantitatively added to the sample cell. As the sample reacts with the titrant, the temperatures of both the sample and reference cells change. The energy of this reaction change can be sensitively detected by the ITC instrument, and a constant temperature is maintained by triggering a thermostat through positive or negative feedback. Thermodynamic parameters involved in the reaction, such as the binding constant, the number of binding sites, the enthalpy change, and the entropy change, can be obtained through software fitting of the isothermal titration calorimeter. This software processing indirectly yields Gibbs free energy, constant-pressure molar fusion, and other parameters. Detailed information about the intermolecular reaction can be obtained from these parameters.

[0076] This embodiment verifies the affinity of the aldosterone aptamer in Example 2 using the ITC method.

[0077] Experimental steps:

[0078] 1. Dilute the synthesized aldosterone aptamer and control sequence to 20 μM with DPBS. Take 200 μl of each, then add 192 μl of DPBS and 8 μl of methanol, mix thoroughly, and the final concentration of the aptamer is 5 μM.

[0079] 2. Dilute aldosterone to 200 μM with DBPS by adding 2 μl of aldosterone to 98 μl of DPBS.

[0080] 3. Place the aptamer into the sample cell, put the aldosterone into the syringe, titrate the nucleic acid aptamer with aldosterone, and titrate 20 times consecutively. Record the heat change during the titration and use Origin7 software to simulate a suitable ligand binding model with aldosterone.

[0081] like Figures 8 to 11 As shown, the ITC results for aldosterone aptamer affinity enzymatic hydrolysis experiments are presented for aptamers with and without affinity. Aptamers with affinity exhibit thermal changes during the ITC experiment, allowing for the plotting of binding curves. This also demonstrates that the affinity aptamers screened through enzymatic hydrolysis have been verified to indeed possess affinity.

[0082] Example 6: Verification of Known Aptamer Affinity

[0083] This embodiment provides two sets of known aptamers with affinity and their corresponding targets for affinity verification experiments to illustrate the versatility of this method. One protein and one small molecule were selected for enzymatic digestion analysis.

[0084] Thrombin: 20mM Tris-HCl, 140mM NaCl, 5mM KCl, 1mM MgCl2, 1mM CaCl2, 10mg / ml tRNA, pH 7.4; the aptamer is the single-stranded deoxynucleotide sequence shown in SEQ ID NO.4.

[0085] ATP: 10mM Tris-HCl, 20mM NaCl, 1.5mM MgCl2, 0.1mg / mL BSA, pH 7.4; the aptamer is the single-stranded deoxynucleotide sequence shown in SEQ ID NO.5.

[0086] Exonuclease: 0.025 U / μL Exo III + 0.05 U / μL Exo I

[0087] Experimental steps:

[0088] 1) Aptamer pre-denaturation;

[0089] 2) Take 5 μL (10 μM) and add it to the corresponding systems (40 μL) containing the target and those without the target for incubation;

[0090] 3) Add 5 μL of the mixed enzyme;

[0091] 4) Take 5 μL samples at different time points and add them to 195 μL of a solution containing quencher, then detect the fluorescence.

[0092] Experimental results are as follows Figure 12 As shown in the figures, Figures a and b represent the relative fluorescence intensity changes of the thrombin aptamer in systems containing and without thrombin; Figures c and d represent the relative fluorescence intensity changes of the APT aptamer in systems containing and without ATP. These two experimental results demonstrate that this method can be used for screening the affinity between thrombin and ATP aptamers.

Claims

1. A method for rapid screening of aptamers based on exonuclease degradation rate, characterized in that... include: Prepare a reaction system containing both the aptamer and the target, and a control system containing only the aptamer; after incubation, add exonuclease to each system and mix well. t Starting from 0, at regular intervals, a portion of the sample is taken out and added to a quenching solution containing fluorescent dye. The fluorescence intensity is detected to obtain a reaction system dataset and a control system dataset based on relative fluorescence intensity. The reaction system dataset and the control system dataset are then compared. The fluorescent dye is selected from anthocyanin-based nucleic acid staining agents or EB; The final concentration of the exonuclease added to the reaction system or control system is 0.01 - 2 U / μl; The quenching buffer consists of 10-50 mM pH 7.4 Tris-HCl, 10-80 v / v % deionized formamide, and 2-100 mM EDTA. The fluorescent dye is then diluted with this quenching buffer, and the final concentration of the detection system is diluted 1-10 times. The quenching solution is obtained by diluting the fluorescent dye with a quenching buffer solution.

2. The method for rapid screening of aptamers based on exonuclease degradation rate according to claim 1, characterized in that: The fluorescent dye is selected from any one of SYBR, Gel Red, GoldView, and EB.

3. The method for rapid screening of aptamers based on exonuclease degradation rate according to claim 1 or 2, characterized in that: The exonuclease is selected from any one or two of T5 Exonuclease, Exonuclease I, Exonuclease III, and Exonuclease T.

4. The method for rapid screening of aptamers based on exonuclease degradation rate according to claim 3, characterized in that: The aptamer is pretreated before being added to the reaction system or control system. The pretreatment involves purifying and dissolving it in ultrapure water, then heating it to 90-95°C for denaturation for 10-20 minutes, and then transferring it to ice water or cooling it at room temperature for later use.

5. A method for rapid screening of aptamers based on exonuclease degradation rate according to any one of claims 1-2, 4, characterized in that: The relative fluorescence intensity R = F t / F 0 × 100%, where F t for t Fluorescence intensity of the reaction system or control system at time points, F 0 The initial fluorescence intensity of the reaction system or control system.

6. The application of the method of claim 1 in the rapid initial screening of affinity between multiple aptamers and targets.

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