CN111445951B - An optimization method for realizing the optimal sequence of low-cost high-throughput nucleic acid aptamers based on a label-free hybridization probe competition method - Google Patents

Abstract

The invention belongs to the field of molecular biology, and relates to an optimization method for realizing the optimal sequence of a low-cost high-throughput nucleic acid aptamer based on a label-free hybridization probe competition method. The selection of the needle position, the optimization of the number of bases of the complementary short-chain probe, and the optimization of the optimal sequence of the nucleic acid aptamer, etc. The optimization method for realizing the optimal sequence of low-cost high-throughput nucleic acid aptamers based on the label-free hybridization probe competition method provided by the present invention can be used for nucleic acid aptamer optimization of any type of target. The present invention is aimed at different targets or different nucleic acid aptamers, and the characterization method is simple, low in cost and high in throughput.

Description

A low-cost, high-throughput nucleic acid adaptation based on a label-free hybridization probe competition method Optimization method for bulk optimal sequence

technical field

The invention belongs to the field of molecular biology, and relates to an optimization method for realizing the optimal sequence of a low-cost high-throughput nucleic acid aptamer based on a label-free hybridization probe competition method.

Background technique

Aptamer (also translated as nucleic acid recognition body, nucleic acid aptamer, aptamer) refers to the systematic evolution of ligands by exponential enrichment (systematic evolution of ligands by exponential enrichment, SELEX), from artificially synthesized DNA Or single-stranded oligonucleotides screened from RNA libraries that can bind to targets with high affinity and recognize them with high specificity. The basic sequence structure of a random library includes: the length of the random sequence in the middle is 35-60 bases, and the lengths of the primers used for amplification at both ends are about 20 bases respectively. The total length of the nucleic acid aptamers finally screened is about 60-100 bases.

Aptamers can form a unique three-dimensional structure, which enables aptamers to better bind to their targets. The targets of aptamers are as small as ions, single molecules, and as large as whole cells. Aptamers have good selectivity and strong affinity for targets, which give them advantages over traditional recognition elements and are comparable to antibodies to some extent. Therefore, nucleic acid aptamers instead of traditional antibodies have broad application prospects in basic research, drug screening, clinical diagnosis and disease treatment of various diseases.

In the reported studies, it has been found that the length of nucleic acid aptamers is not conducive to the formation of stable structures, and it is easy to cause unnecessary base mismatches, thereby affecting the recognition process. By removing the non-essential sequences that are not related to the recognition region, and retaining only the recognized core region, the length of the nucleic acid aptamer can be greatly shortened; and then by analyzing the possible spatial structure, increasing the structural domain to improve the structural stability, so as to achieve improved The affinity and specificity of nucleic acid aptamers to target binding. The high-affinity nucleic acid aptamer obtained by optimization can make full use of bases and reduce the synthesis cost. Currently, nucleic acid aptamers that have been widely used, such as thrombin aptamer, ATP aptamer, IgE aptamer, etc., have a core sequence length of 15-30 bases. A large number of nucleic acid aptamers that have been screened cannot be fully utilized due to their long sequences. Therefore, the sequence optimization of the screened nucleic acid aptamers has become the only way for their further application, and it is an urgent problem to be solved. problem.

Methods for sequence optimization of nucleic acid aptamers include bioinformatics prediction methods, enzyme cleavage protection experiments, chip analysis methods, and the like. The bioinformatics prediction method predicts the secondary structure of nucleic acid aptamers based on algorithms such as thermodynamic minimum free energy, but the reliability of the nucleic acid secondary structure that can be predicted by each algorithm is very limited, and its reliability is difficult to guarantee. Ribozyme hydrolyzes the complex of the aptamer and the target, so that the partial sequence of the aptamer and the target can be obtained, but this method cannot know the non-binding sequence that plays a key role in the stability of the aptamer. ; Chip technology is to make the truncated fragment of nucleic acid aptamer into a chip, and use fluorescence to modify the binding signal between the target and the chip, and optimize the sequence with better affinity. This method has high accuracy, but the cost is too expensive. Due to the lack of fast, accurate and satisfactory methods for sequence optimization of nucleic acid aptamers, especially the two major difficulties of throughput and cost.

Patent CN108387561A discloses an optimization method for realizing the optimal sequence of low-cost high-throughput nucleic acid aptamers based on the principle of base quenching fluorescence. Nucleic acid for high-throughput screening and optimization of unmodified aptamers. The fluorescein-labeled short-chain nucleic acid used in the patent CN108387561A has a high synthesis cost. Although the optimization cost is reduced by half compared with the double fluorescent labeling method, the cost is still relatively high. For example, the design difficulty and synthesis cost of complementary short-chain probes Relatively high, not suitable for high-throughput optimization.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to overcome the problems of complex operation, low throughput and high cost in the prior art, and to provide a low-cost and high-throughput nucleic acid aptamer optimal sequence based on a label-free hybridization probe competition method. optimization method.

In order to achieve the above object, the technical scheme provided by the invention is:

A method for optimizing the optimal sequence of low-cost high-throughput nucleic acid aptamers based on a label-free hybridization probe competition method, comprising the following steps:

Step 1. Select the optimal complementary region and the optimal complementary short-chain probe:

Using the obtained long-chain nucleic acid aptamers as candidate nucleic acid aptamers, according to the number of bases in the full-length sequence, plan n regions, each region contains 20 bases, and each region is designed with 7-20 bases The complementary short-chain probe; after the long-chain nucleic acid aptamer to be characterized and the target molecule with a concentration of 0-1000nM were reacted in the binding solution for 5-30 minutes at room temperature, each complementary short-chain containing SYBR Green I was added The probe reacts, and the experimental results are fitted by a four-parameter equation. The four-parameter equation is:

Among them, _IF is the fluorescence response value; C _OTA is the concentration of target molecule; A _max represents the maximum value obtained after fitting the four-parameter equation; A _min represents the minimum value obtained after fitting the four-parameter equation; IC ₅₀ represents the four-parameter equation Half-inhibition value obtained after fitting; p in the formula is the slope of the fitted curve when C _OTA is IC ₅₀ ;

The (A _max -A _min )/IC ₅₀ value of each complementary short-chain probe was obtained through the analysis of the working curve, and the complementary short-chain probe with the largest (A _max -A _min )/IC ₅₀ value was selected as the optimal complementary short-chain probe. Needle;

Step 2. Preliminarily obtain better nucleic acid aptamers:

The corresponding region of the optimal complementary short-chain probe determined in step 1) is the core recognition region of the nucleic acid aptamer, and the two ends of the original nucleic acid aptamer are reduced by 3 bases each time to the core region, and a series of new aptamers are designed. For nucleic acid aptamers, the IC ₅₀ value of the new nucleic acid aptamer is obtained by the method of step 1), and the optimal nucleic acid aptamer is selected with the smallest IC ₅₀ value;

Step 3. Obtain the shortest aptamer sequence:

The optimal nucleic acid aptamer obtained in step 2) is reduced by one base at a time from both ends of the nucleic acid aptamer to the core region, and a series of one-base truncated nucleic acid aptamers are designed. The optimal complementary short-chain probe, target molecule and each one-base truncated nucleic acid aptamer determined in 1) were reacted to obtain the corresponding IC ₅₀ value of each one-base truncated nucleic acid aptamer, and the IC was selected. The core sequence of the nucleic acid aptamer with the smallest ₅₀ value or the smaller IC ₅₀ value but the structure is complementary to the tail end, that is, the shortest nucleic acid aptamer sequence;

Step 4. Construct nucleic acid aptamers with higher affinity:

The shortest nucleic acid aptamer sequence obtained in step 3) is subjected to structural analysis and the method of increasing the structural stability to obtain a nucleic acid aptamer with higher affinity, that is, the analyzed sequence.

Further, the candidate nucleic acid aptamer described in step 1) is one sequence or multiple sequences, including 60-100 bases.

Further, the length of the complementary short-chain probe described in step 1) is 7-20 bases.

Further, the length of the analyzed sequence described in step 4) is 14-45 bases.

Further, the concentration of NaCl in the binding solution described in step 1) is 80-200nM; the concentration of calcium ions or magnesium ions is 1-30mM; the concentration of Tris-HCl buffer solution is 10-30mM, and the pH is 5-10; The surfactant is Tween20, and its mass fraction is 0.01%-0.2%.

Further, the target molecules in step 1) are small molecules, macromolecules, cells and pathogens.

Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention utilizes the characteristic of fluorescence response when Sybr Green I is only embedded in double-stranded nucleic acids, and uses label-free complementary short-chain probes for high-throughput optimization of unmodified nucleic acid adaptors. Ligand. The method is based on the secondary sequence of nucleic acid aptamer, which avoids the uncertainty of bioinformatics analysis, and the accuracy of sequence optimization is significantly improved; compared with the chip method and the fluorescent labeling method, the method of the present invention significantly reduces the synthesis cost. This method can be used for nucleic acid aptamer optimization for any type of target. The present invention is aimed at different targets or different nucleic acid aptamers, and the characterization method is simple, low in cost and high in throughput. To sum up, compared with the prior art, the present invention further greatly reduces the optimization cost, obtains signals through the label-free fluorescent dye SGI, greatly reduces the design difficulty and synthesis cost of complementary short-chain probes, and is more suitable for high-throughput optimization.

Description of drawings

Fig. 1 is a schematic diagram of the method for optimizing the sequence of nucleic acid aptamers according to the present invention.

Figure 2A and Figure 2B are graphs of (A _max - A _min )/IC ₅₀ values when OTA Ap42 is added with short-chain probes of different lengths.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the above solutions are further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments are used to illustrate the present invention and not to limit the scope of the present invention. The implementation conditions used in the examples can be further adjusted according to the difference of candidate nucleic acid aptamers and targets, and the unremarked implementation conditions are usually the conditions in routine experiments.

An optimization method for realizing the optimal sequence of low-cost high-throughput nucleic acid aptamers based on a label-free hybridization probe competition method includes proposing affinity evaluation criteria, selection of complementary short-chain probe positions, and complementary short-chain probe bases. The optimization of the number of nucleic acid aptamers and the optimization of the optimal sequence of nucleic acid aptamers specifically include the following steps:

(1) Propose the affinity evaluation criteria. In the recognition system of this nucleic acid aptamer, ochratoxin A (OTA) and the complementary short-chain probe are in a competitive relationship, so the affinity of the complementary short-chain probe and the nucleic acid aptamer will affect the relationship between the nucleic acid aptamer and the complementary short-chain probe. OTA interaction. In order to obtain complementary short-chain probes with similar competitiveness to OTA, the present invention proposes the following evaluation criteria:

A. When evaluating complementary short-chain probes, the (A _max -A _min )/IC ₅₀ value is obtained through the analysis of the working curve,

The larger the ( _Amax - _Amin )/ _IC50 , the better the complementary short-strand probe.

B. After the complementary short-chain probe is immobilized, when evaluating the new nucleic acid aptamer, the IC ₅₀ value is obtained through the analysis of the working curve. The smaller the IC ₅₀ , the better the nucleic acid aptamer.

The targets are small molecules, macromolecules, cells and pathogens. After reacting the nucleic acid aptamer to be characterized with the target molecule with a concentration of 0-1000nM in the binding solution for 5-30 minutes at room temperature, a complementary short-chain probe containing SYBR Green I (SGI) was added, and the binding solution was Including solvent and solute, wherein the solvent is water; the solute includes 80-200nM NaCl, 1-30 mM calcium ion or 1-30 mM magnesium ion, 10-30 mM Tris-HCl buffer solution, buffer The pH of the solution is 5-10, and the mass fraction is 0.01%-0.2% surfactant Tween20.

The experimental results are fitted by a four-parameter equation, and the four-parameter equation is:

Among them, _IF is the fluorescence response value; C _OTA is the concentration of target molecule, here is the concentration of OTA; A _max represents the maximum value obtained after fitting the four-parameter equation; A _min represents the minimum value obtained after fitting the four-parameter equation; IC ₅₀ represents the half-inhibition value obtained after fitting a four-parameter equation; p in the formula is the slope of the fitted curve when C _OTA is IC ₅₀ .

(2) Select the optimal complementary region and the optimal complementary short-chain probe. Taking the obtained long-chain nucleic acid aptamers as candidate nucleic acid aptamers, according to the number of bases in the full-length sequence, plan n regions, each region contains 20 bases, and each region is designed with 7-20 bases. Complementary short-strand probe, the position of the complementary region can be adjusted if necessary. The (A _max -A _min )/IC ₅₀ values of different complementary short-chain probes were obtained by the method of step (1). The maximum value is selected by comparing (A _max -A _min )/IC ₅₀ value, so as to select the optimal complementary region and the optimal complementary short-chain probe.

(3) Preliminary acquisition of better nucleic acid aptamers. The corresponding region of the optimal complementary short-chain probe is the core recognition region of the nucleic acid aptamer. The two ends of the original nucleic acid aptamer are reduced by 3 bases each time to the core region, and a series of new nucleic acid aptamers are designed. The method of step (1) obtains the IC ₅₀ value of the new nucleic acid aptamer, and selects the better nucleic acid aptamer with the smallest IC ₅₀ value.

(4) Obtain the shortest nucleic acid aptamer sequence. The optimal nucleic acid aptamer obtained in step (3) is reduced by one base at a time from the two ends of the nucleic acid aptamer to the core region, and a series of new nucleic acid aptamers are designed, and the new design is obtained by the method of step (1). The IC ₅₀ value corresponding to the nucleic acid aptamer is selected, and the core sequence of the nucleic acid aptamer is selected with the smallest IC ₅₀ value or with a smaller IC ₅₀ value but the structure is complementary to the tail.

(5) Construct nucleic acid aptamers with higher affinity. The shortest nucleic acid aptamer core sequence obtained in step (4) is subjected to structural analysis and a method of increasing structural stability to obtain a nucleic acid aptamer with higher affinity. The length range of the analyzed sequence is adjusted according to the different candidate nucleic acid aptamers, and is specifically 14-45 bases.

Example

Example of a sequence optimization method for implementing an ochratoxin A nucleic acid aptamer: please refer to FIG. 1 , which is an optimization of the optimal sequence of a low-cost high-throughput nucleic acid aptamer based on a label-free hybridization probe competition method according to the present invention. Schematic diagram of the method flow. It mainly includes the following steps:

1. Select the optimal complementary region and the optimal complementary short-chain probe

The obtained OTA aptamer sequence is TGGTGGCTGTAGGTCAGCATCTGAT CGGTGTGGGTGGCGTAAAGGGAGCATCGGACAACG (SEQ ID NO. 1), a total of 61 bases, which are divided into 3 regions, each region contains 20 bases.

In the actual experiment, it was found that the first 20 bases of this sequence were primers, and its complementary sequence did not respond to different concentrations of OTA, so the complementary region was set within the last 42 bases (in order to meet the requirements of each shearing 3 1 base, and 1 T base at the 5' end), because the number of bases is small and the core region is in the middle, 10 probe sequences located in the middle are designed in this example.

According to the experimental results of Figure 2A and Figure 2B, the (A _max -A _min )/IC ₅₀ value of B13 changed the most. In this system, the dissociation constant of the nucleic acid aptamer and OTA was 200 nM, so its competitor molecule ( The dissociation constants of both complementary short-chain probes) are also in this order of magnitude. Because the complementary short-chain probe will inhibit the interaction between OTA and the nucleic acid aptamer, for small molecules with weak binding ability, the length of the complementary chain is 7-20 bases.

2. Preliminary acquisition of better nucleic acid aptamers.

From the 5' end of the original nucleic acid aptamer to the core region, reduce three bases each time, design a series of new nucleic acid aptamers, obtain the IC ₅₀ value of the new nucleic acid aptamer by the method of step (1), and select The aptamer with the smallest IC ₅₀ value is the better aptamer. After analyzing the experimental data, OTA Ap39L is the better aptamer truncated from 5'.

3. Reduce three bases each time from the 3' end of OTA Ap39L to the core region, design a series of new nucleic acid aptamers, obtain the IC ₅₀ value of the new nucleic acid aptamers by the method of step (1), and select The aptamer with the smallest IC ₅₀ value is the better aptamer. After analyzing the experimental data, OTA Ap39L-6 is the better aptamer truncated from 3'.

Fourth, obtain the shortest nucleic acid aptamer sequence.

After analyzing the experimental data, the nucleic acid aptamers OTA Ap39L-6, OTA Ap39L-7 and OTA Ap39L-8 still have good recognition ability compared to ochratoxin A, while OTA Ap38L, OTA Ap39L-9, OTA Ap39L- The binding ability of nucleic acid aptamers such as 10 was significantly weakened. So OTA Ap39L-8 is the shortest sequence available.

Fifth, construct nucleic acid aptamers with higher affinity.

According to the shortest nucleic acid aptamer core sequence obtained in step (4), according to the experimental results and previous work, it is considered here that the nucleic acid aptamer recognizes ochratoxin A and forms an antiparallel G quadruplex and the analysis of 31 bases. The structure will form a G quadruplex and the tail chain will form three pairs of complementary sequences. The complementary tail chain structure is beneficial to the stability of the whole system. Therefore, when a few pairs of complementary bases are appropriately added to the tail chain, the IC ₅₀ value is further reduced, which further proves that the formed aptamer recognizes OTA and forms a complementary tail chain, and extending the complementary tail chain is conducive to stabilizing the structure. Finally, new nucleic acid aptamers GG29CC, TATG29CATA and TATGG29CCATA with better affinity were obtained.

sequence listing

<110> Dalian University of Technology

<120> An optimal method for realizing the optimal sequence of low-cost high-throughput nucleic acid aptamers based on a label-free hybridization probe competition method

method

<130> 2020

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<170> PatentIn version 3.5

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Claims (10)

1. An optimization method for realizing the optimal sequence of a low-cost high-throughput aptamer based on a label-free hybridization probe competition method is characterized by comprising the following steps:

step 1, selecting an optimal complementary region and an optimal complementary short-chain probe:

planning n regions by using the obtained long-chain aptamer as a candidate aptamer according to the base number of the full-length sequence, wherein each region comprises 20 bases, and a complementary short-chain probe with 7-20 bases is designed in each region; reacting a long-chain aptamer to be characterized with a target molecule with the concentration of 0-1000nM in a binding solution for 5-30 minutes at room temperature, adding each complementary short-chain probe containing SYBR Green I for reaction, and fitting an experimental result through a four-parameter equation, wherein the four-parameter equation is as follows:

wherein, I _F Is a fluorescence response value; c _OTA Is a targetThe concentration of the target molecule; a. the _max Expressing the maximum value obtained after fitting the four-parameter equation; a. the _min Expressing the minimum value obtained after fitting the four-parameter equation; IC (integrated circuit) ₅₀ Expressing a semi-inhibition value obtained after fitting of a four-parameter equation; in which p is C _OTA Is an IC ₅₀ The slope of the time-fitted curve;

each complementary short-chain probe (A) was obtained by analysis of the working curve _max -A _min )/IC ₅₀ Value, select (A) _max -A _min )/IC ₅₀ The complementary short-chain probe with the maximum value is the optimal complementary short-chain probe;

step 2, obtaining a better aptamer preliminarily:

the region corresponding to the optimal complementary short-chain probe determined in the step 1) is a core recognition region of the aptamer, 3 bases are reduced from two ends of the original aptamer to the core region each time, a series of new aptamers are designed, and the IC of the new aptamers is obtained by the method of the step 1) ₅₀ Value, select IC ₅₀ The smallest value is the preferred aptamer.

Step 3, obtaining the shortest aptamer sequence:

reducing the optimal nucleic acid aptamer obtained in the step 2) from two ends of nucleic acid aptamer to a core region by one base each time, designing a series of base truncated nucleic acid aptamers, adopting the method of the step 1), reacting the optimal complementary short-chain probe determined in the step 1), the target molecule and each base truncated nucleic acid aptamer to obtain IC corresponding to each base truncated nucleic acid aptamer ₅₀ Value, select IC ₅₀ Minimum value OR IC ₅₀ Smaller but tailend-complementary structures are aptamer core sequences, i.e., the shortest aptamer sequence.

Step 4, constructing the aptamer with higher affinity:

and 3) obtaining the aptamer with higher affinity, namely the resolved sequence, by a method of analyzing the structure and increasing the structural stability of the shortest aptamer sequence obtained in the step 3).

2. The method for optimizing the optimal sequence of the aptamer with low cost and high throughput based on the label-free hybridization probe competition method as claimed in claim 1, wherein the candidate aptamer in step 1) is one or more sequences comprising 60 to 100 bases.

3. The method for optimizing the optimal sequence of the aptamer with low cost and high throughput based on the label-free hybridization probe competition method according to claim 1 or 2, wherein the length of the complementary short-chain probe in the step 1) is 7-20 bases.

4. The method for optimizing the optimal sequence of the aptamer with low cost and high throughput based on the label-free hybridization probe competition method according to claim 1 or 2, wherein the length of the resolved sequence in the step 4) is 14-45 bases.

5. The method for optimizing the optimal sequence of the aptamer with low cost and high throughput based on the label-free hybridization probe competition method as claimed in claim 3, wherein the length of the sequence analyzed in step 4) is 14-45 bases.

6. The optimization method for realizing the optimal sequence of the aptamer with low cost and high throughput based on the label-free hybridization probe competition method according to claim 1, 2 or 5, wherein the concentration of NaCl in the binding solution in the step 1) is 80-200 nM; the concentration of calcium ion or magnesium ion is 1-30 mM; the concentration of the Tris-HCl buffer solution is 10-30mM, and the pH value is 5-10; the surfactant is Tween20 with a mass fraction of 0.01-0.2%.

7. The optimization method for realizing the optimal sequence of the low-cost high-throughput aptamer based on the label-free hybridization probe competition method according to claim 3, wherein the concentration of NaCl in the binding solution in the step 1) is 80-200 nM; the concentration of calcium ion or magnesium ion is 1-30 mM; the concentration of the Tris-HCl buffer solution is 10-30mM, and the pH value is 5-10; the surfactant is Tween20 with a mass fraction of 0.01-0.2%.

8. The optimization method for realizing the optimal sequence of the low-cost high-throughput aptamer based on the label-free hybridization probe competition method as claimed in claim 4, wherein the concentration of NaCl in the binding solution in the step 1) is 80-200 nM; the concentration of calcium ion or magnesium ion is 1-30 mM; the concentration of the Tris-HCl buffer solution is 10-30mM, and the pH value is 5-10; the surfactant is Tween20 with a mass fraction of 0.01-0.2%.

9. The optimization method for realizing the optimal sequence of the aptamer with low cost and high throughput based on the label-free hybridization probe competition method according to claim 1, 2, 5, 7 or 8, wherein the target molecules in the step 1) are small molecules, large molecules, cells and pathogens.

10. The method for optimizing the optimal sequence of the aptamer with low cost and high throughput based on the label-free hybridization probe competition method according to claim 6, wherein the target molecules in step 1) are small molecules, large molecules, cells and pathogens.

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