阅读说明：本技术 一种筛选获得极短序列核酸适配体的方法 (method for obtaining ultra-short sequence aptamer through screening ) 是由 郑江 江兴龙 鄢庆枇 于 2019-09-03 设计创作，主要内容包括：本发明公开了一种筛选获得极短序列核酸适配体的方法,在SELEX筛选方法基础上,结合高通量测序技术,对每轮SELEX筛选出的寡核苷酸文库都进行高通量测序,通过统计分析每个随机文库对应的高频序列数,进而对随机文库的随机序列进行增减,并通过循环逼近法最终获得最短的适配体序列。通过构建不同的筛选文库,采用高通量测序逼近的方法来获得极短序列的核酸适配体,省去了后期截短加工和改造的麻烦。(the invention discloses a method for obtaining a very short sequence aptamer through screening, which is characterized in that on the basis of a SELEX screening method, a high-throughput sequencing technology is combined, high-throughput sequencing is carried out on oligonucleotide libraries screened by each round of SELEX, the number of high-frequency sequences corresponding to each random library is statistically analyzed, then the random sequences of the random libraries are increased and decreased, and the shortest aptamer sequence is finally obtained through a circular approximation method. By constructing different screening libraries and adopting a high-throughput sequencing approximation method to obtain the aptamer with an extremely short sequence, the troubles of late truncation processing and modification are saved.)

1. A method for screening and obtaining an extremely short sequence aptamer is characterized by comprising the following steps:

(1) Synthesis of random oligonucleotide libraries: synthesizing a random oligonucleotide library with fixed sequences at two ends and a random sequence in the middle, wherein the fixed sequences at two ends are used for combining with a primer to carry out PCR amplification; the number of the bases of the intermediate random sequence is N, N is more than or equal to 5 and less than or equal to 35, and N is a positive integer;

(2) combining: binding a random oligonucleotide library to a target;

(3) Separation: isolating the oligonucleotide bound to the target;

(4) And (3) PCR amplification: performing PCR amplification on the separated oligonucleotides capable of binding to the target, wherein one part of the PCR product is used as an oligonucleotide library for the next round of screening, and the other part is used for high-throughput sequencing in the subsequent step (5);

(5) High-throughput sequencing: performing high-throughput sequencing on the oligonucleotide library amplified by PCR, and selecting a high-frequency oligonucleotide sequence of a non-primer sequence with the occurrence frequency or frequency more than or equal to 2 times, wherein the high-frequency oligonucleotide sequence is a candidate sequence for the 1 st round of SELEX screening;

(6) And (3) repeated circulating screening: selecting the oligonucleotide library amplified in the 4 th step, binding with a target object, and repeating the steps (2) - (5) to obtain a candidate sequence for the 2 nd round SELEX screening; repeating the cycle for m times, wherein m is more than or equal to 1 and less than or equal to 10, and obtaining m rounds of SELEX screening candidate sequences; counting the candidate sequences from the 1 st round to the m th round, and using k to represent the number of the high-frequency candidate sequences obtained through accumulation;

When the following situation occurs during the screening process,

if the number of intermediate random sequences in the initial random library is N, when the number m of repeated screening cycles meets the condition that m is more than or equal to 1 and less than or equal to 10 and the number k of candidate sequences meets the condition that k is more than or equal to 0 and less than 5, the step of repeating (2) to (6) continues to be carried out for m +1 rounds of cycle screening;

if the number of the random sequences in the initial random library is N, stopping screening when the number of the cycles m satisfies that m is more than or equal to 1 and less than or equal to 10 and the number k of the high-frequency sequences is more than or equal to 5; re-synthesizing an initial random library with the intermediate random sequence number of N-1, and carrying out new SELEX screening by taking the initial random library as a starting point according to the steps (2) to (6);

thirdly, if the random sequence in the initial random library is N, when the cycle number m is greater than 10 and k =0, namely 10 cycles of screening are completed, the number k of the high-frequency sequences is still zero, namely no high-frequency sequence is found, and then screening is stopped; re-synthesizing an initial random library with the intermediate random sequence number of N + 1; then using the new random library of the N +1 as an initial library, and carrying out new SELEX screening according to the steps (2) to (6); if the initial random library with the random sequence of N +1 is screened before and a certain number of high-frequency sequences are obtained, the high-frequency sequences are the required extremely short sequences and do not need to be screened again; finishing all screening;

If the random sequence in the initial random library is N, stopping the screening when the cycle number m is greater than 10 and the number k of the high-frequency sequences is greater than or equal to 1, re-synthesizing the initial random library with the intermediate random sequence number of N-1, and then performing new SELEX screening according to the steps (2) - (6); if the initial random library with the random sequence N-1 is screened and no high-frequency sequence is obtained, stopping all screening, wherein k high-frequency sequences obtained by the screening are the required extremely short sequences; and finishing all screening.

Technical Field

The present invention relates to oligonucleotide sequences and aptamer sequences, and in particular to a screening technique for obtaining aptamers with shorter sequences.

Background

The Exponential Enrichment ligand Evolution technology (systematic Evolution of Ligands by expression Evolution), referred to as SELEX technology for short, is a system screening technology newly developed in recent years. It utilizes oligonucleotide molecule to form various three-dimensional structures in space, and utilizes the constructed random oligonucleotide library to screen out the oligonucleotide molecule-nucleic acid aptamer with high affinity and specific recognition action with target molecule, and its molecular recognition capability can be up to or even surpass the level of monoclonal antibody, and its preparation technology is simple and quick compared with monoclonal antibody. The aptamer of tumor cells, bacillus anthracis and the like which are screened out by the technology at present can be used for detecting and identifying the tumor cells and the bacillus anthracis.

However, in many cases, the aptamers obtained by screening are not satisfactory, and usually, the length of the aptamers is too long, which results in higher synthesis cost, and in addition, the number of unnecessary sequences in the too long aptamers is also increased, which causes some side reactions and interference factors to easily occur in the detection process. Therefore, most people carry out truncation processing and modification on the obtained aptamer so as to obtain the aptamer with shorter sequence and better affinity. However, the effect of the chopping process is not preferable in many cases.

Disclosure of Invention

The invention aims to provide a SELEX screening method for obtaining a nucleic acid aptamer with a shorter sequence, which optimally controls the sequence length of the nucleic acid aptamer during screening to reduce the sequence length as much as possible, avoids the troubles of subsequent processing modification and truncation, and is easy to realize automatic operation, and the method specifically comprises the following steps:

A method for screening and obtaining an extremely short sequence aptamer is characterized by comprising the following steps:

(1) Synthesis of random oligonucleotide libraries: synthesizing a random oligonucleotide library with fixed sequences at two ends and a random sequence in the middle, wherein the fixed sequences at two ends are used for combining with a primer to carry out PCR amplification; the number of the bases of the intermediate random sequence is N, N is more than or equal to 5 and less than or equal to 35, and N is a positive integer;

(2) Combining: binding a random oligonucleotide library to a target;

(3) separation: isolating the oligonucleotide bound to the target;

(4) And (3) PCR amplification: performing PCR amplification on the separated oligonucleotides capable of binding to the target, wherein one part of the PCR product is used as an oligonucleotide library for the next round of screening, and the other part is used for high-throughput sequencing in the subsequent step (5);

(5) high-throughput sequencing: performing high-throughput sequencing on the oligonucleotide library amplified by PCR, and selecting a high-frequency oligonucleotide sequence of a non-primer sequence with the occurrence frequency or frequency more than or equal to 2 times, wherein the high-frequency oligonucleotide sequence is a candidate sequence for the 1 st round of SELEX screening;

(6) And (3) repeated circulating screening: selecting the oligonucleotide library amplified in the 4 th step, binding with a target object, and repeating the steps (2) - (5) to obtain a candidate sequence for the 2 nd round SELEX screening; repeating the cycle for m times (m is more than or equal to 1 and less than or equal to 10) to obtain m rounds of SELEX screening candidate sequences; counting the number of the candidate sequences from the 1 st round to the mth round, and using k to represent the number of the high-frequency candidate sequences obtained through accumulation;

When the following situation occurs during the screening process,

If the number of intermediate random sequences in the initial random library is N, when the number m of repeated screening cycles meets the condition that m is more than or equal to 1 and less than or equal to 10 and the number k of candidate sequences meets the condition that k is more than or equal to 0 and less than 5, continuing screening m +1 rounds, namely repeating the steps (2) to (6) for performing the repeated screening;

if the number of the random sequences in the initial random library is N, stopping screening when the number of the cycles m satisfies that m is more than or equal to 1 and less than or equal to 10 and the number k of the high-frequency sequences is more than or equal to 5; re-synthesizing an initial random library with the intermediate random sequence number of N-1, and carrying out new SELEX screening according to the steps (2) - (6) by taking the initial random library as a starting point;

thirdly, if the random sequence in the initial random library is N, when the cycle number m is greater than 10 and k =0, namely 10 cycles of screening are completed, the number k of the high-frequency sequences is still zero, namely no high-frequency sequence is found, and then screening is stopped; re-synthesizing an initial random library with the intermediate random sequence number of N + 1; then using the new random library of the N +1 as an initial library, and carrying out new SELEX screening according to the steps (2) - (6); if the initial random library with the random sequence of N +1 is screened before and a certain number of high-frequency sequences are obtained, the high-frequency sequences are the aptamer of the required extremely short sequence and do not need to be screened again; finishing all screening;

if the random sequence in the initial random library is N, stopping the screening when the cycle number m is greater than 10 and the number k of the high-frequency sequences is greater than or equal to 1, re-synthesizing the initial random library with the intermediate random sequence number of N-1, and then performing new SELEX screening according to the steps (2) - (6); if the initial random library with the random sequence N-1 is screened before and no high-frequency sequence is obtained, stopping all screening, wherein k high-frequency sequences obtained by the screening are the aptamer of the required extremely short sequence; and finishing all screening.

(7) and finally, measuring parameters such as affinity constant and the like of the obtained extremely-short-sequence aptamer so as to verify the affinity of the aptamer.

the extremely short sequence reduces to the maximum extent, even completely eliminates the occurrence of unnecessary sequences, reduces the generation of side reactions and interference factors in the detection process to the maximum extent, improves the detection quality and reduces the detection cost; on the basis of a SELEX screening method, a high-throughput sequencing technology is combined, high-throughput sequencing is carried out on oligonucleotide libraries screened by SELEX in each round, the number of high-frequency sequences corresponding to each random library is statistically analyzed, the random sequences of the random libraries are increased and decreased, and the shortest sequence of the aptamer is finally obtained by a circular approximation method. According to the invention, by constructing random screening libraries with different lengths and adopting a high-throughput sequencing approximation method, the aptamer with an extremely short sequence is obtained, so that the troubles of truncation processing and modification in the later period are eliminated, the detection cost can be well reduced, and the detection efficiency is improved.

Drawings

FIG. 1 is a screening flow chart of the method of the present invention.

Detailed Description

The invention is further described below with reference to examples.