SELEX technology is the exponential enrichment of ligand system evolution technology (Systematic Evolution of Ligands by Exponential Enrichment, SELEX). Using this technology, nucleic acid aptamers (Aptamers) with high specificity and high affinity to the target substance can be screened from the random single-stranded nucleic acid sequence library. SELEX technology has become an important research method and tool after more than ten years of development since Tuerk et al.
Principle of SELEX Technology
The basic idea of SELEX technology is to chemically synthesize a single-stranded oligonucleotide library in vitro, use it to mix with the target substance, there is a complex between the target substance and nucleic acid in the mixture, wash away the nucleic acid that is not bound to the target substance, and separate the target substance from the target substance. The nucleic acid molecule bound by the substance is amplified by PCR using the nucleic acid molecule as a template, and the next round of screening process is performed. Through repeated screening and amplification, some DNA or RNA molecules that do not bind to the target substance or have low or medium affinity with the target substance are washed away, while the aptamer (Adaptorprotein) is the DNA or RNA molecules with high affinity to the target substance. RNA is isolated from a very large random library and the purity increases as the SELEX process progresses, from p to n moles, eventually occupying the majority (>90% or so) of the library. SELEX firstly synthesized a single-stranded random oligonucleotide library based on molecular biology technology. Usually the library capacity is 10″ to 10 single-stranded oligonucleotide sequences. The library includes DNA library, RNA library, and modified RNA library. The middle of the library is a random sequence, and the sequence length is often between 20 and 40 bp. The random sequence two The end is a fixed sequence with a restriction endonuclease site, which is the binding site for primers related to polymerase chain reaction and other enzymatic reactions. In random libraries, single-stranded random oligonucleotide molecules are easy to form multiple A kind of three-dimensional structure, which can interact with almost all kinds of molecules in nature. Especially RNA molecules are easy to form secondary structures such as hairpins, pseudo-sections, bulges, stem-loops, G-tetramers, etc., and are more likely to interact with proteins, nucleic acids and nucleic acids. However, it is easily degraded by nucleases in vivo, and must be modified such as 2’2 F and 2’2 NH modification of pyrimidine ring. Compared with RNA, DNA has low production cost, is relatively stable in vivo, and is not easy to be degraded. The aptamers (Adaptorprotein) screened from the constructed library are more suitable for in vitro diagnosis and in vivo treatment.
SELEX technical features
(1) The library has a large capacity and a wide range of adaptations; there are no restrictions on targets, including proteins, nucleic acids, small molecular organics, and even metal ions.
(2) High resolution. According to the research results of Jenison et al., the dissociation constant Kd of aptamer combined with theophylline obtained by screening with theophylline is 0.1 mol/L, which is 1000 times that of caffeine, although the difference between theophylline and caffeine is only in With or without a methyl group at the N7 position of the purine ring.
(3) High affinity. Taking protein as the target substance, the dissociation constant of aptamer can reach the level of nmol/L, or even the level of pmol/L. For the target substance with small Mr, the dissociation constant can also reach the level of mol/L—nmol/L.
(4) The screening process is relatively simple, fast and economical. Generally, a typical SELEX screening process can be completed in 2 to 3 months, and the small-scale synthesis and purification of the screened aptamers does not exceed 3 days. No special instruments and reagents are required, and it can be done in general laboratories. Screening antibodies takes at least 3 to 6 months, which is laborious and costly. Automation and scale-up of screening enables more, faster, and more economical production of aptamers. Recently Men-donsa et al. reported that when SELEX is combined with capillary electrophoresis, it takes only a few days to screen out oligonucleotide aptamers with high affinity and high specificity for target molecules.
(5) The practicality of screening. Screening aptamers (Adaptorprotein) can be timed, quantitative and quality-preserving, and the denaturation and renaturation of aptamers are fast, reversible, reusable, long-term storage and transport at room temperature, and have strong practicability relative to antibodies and other protein molecules. The screening conditions can be artificially set according to the different properties of the screened aptamers, and can be linked with other functional groups or molecules (such as biotin, sulfhydryl, methyl) at will during synthesis to meet the needs of users. needs.
(6) The aptamer is small in size. When administered as a drug, it only produces very low immunogenicity, but it has strong tumor penetration and is easy to clear in the body.