Looking back at the 20th century, the development of molecular biology and related disciplines has brought rapid changes to scientific research and human life, and also updated some traditional concepts. For example, since the 1930s, it has been thought that “all enzymes are proteins”. In 1986, Lerner et al. first developed an antibody that successfully catalyzed the hydrolysis of carboxylate, called Abzyme. In 1987, Cech and others discovered RNA molecules with catalytic activity, which they called ribozymes (Ribizymes). Recently, DNA molecules that specifically cleave RNA have been discovered, called deoxyribozymes (DNAzymes). Now, an enzyme can be defined as follows: “Enzymes are a class of biological macromolecules with catalytic activity and special spatial conformation in organisms, including proteins and nucleic acids.”
Antibodies are the most important effector molecules in the body’s immune system, with functions such as binding antigens, binding complements, neutralizing toxins, mediating cytotoxicity, promoting phagocytosis and passing through the placenta, and exerting functions such as anti-infection, anti-tumor, immune regulation and surveillance. Antibody research began at the end of the 18th century, that is, in 1890, German scholar Von Behring proved the existence of antitoxin (antibody) in the serum of immunized animals. Subsequently, agglutinin, precipitin, hemolysin, lysin, and complement fixin were successively discovered.
With the deepening of DNA-binding protein research, inspired by combinatorial chemistry, antibody library and random phage peptide library technology, Gold et al. constructed a random nucleic acid library in 1990 and screened out the nucleic acid ligands that specifically bind to the target protein, named index The enriched ligand system evolution technology, referred to as SELEX technology (Systematic Evolution of Ligands by Exponential Enrichment). At present, a variety of ligands that specifically bind to proteins, nucleic acids, small peptides, amino acids, organics, and metal ions have been screened from nucleic acid libraries and used in clinical treatment and diagnosis. To this end, we propose the following assumption: Can nucleic acids (deoxynucleic acids) be used as antibody molecules to replace immunoglobulins? Is it possible to synthesize antibody molecules in vitro to change the traditional way of antibody production and create a new era of antibody engineering?
The production route of antibodies
There are three ways to produce antibodies: the first is the classical way, that is, polyclonal antibodies are produced by immunizing animals; the second is the cell engineering way, that is, the production of monoclonal antibodies by hybridoma technology; the third is the use of genetic engineering ways to express and transform antibodies.
However, the production and application of protein antibodies have the following limitations:
Animals immunized with toxic antigens cannot bear it, and those with weak immunogenicity are difficult to produce antibodies;
Hybridomas are produced in mice, with limited therapeutic application (HAMA); heterologous antibodies produce non-specific reactions (false positives) in diagnosis, and are also interfered with by rheumatoid factors and autoantibodies;
High cost, time-consuming and labor-intensive, and rare antibodies require extensive screening to obtain;
The effective preservation of cloned strains (cells) is not easy, and some hybridomas are difficult to grow in vivo;
The quality varies between batches, and should be re-optimized during diagnosis;
The recognition specificity is different between in vivo and in vitro;
Kinetic parameters of antibody-target interactions cannot be changed as required;
Limited lifetime; temperature sensitive and irreversible.
2. SELEX technology and its advantages
The screening process of nucleic acid antibodies (ligands) is called SELEX technology. The basic principle is to use molecular biology technology to construct artificially synthesized single-stranded random oligonucleotide libraries. The capacity is between 1014 and 1015. Because single-stranded random oligonucleotide fragments, especially RNA, are prone to form secondary structures such as hairpins, pockets, pseudosections, and G-tetramers, they can combine with proteins, nucleic acids, small peptides, amino acids, organics, and even metal ions to form Complex with strong binding force. Using this principle, the random oligonucleotide library interacts with target molecules such as antigens or drugs, elutes and selects specific oligonucleotide ligands (aptemer), and generates a new secondary level by RT-PCR and in vitro transcription. library, and then bind to the target. After several cycles, the oligonucleotide fragments that can specifically bind to the target can be screened (Figure 1).
Random oligonucleotide libraries, especially random RNA libraries, have the following advantages compared with the commonly used protein, peptide and synthetic small molecule organic compound libraries:
A wider range of target molecules: no restrictions on targets, including metal ions, organic dyes, drugs, amino acids, complex factors, sugars, antibiotics, nucleic acids, base analogs, nucleotides, polypeptides, enzymes, growth factors, antibodies , gene regulatory factors, cell adhesion factors, phytohemagglutinin, intact virus particles and pathogenic bacteria, etc.;
The selected ligands have stronger binding ability to target molecules: even stronger than natural ligands, and the dissociation constant (kd) is mostly between pMol/L～nMol/L;
The specificity of binding to the target molecule is stronger: it can distinguish the subtle differences in the structure of the target molecule, and can distinguish the difference of a methyl group or a hydroxyl group. For example, the structure of theophylline is very similar to other xanthine analogs caffeine and theobromine. The conventional monoclonal antibody detection of theophylline has cross-reaction with the latter two, while the oligonucleotide ligands screened by SELEX technology only binds specifically Theophylline, which is unreactive with the other two substances, has a 10,000-fold higher affinity with theophylline than with caffeine. Through reverse SELEX screening, oligonucleotide ligands that bind to both target molecules and target molecule analogs can be effectively weakened or eliminated, thereby screening oligonucleotide ligands that specifically bind to the target molecule. Screening oligonucleotide ligands of a known or unknown target molecule from mixed systems, reverse SELEX technology has shown its unique value, for example, used to find oligonucleotide ligands that specifically bind to a tumor marker The target oligonucleotide ligands can be screened out by pre-screening the tissue cells of healthy individuals to remove the sequences that bind to the background.
Higher degree of heterogeneity: For each random oligonucleotide in the random oligonucleotide library, there are four possibilities for each nucleotide position in the random region. If the random region has n nucleotides, then There are n4 kinds of random sequence diversity, plus rare bases or artificially modified bases, the diversity of random sequences will be more. Generally, the length of the random region is about 30 nucleotides, and the capacity of the library can reach 1014-15. There are more thermal temperature structures than peptides of the same length, and 10 nt can form structural units such as hairpins and ring climbing, especially molecules such as iso G/iso C can be introduced to further increase the diversity. However, the diversity of random peptide libraries is limited due to the restriction of the gene bias of encoding peptides, the transformation efficiency of E. coli and the selection of biological systems.
The screening cycle is shorter: generally only 8 to 15 cycles are required, which takes about 2 to 3 months, and the screening process can be automated at present. The preparation of monoclonal antibodies, if successful, will take at least 3 to 6 months.
Compared with protein antibodies, nucleic acid antibodies (ligands) have the following advantages:
Screening under in vitro rather than in vivo (animal, cell) conditions, properties can be changed as required;
Different screening conditions result in different results (target substances), and kinetic parameters can be changed according to the requirements of in vitro diagnostic conditions;
Overcome the limitations of toxic and less immunogenic antigens;
In vitro chemical synthesis, time, quality and quantity can be guaranteed;
Specificity and affinity are not interfered by non-target proteins in tissues or samples;
Other functional groups and molecules, such as sulfhydryl groups, amino groups, and fluorescein, biotin, enzymes, etc., can be linked precisely, site-specifically, and at will during synthesis.
The molecule is smaller than the antibody, which is convenient for in vivo imaging diagnosis and treatment. If connected with phosphorothioate, it can be used for intracellular diagnosis and treatment;
Denaturation and renaturation are reversible and fast, and can be used repeatedly, stored for a long time, and shipped at room temperature.
3. Application status and prospect of SELEX technology in diagnostics
In the less than 10 years since the establishment of SELEX technology, a large number of ligands have been screened and researched for clinical diagnosis. Almost all diagnostic fields involving antibodies can be replaced by nucleic acid ligands, and their application modes include the following aspects.
1. Two-site binding assay (sandwich method)
The double-antigen/antibody sandwich method is currently the most commonly used diagnostic modality. In the meantime, the antigen/antibody serves as both capture molecule and detection molecule. Studies have shown that nucleic acid ligands can also be used as capture molecules and detection molecules, such as the detection of vascular endothelial growth factor (VEGF) and CD4. However, nucleic acid ligands cannot perform both capture and detection functions at the same time, because they compete for the same site of the binding ligand (antigen). The solution is to apply nucleic acid ligands against two different epitopes (non-overlapping sites). It can be obtained by changing the screening conditions and methods or the type of nucleic acid library (RNA/DNA); it can also be obtained by further screening the ligand-target complex to obtain a second different ligand, especially for small molecule antigens. In addition, this mode can also be applied to the detection of antigen-antibody complexes.
Nucleic acid ligands may be more suitable for differential diagnosis of structural analogs or cross-antigens that are indistinguishable from monoclonal antibodies, because the former binds more specifically to the target antigen. For example, the RNA ligands screened have no cross-reactivity among the four estrogens, hTSH, hLH, hCG, and hFSH, although the α chains of the 4 hormones are the same and the structures of the β chains are similar. Another example is the distinction between glycosylated hemoglobin and normal hemoglobin, the differential diagnosis of some drugs, etc., the application of monoclonal antibodies is difficult to solve.
It is very meaningful to screen out nucleic acid ligands with catalytic activity for homogeneous detection. Recently, Wilson and Szostak screened a DNA library for ligands that specifically bind to a fluorescein precursor and cause it to oxidatively emit light.
2. Flow Cytometry
Flow cytometry is a powerful tool for multiparametric analysis of cells and microsphere particles, and has previously been performed by differentially staining fluorescein-conjugated mAbs. The advantages of nucleic acid ligands instead of monoclonal antibodies in flow cytometry are reflected in two aspects: one is that the half-life of nucleic acid ligands bound to the cell surface is longer than that of monoclonal antibodies; the other is that they are not affected by Fc on the cell surface. body interference. For example, anti-CD4 RNA ligands labeled with fluorescein or phycoerythrin are used to detect CD4 molecules expressed on the cell surface on a flow cytometer.
3. sensor
Sensors are valued for their fast, simple, and quantitative detection, but the reuse of antibody-mediated immunosensors is limited and requires mild conditions so as not to destroy the function of antibodies. The nucleic acid ligands can be repeatedly denatured and renatured under conditions such as heat, different salt concentrations, metal chelating agents, etc., and can be modified and solidified, and the reporter group can be easily labeled. There have been reports of fluorescently labeled anti-human hemagglutinin nucleic acid ligands for in vivo diagnosis. Another advantage of this sensor is that it does not require labeling of the target molecule and can also be used for in vivo diagnostics.
4. Fluorescence polarization
In the simple and rapid detection method of small molecule hormones based on the principle of fluorescence polarization, the competitive mode is used, which has the disadvantage of low sensitivity and narrow kinetic range of antibody-mediated fluorescence polarization detection, and requires precise reagent control. The fluorescent labeling on nucleic acid ligands is 1/10 smaller than that of antibody molecules, rolls faster, and has large fluorescence polarization; it causes conformational changes after binding to small molecules, which is more suitable for non-competitive fluorescence polarization assays. The application of this technology to detect human neutrophil elastase and prothrombin has been reported.
5. Fluorescence quenching
In recent years, a new qualitative and quantitative genetic diagnosis method has been established by applying the principle of fluorescence quenching, which is characterized by sensitivity, specificity and easy automation. But so far, fluorescence quenching can only be used for nucleic acid detection. Nucleic acid ligands can be used as bridges to detect non-nucleic acids such as proteins. The basic idea is that when there is no target molecule, the cyclic ligand beacon labeling two mutually quenched fluorescein is base-paired with the nucleic acid ligand, the ligand beacon chain is straightened, and the fluorescence quenching disappears. When a target molecule exists, the binding of the target molecule to its corresponding nucleic acid ligand causes its configuration change (non-linear), which hinders the binding of the ligand beacon to the nucleic acid ligand, and fluorescence quenching still exists. Thereby, the purpose of detecting the target molecule can be achieved. Currently, this research is ongoing.
6. capillary electrophoresis
Capillary electrophoresis technology is increasingly used due to its advantages of rapidity, sensitivity, automation and multiparameter analysis, including immunological detection. It is difficult to establish a non-competitive method for antibody-based capillary electrophoresis detection, because it is difficult to separate labeled antibody and labeled antibody/antigen complexes, and it is difficult to obtain uniform antibody-labeled small molecule complexes. In addition, the abnormal electrophoretic behavior due to the glycosylation of the antibody is not conducive to the analysis of the results. German et al. invented an affinity probe capillary electrophoresis (APCE) technique, which uses fluorescently labeled nucleic acid ligands to detect IgE. The advantage is that the binding of nucleic acid ligands to target molecules changes its conformation and mass. , resulting in a significant change in its electrophoretic behavior.
7. molecular switch
Nucleic acid ligands can be repeatedly denatured and renatured under various environmental conditions and can cause configuration changes after binding to target molecules, indicating that they can become good molecular switches. For example, screening ribozymes using AMP-nucleic acid ligands, called aptazymes, is a sensitive molecular sensor. The nucleic acid ligand region recognizes the ligand, and the catalytic region amplifies the signal. Anti-DNA polymerase nucleic acid ligands are more effective than mAbs in PCR hot-start applications: they inactivate Taq enzyme at 40, and are effective against Taq, Tth, and Stoffel fragments. Some people also use nucleic acid ligands as a medium for affinity chromatography, which has the advantage of strong affinity and can be used repeatedly.
8. Proteome Research
With the progress of the Human Genome Project, the research on its function has been mentioned on the daily agenda, and the Proteome Project has quietly emerged. While 2-D electrophoresis and immunoarrays are the primary tools for proteomic research, nucleic acid ligand arrays have unique advantages:
(1) Simple and fast, easy to form an automation platform;
(2) High density, can be cured accurately;
(3) Homogeneous nucleic acid ligands can be obtained from chemical synthesis;
(4) firmness and longevity;
(5) The nucleic acid ligand containing 5-ring uracil can reversibly lock the bound protein;
(6) In addition to affinity specificity, there is also cross-linking specificity.
Since the advent of SELEX technology, its technology itself has developed very rapidly. The successive establishment of improved SELEX technologies such as blended SELEX (blended SELEX), complex targets SELEX (complex targets SELEX), and genomic SELEX (genomic SELEX) has made the application of SELEX technology. The prospects are even broader. As diagnostic reagents, oligonucleotide ligands, alone or in combination with antibodies, have shown their unique advantages in various diagnostic modes, especially to make up for the deficiencies of antibodies in the field of diagnosis. It is believed that in the near future, oligonucleotide ligand arrays will become one of the main tools in proteomic research, which will not only greatly facilitate the diagnosis of diseases, but also help to discover new therapeutic methods.
