Since it was first proposed in 1975, monoclonal antibody technology has quickly become a revolutionary breakthrough in the field of life sciences. This technology has not only promoted the in-depth development of basic research, but also achieved remarkable results in many fields such as drug development, biosensors, and diagnostic tools. Especially in the medical field, it has broad application potential. Initially, monoclonal antibodies were mainly used for basic scientific research in laboratories, such as protein identification and cell labeling, but with the advancement of technology, monoclonal antibodies were gradually introduced into clinical medicine and became a powerful weapon for the treatment of many complex diseases. In 1986, the world's first therapeutic monoclonal antibody was successfully developed and used for the prevention and treatment of rejection reactions in kidney transplantation, marking an important step in the clinical application of antibody drugs.

Since then, the development of monoclonal antibody drugs has entered a golden period. Through targeted recognition of specific antigens, antibody drugs provide new treatment options for cancer, immune diseases, infectious diseases, etc. For example, in recent years, antibody drugs have been successfully used in the treatment of diseases such as asthma, rheumatoid arthritis, and breast cancer, greatly improving the quality of life and cure rate of patients. However, despite the great success of monoclonal antibody drugs in clinical practice, they still have certain limitations in the treatment of certain specific diseases.

A significant challenge is that many biomacromolecules, such as lipids, carbohydrates, and some organic macromolecules, are often difficult to bind to monoclonal antibodies because these molecules do not have clear antigenic epitopes and antibodies have low affinity for them. For example, in targeted drug design, the coupling efficiency and specificity of antibodies are often limited, especially in certain cases with complex structures or small molecule targets, where the effect of antibodies may be far less than expected. Therefore, traditional monoclonal antibodies face problems such as insufficient affinity and low targeting efficiency in certain treatments.

As the limitations of monoclonal antibody technology gradually emerge, artificial ligand technology has emerged, among which new artificial ligands represented by aptamers have received widespread attention. Aptamers are a class of small molecules composed of single-stranded nucleic acids that can specifically bind to a variety of targets. Compared with traditional antibodies, aptamers have many unique advantages: their small molecular weight makes them more efficient in target recognition and cell penetration; their synthesis cost is low and they can be easily obtained through chemical synthesis; the synthesis process of aptamers is standardized, easy to mass produce, and their structure can be customized and modified according to needs. Most importantly, aptamers have flexible nucleic acid template properties and can adapt to different targets by modifying their sequences, thus playing a role in a wide range of applications.

The advantages of aptamers have made them show great potential in the application of supplementing and replacing traditional antibodies. Especially when targeting small molecules, metal ions, nucleic acids, and even some macromolecules that are difficult to recognize with antibodies, the affinity and specificity of aptamers often exceed those of traditional antibodies. They can be used in targeted therapy, diagnosis, sensor development and other fields, becoming a new generation of molecular tools. For example, aptamers can be used to prepare highly specific biosensors, which are not only small in size and low in cost, but also easy to operate and can quickly detect target molecules. They are widely used in food safety, environmental monitoring, early disease diagnosis and other fields.

Nucleic acid aptamers, as a small molecule composed of single-stranded DNA or RNA, are usually composed of 20 to 110 nucleotides. They form a stable three-dimensional structure through a certain random sequence and a fixed sequence combination through a specific folding structure. This three-dimensional structure gives nucleic acid aptamers high affinity and specificity, enabling them to accurately bind to target molecules. Aptamers can be used not only for targeted therapy of small molecules, but also for complex macromolecules that cannot be recognized by antibodies, such as certain membrane proteins and peptide molecules. Unlike traditional antibodies, aptamers have higher flexibility and plasticity. They can be screened and optimized multiple times during the synthesis process to ensure the best binding ability under specific conditions.

In addition, aptamers have very broad application potential. For example, in gene therapy, aptamers can be used as carriers to deliver therapeutic molecules into cells; in the process of drug discovery, aptamers can be used as screening tools to quickly identify potential drug targets, and can even be used for the design and development of targeted drugs. In the fields of virus detection and immune monitoring, aptamers can be used as specific probes to provide more sensitive and efficient detection methods.

With the continuous advancement of technology, the application fields of aptamers are rapidly expanding. In the future, they are expected to become an important molecular tool in the field of biomedicine, further promoting the development of precision medicine. By combining the unique advantages of aptamers with existing technologies, we will be able to develop more efficient, flexible, and low-cost treatment solutions in the future, solve the shortcomings of traditional antibodies in certain fields, and promote the sustainable development of biomedical research and clinical treatment.

Advantages of nucleic acid aptamers: ① It has the advantages of high thermal stability, easy chemical synthesis and modification, and low immunogenicity, and is used in bioanalysis, biomedicine, biotechnology, sensor technology and other fields. ② It has the advantages of short production time, low cost, and high specificity, and is used in the medical field.

Disadvantages of nucleic acid aptamers: screening is time-consuming and labor-intensive, with high failure rate and high cost.

Aptamer Design

Aptamers are divided into RNA aptamers and DNA aptamers. Although these two types of molecules show high specificity and affinity in applications, they also have the disadvantage of being easily degraded by nucleases. Therefore, in response to this problem, scientists have improved their ability to resist enzyme degradation and prolonged their stability in vivo and in vitro by chemically modifying aptamers. Common chemical modification methods include methylation, phosphorylation, glycosylation and other treatments on nucleic acid chains. These modifications can not only enhance the stability of aptamers, but also increase the specificity of aptamer binding to target molecules while improving affinity.

In the screening of aptamers, SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology is widely used. This technology can be used to screen aptamers that specifically bind to target molecules from a large nucleic acid library. The success of SELEX is closely related to the design of the library. Therefore, reasonable aptamer sequence design is particularly important. Aptamer sequence design mainly includes the design of fixed regions and random regions. Fixed regions are usually used for PCR amplification of libraries. When designing, it is necessary to avoid the formation of self-dimers to ensure that non-specific PCR products are not generated during the amplification process. Generally speaking, the length of the primer region is controlled between 18 and 21 nucleotides. Shorter primer regions have less impact on the subsequent screening process and can help screen out more diverse sequences. The design of partially complementary fixed sequences at both ends helps to obtain structurally stable nucleic acid aptamers, but may affect the probability of screening highly specific aptamers.

The design of the random region is also crucial, and its length is usually 30 to 60 nucleotides, with a random region of 40 nucleotides being the most common. The design of short random regions facilitates the subsequent shearing and application of aptamers, especially for aptamers that require a smaller volume. Relatively long random regions are more suitable for large aptamers that require complex three-dimensional structures. This type of design helps to improve the specificity of aptamers and is suitable for binding to large or structurally complex targets.

At present, there are many methods for the design and optimization of nucleic acid aptamers. For example, the design of RNA aptamers often adopts strategies such as scaling clustering and finding motifs, aiming to screen the most affinity and specific aptamers based on the characteristics of the target molecule. In addition, the optimization of aptamers is not limited to sequence design, and computational chemistry methods are also widely used in the design of aptamers. By using scoring functions, molecular docking, kinetic simulation and other technologies, scientists can predict the affinity and stability of aptamer binding to the target, thereby further optimizing the design of aptamers. Quantum mechanics/molecular mechanics (QM/MM) methods and quantitative structure-activity relationship (QSAR) models can also be used to screen and optimize aptamers to enhance their targeting ability and drug development potential.

The combination of these technologies and methods makes aptamer design not only highly flexible and innovative, but also achieves excellent results in a variety of applications, such as targeted therapy, disease diagnosis, and biosensing.

Methods for Screening Nucleic Acid Aptamers: SELEX Screening

SELEX (Systematic Evolution of Ligands by Exponential Enrichment) screening technology is a method for screening high-affinity and high-specificity nucleic acid aptamers. Its principle is based on the gradual enrichment of nucleic acid sequences that can specifically bind to target molecules through cyclic screening.

1. Design of Nucleic Acid Ligand Library

The design of nucleic acid ligand library is the first step in SELEX screening. The ligand library consists of a large number of single-stranded nucleic acid molecules (DNA or RNA), which usually have random nucleotide sequences and fixed sequences of a certain length. The fixed sequence part is used for primer binding and PCR amplification, while the random sequence part is used for specific binding to the target molecule. Nucleic acid ligand libraries can be prepared by chemical synthesis (for DNA aptamers) or biosynthesis (for RNA aptamers), and the resulting libraries contain millions to billions of different sequences, ensuring that a wide range of binding properties and structures can be covered.

2. Affinity Screening

Affinity screening is the most critical step in the SELEX process, which aims to screen aptamers that can specifically recognize target molecules from the nucleic acid ligand library. First, the nucleic acid ligand library containing random sequences is mixed and incubated with target molecules (such as proteins, small molecules, cells or viruses, etc.). By selecting appropriate screening methods, such as solid phase adsorption, magnetic bead method, filtration method, gel electrophoresis, etc., the nucleic acid/target molecule complex bound to the target molecule can be separated from the unbound free nucleic acid. The solid phase adsorption method usually uses materials with strong affinity (such as metal ions or antigen antibodies) to fix the target molecule, and the magnetic bead method uses magnetic bead carriers to capture the target molecule. Through these methods, the target molecule can be efficiently separated from the unbound nucleic acid ligand, providing a suitable sample for subsequent screening.

3. Elution

The elution step is a key process for separating nucleic acid ligands from the complex. By changing the conditions of the solution (such as salt concentration, pH value or competitive molecules) to break the binding between nucleic acid/target molecules, the nucleic acid sequence bound to the target molecule is released. The released nucleic acid sequence will be collected and used for subsequent PCR amplification. In this step, accurate elution conditions are very important. Excessive elution may cause non-specifically bound nucleic acids to be released, while too strict elution may cause the loss of aptamers with weaker target molecule binding.

4. PCR Amplification

The eluted nucleic acid sequence will be amplified by PCR to prepare enough nucleic acid library for the next round of screening. PCR amplification is a key step to improve screening efficiency, which can enrich and rapidly proliferate nucleic acid ligands with strong affinity. At this time, the PCR primer design needs to correspond to the fixed sequence region of the nucleic acid library and ensure the specificity and efficiency of the PCR amplification process. During the amplification process, the nucleic acid sequences that have undergone affinity screening will increase exponentially, while the sequences that fail to bind to the target molecule will gradually decrease, thereby ensuring that only high-affinity aptamers are screened in the subsequent steps.

5. Cyclic Screening

The above steps are usually repeated for multiple rounds to further improve the screening effect and accuracy. Each round of screening and amplification will gradually enhance the affinity and specificity of the target molecule. Usually, in the initial screening, the nucleic acid aptamer sequences in the library are random. After several rounds of screening, the sequences that can specifically recognize and bind to the target molecule will gradually dominate, and finally form an aptamer library with significant affinity. The number of cycles depends on the characteristics of the target molecule and the level of affinity required. Through multiple rounds of screening, researchers can gradually improve the selectivity and affinity of the aptamer, and finally screen out nucleic acid aptamers with high specificity and low non-specific binding.

Fig.1 Schematic model of aptamer screening technology

TekBiotech provides comprehensive nucleic acid aptamer screening services, which can be customized for different types of targets, including proteins, small molecules, metal ions, etc. Through advanced SELEX technology, TekBiotech can screen out aptamers with high affinity and specificity from a huge nucleic acid library. These aptamers are not only suitable for targeted therapy and drug development, but also for precision diagnosis, sensor construction and other fields. Whether it is for complex protein targets or small targets such as small molecule drugs or metal ions, TekBiotech can provide efficient and customized screening services to meet customers' diverse needs in biomedicine, environmental monitoring, food safety and other aspects.