Introduction to Nucleic Acid Aptamers

Monoclonal antibody production technology was developed in 1975. It can be used not only in basic science, but also in pharmaceuticals, biosensors and other fields, and is of great significance in medicine. The world's first therapeutic antibody was discovered in 1986 to prevent kidney transplant rejection. Since then, many antibody drugs have been found to be used to treat various diseases, such as asthma. However, monoclonal surgery has certain limitations in treating diseases. For example, antibodies against lipids, carbohydrates, and organic macromolecules have lower affinity, and their affinity for drug conjugation is affected to a certain extent. Therefore, artificial ligand-aptamers are gradually emerging.

Aptamers are a type of single-stranded nucleic acid (DNA or RNA) molecules that can highly bind to specific target molecules. Their unique structure and properties make them gradually become an important technology in biomedical research and applications. Compared with traditional antibodies, aptamers have a series of significant advantages such as small size, low synthesis cost, high uniformity, and easy customization and modification. Since aptamers are composed of short-chain nucleic acids, their synthesis does not rely on cell culture and can be rapidly produced on a large scale through chemical synthesis, which makes aptamers more advantageous in commercial production.

The binding of aptamers to targets usually relies on their unique three-dimensional structure, which can efficiently recognize and bind specific small molecules, ions, proteins, viruses or other biomolecules. These properties enable aptamers to be widely used in various fields, such as targeted drug delivery, clinical diagnosis, environmental monitoring, food safety, etc. In terms of expanding the target range, aptamers are particularly suitable for small molecules and metal ions that cannot be effectively recognized by traditional antibodies, which is extremely important in some special application scenarios. In addition, aptamers are highly customizable, and researchers can enhance their stability, affinity, and selectivity through precise chemical modifications. This customization capability allows aptamers to provide customized solutions for different experimental needs. Optimization and modification based on nucleic acid templates provide broad prospects for the application of aptamers in precision medicine and drug development.

Nucleic acid aptamers (aptamers) are a type of highly specific small molecule ligands consisting of 20 to 110 nucleotides and usually exist in the form of single-stranded DNA or RNA. Its basic structure consists of two parts: one part is a fixed sequence, and the other part is a random sequence. Random sequences are mutated within a specific range to form nucleic acid fragments with different spatial structures. These fragments can bind to target molecules (such as proteins, ions, small molecules, etc.) after screening. The design and screening process of nucleic acid aptamers usually relies on a method called SELEX (systematic evolutionary in vitro ligand selection). Through multiple rounds of selection, aptamers that can bind target molecules efficiently and specifically are gradually screened out.

The unique property of aptamers is their ability to fold into specific tertiary structures, which are usually determined by hydrogen bonds, π-π interactions, and other non-covalent interactions between nucleotide sequences. These structures not only enable aptamers to possess high binding affinity, but also enable them to recognize and bind target molecules very accurately. The "molecular recognition" ability of nucleic acid aptamers is similar to that of antibodies, but it has unique advantages in terms of molecular weight, production methods, synthesis costs, and modifiability.

Due to their small molecular weight and high flexibility, nucleic acid aptamers can bind to a variety of target molecules, including not only macromolecular proteins, but also small molecule drugs, metal ions, and even pathogens such as viruses. In addition, the design of aptamers can precisely control their affinity, stability and selectivity, so they can be optimized according to different needs. These advantages enable nucleic acid aptamers to show broad application potential in many fields, especially in the fields of precision medicine, disease diagnosis, drug delivery, and biosensors. For example, aptamers can achieve specific recognition and binding of target molecules in complex pathological environments through precise structural design and modification, which makes them of great significance in targeted therapy. In the process of drug development, aptamers can not only serve as targeting ligands to replace antibodies, but can also be used to screen new drugs, develop new biomarkers and sensors, and even be used in vaccine research and development.

Advantages of nucleic acid aptamers: ① It has the advantages of high thermal stability, easy chemical synthesis and modification, and low immunogenicity. It is used in fields such as bioanalysis, biomedicine, biotechnology, and sensing technology. 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, has a high failure rate and is costly.

In Vitro Screening and Optimization of Aptamers

1.Quick Screening Method

Rapid screening methods play a vital role in the development process of nucleic acid aptamers, which can significantly improve the binding efficiency of targets and aptamers, reduce screening rounds, and thus accelerate the research process. Among them, SELEX technology based on electrophoresis (CE), microfluidic chips and magnetic beads is currently the most widely used and influential rapid screening method.

①. CE-SELEX (Capillary Electrophoresis-SELEX) is a screening method combined with electrophoresis technology. By efficiently separating aptamers and target complexes, high-affinity aptamers can be quickly obtained in only 1 to 3 screening cycles. . This method is usually used for nucleic acid aptamer screening of protein targets, and is widely used in the field of biomedicine due to its rapid and efficient characteristics.

②.Microfluidic chip technology (M-SELEX) provides a more compact and efficient solution for rapid screening of nucleic acid aptamers. This method relies on microfluidic technology and can achieve higher automation, miniaturization and integration, so that the screening process is not only accelerated, but also large-scale, high-throughput screening can be carried out. This provides new possibilities for precise screening and optimization of aptamers, especially in the fields of drug development and biosensing, showing great potential.

③. Magnetic bead-SELEX (MB-SELEX) is a screening method that immobilizes targets on functionalized magnetic beads through chemical methods. The target-bound aptamer is separated from the unbound sequence using a magnetic field. This method is more dependent on experimental conditions in terms of separation efficiency. Although aptamers can be screened relatively quickly, the overall screening efficiency is low, so there may be some limitations when high-efficiency screening is required.

These rapid screening technologies not only improve the efficiency of aptamer screening, but also provide more flexible options for subsequent high-throughput screening and precise optimization, laying the foundation for the application and development of nucleic acid aptamers.

2. Methods Suitable for Screening Small Molecule Target Nucleic Acid Aptamers

With the continuous development of aptamer screening technology, in vitro screening methods based on target immobilization have effectively solved a series of technical problems such as non-specific binding of libraries, exposure of target binding sites, low affinity and chemical modification, and significantly improved the aptamer screening method. Accuracy and efficiency of ligand screening.

Capture SELEX (library immobilization-based screening technology) is a representative method to break through these technical bottlenecks. The aptamer library used in this method consists of three parts: random sequence, docking sequence and primer sequence. During the screening process, the aptamer library binds to the target through specific docking sequences, thereby effectively avoiding the problem of non-specific binding, while also ensuring the exposure of the target binding site, improving the affinity and affinity of the screened aptamers. Specificity. The application of this technology has greatly improved the screening efficiency and the reliability of the results, making the aptamer screening process more accurate.

On the other hand, homogeneous screening technology further improves the efficiency of aptamer screening by simplifying the screening steps. This technology can quickly screen out nucleic acid aptamers with high affinity to the target from a large number of libraries, and the screened aptamers can not only be used in basic research, but can also be transformed into biosensors for environmental monitoring and medical treatment. testing and other fields. Homogeneous screening makes the screening process more efficient and flexible by reducing operational complexity, which greatly promotes the application of aptamer technology.

3. Screening of Highly Stable Nucleic Acid Aptamers in Vivo

Chemical modification is one of the key technologies to improve the stability of aptamers in vivo. From the initial modification of the phosphate backbone, pentose sugar and bases to the introduction of artificial bases, chemical modification has become a common method to improve the stability of aptamers. These chemical modifications can effectively enhance the biological stability of aptamers and extend their half-life in the body. Through these modifications, aptamers can resist enzymatic degradation in vivo and in vitro, thus improving their application potential as drug delivery tools or diagnostic reagents.

During the screening process, chemically modified aptamer libraries can be used directly for screening, or libraries containing natural bases can be screened to find the optimal aptamer. This enables researchers to select appropriate screening strategies based on needs.

Mirror-image nucleic acid aptamers (L-shaped nucleic acids) are a special form of nucleic acid that is opposite to the natural D-shaped nucleic acid structure and therefore are not easily degraded by natural nucleases. Due to their high chemical stability, mirror-image aptamers have a long biological half-life in the body and show great application potential in areas such as drug delivery and biosensors.

Circular nucleic acid (CNA) is a molecule that forms a ring structure by connecting the two ends of nucleic acid. This structure can effectively resist the degradation of exonuclease and has extremely high stability. Circular nucleic acids are widely used in biosensors based on roller amplification. Due to their unique stability and resistance to enzymatic degradation, CNA has important application value in biological detection and precision medicine.

4. Screening Methods to Improve the Specificity of Nucleic Acid Aptamers

Currently, there are two main methods used for aptamer screening. One is through negative screening. Although this method can effectively remove non-specific binding molecules, it is usually affected by background interference and high cross-reactivity, resulting in certain limitations in the accuracy of the screening results. Another method is to combine 2-3 SELEX technologies. By combining different screening strategies, the non-specific sequences that may be introduced by a single SELEX technology can be effectively eliminated, thereby improving the accuracy and reliability of the screening results. This multiple screening strategy can further optimize the specificity and affinity of aptamers in multiple screening steps to ensure the acquisition of high-quality aptamers.

5. Screening Methods to Improve the Affinity of Nucleic Acid Aptamers

Conventional screening methods to improve aptamer affinity include reducing the concentration of positive screening targets, using chemically modified libraries, and increasing screening pressure for screening. With the development of technology, engineering design or in vitro screening of high-priced nucleic acid aptamers has become an important method to improve nucleic acid aptamers. A technical means of ligands.

Peptide Aptamer Screening

The choice of peptide aptamer screening method depends on the subsequent experimental use of the peptide aptamer, which can be divided into in vivo non-display systems, in vitro display systems and emerging molecular docking simulation methods based on bioinformatics.

① Yeast Two-hybrid Technology

With the development of technology, the Y2H system has been integrated with next-generation sequencing (NGS) to create the Y2H-seq assay, which requires only one step of PCR to comprehensively identify interacting proteins, significantly improving experimental efficiency.

② Phage Display Technology

This technology can preserve the activity of peptide aptamers throughout the screening process, and the competitive environment promotes the improvement of affinity to the screened peptide aptamers. But when dealing with small molecule targets on the solid phase, time-consuming inverse screening is required.

③ Molecular Docking Technology

Molecular docking is a simulation technology used to predict the binding mode of receptors and ligands (including docking position, docking size, binding affinity, etc.), and can be combined with bioinformatics to design and screen peptide aptamers.

TekBiotech provides professional nucleic acid aptamer screening services and is committed to screening out nucleic acid aptamers with high affinity and high specificity for customers. Through advanced SELEX technology, combined with negative screening, combined screening and multi-round optimization strategies, TekBiotech can effectively eliminate non-specific binding and ensure the accuracy of screening results. In addition, TekBiotech also provides chemical modification, stability optimization and other services to help customers obtain highly stable and efficient aptamers to meet the needs of various biomedical and diagnostic applications.