Aptamer Synthesis: Revolutionizing Biotech Solutions Today

Introduction of Nucleic Acid Aptamers

Monoclonal antibody production technology was introduced in 1975 and is of great significance in medicine, not only for basic science but also for drugs, biosensors, and other fields. The world's first therapeutic antibody was discovered in 1986 to prevent kidney transplant rejection, and since then, many antibody drugs have been found to treat various diseases, such as asthma. However, the monoclonal technique has certain limitations in the treatment of diseases. For example, antibodies that target lipids, carbohydrates, and organic macromolecules have a low affinity, and the affinity for drug coupling is somewhat affected. Therefore, artificial ligand-aptamers are emerging.

The aptamer is a single-strand nucleic acid chain that can be combined with various targets. It has the unique advantages of small size, low cost, uniform synthesis, customized modification, and nucleic acid template property, which can expand the range of potential targets in the selection scheme. For example, small molecules and ionic aptamers can supplement antibodies and are emerging as artificial ligands.

Nucleic acid aptamers are a class of single-stranded DNA or RNA molecules with specific molecular recognition ability. They are screened by SELEX in vitro and consist of 20-110 nucleotides. The sequences include random and fixed sequences and can be folded to form small molecular groups with tertiary structure, high affinity, and specific binding to target molecules. The target types of nucleic acid aptamers are broad, including small molecules, proteins, ions, cells, bacteria, tissue sections, etc.

Advantages of nucleic acid aptamer: ① It has the advantages of high thermal stability, easy chemical synthesis and modification, and low immunogenicity, and is used in biological analysis, biomedicine, biotechnology, sensing technology, and other fields. ② It has the advantages of short production time, low cost, and high specificity which has broad application prospects in the orthopedics field.

Disadvantages of nucleic acid aptamer: screening time and effort, high failure rate, and high cost.

Aptamer Design

Natural nucleic acids are composed of four nucleotides (A, C, G, T/U), and have less chemical diversity. Functional groups with the target can be introduced to improve diversity by chemically modifying nucleic acid aptamers. Libraries containing more guanine (G) were more likely to be screened for aptamers with G-quadruplex (G4).

The aptamer class includes RNA and DNA aptamers, easily degraded by nucleases in cells or biological samples. The aptamers were improved with anti-enzyme degradation ability, affinity, and specificity by chemical modification. Many chemically modified nucleic acid libraries have been used for aptamer screening. For example, circular DNA libraries for aptamer screening are simple and widely.

The screening results are related to the design of the initial library, so the aptamer sequence design is important. Aptamer sequence design includes the design of fixed regions and random regions.

The fixed region is related to the efficiency of PCR amplification of the library, and the design should ensure that no self-dimerization is formed during amplification, to avoid non-specific PCR products during amplification. The average length of the primer region was 18~21 nucleotides, and the shorter primer region had little influence on the subsequent screening, which was conducive to the selection of diverse sequences. The partially complementary library design with fixed sequences at both ends is beneficial to obtain stable secondary structure aptamers, but not conducive to screening high specificity aptamers.

The length of the random area is 30-60 nt, of which the 40 nt random area is the most commonly used. The design of the short random region is conducive to the subsequent truncation and application. The longer random region is suitable for the complex structure of the aptamer, which can obtain high specificity of the aptamer.

At present, there are several methods for designing oligonucleotide aptamers: scaling clustering of RNA aptamers, mode-finding, and aptamer optimization methods. In addition, scoring function, docking, dynamic simulation, QM/MM, QSAR, and others related to aptamer docking can be used for aptamer design.

The Method of in Vitro Screening of Aptamer SELEX

Aptamer SELEX is a method for in vitro screening of aptamers. In the early stage, SELEX mainly screened protein-target nucleic acid aptamers. With the aptamer development, it adapted to screen small molecule targets nucleic acid aptamers.

The method of SELEX in nucleic acid aptamer development that the target range has expanded from the initial purification of proteins and small organic molecules to almost all types of targets such as cells, viruses, tissue slices, ions, etc. and its principle is as follows:

Nucleic acid ligand library design

Nucleic acid ligand libraries can be prepared by chemical synthesis or biosynthesis. There are two types of nucleic acid ligand libraries: DNA aptamers and RNA aptamers.

Affinity screening

The nucleic acid ligand library was incubated with the target molecule, and the nucleic acid/target molecule complex was separated from the free nucleic acid sequence by solid phase adsorption, magnetic bead, filtration, and gel electrophoresis.

Elution

The nucleic acid/target molecule complex is obtained and the nucleic acid sequence binding to the target molecule is released.

PCR amplification

By PCR amplification, a nucleic acid library was obtained for the next round of screening.

SELEX screening

Repeat the above steps for multiple rounds of screening to increase the proportion of nucleic acid sequences binding target molecules.

According to the separation methods of the SELEX screening process, it can be divided into magnetic bee-based SELEX, library fixed SELEX, microfluidic SELEX, or capillary electrophoresis (CE) based SELEX.

 With the rapid development of artificial intelligence, high-throughput sequence analysis and high-performance nucleic acid aptamer identification and sequence optimization design are improved. The data utilization rate of high-throughput sequencing is low, and the tools for rapid and accurate analysis of sequence secondary structure are lacking. The selection of candidate sequences also mainly depends on the abundance ordering and the frequency of occurrence of conserved sequences in the primary structure.

Fig.1 Schematic model of aptamer screening techniques. (Reference source: An overview of aptamer: Design strategy, prominent applications, and potential challenge in plants)

KMD Bioscience has completed several nucleic acid aptamer projects and has a wealth of experience and mature technology. In addition, KMD Bioscience can also provide customized accounting aptamer design (DNA aptamer, RNA aptamer), antibody expression, affinity determination, antibody sequencing, etc., to meet customer needs.

References:

[1] Chai C, Xie Z, Grotewold E. SELEX (Systematic Evolution of Ligands by Exponential Enrichment), as a powerful tool for deciphering the protein-DNA interaction space [J]. Methods Mol Biol. 2011, 754: 249-258.

[2] Zhou J, Rossi J. Aptamers as targeted therapeutics: current potential and challenges [published correction appears in Nat Rev Drug Discov [J]. 2017, 16(6): 440.

[3] Kohlberger M, Gadermaier G. SELEX: Critical factors and optimization strategies for successful aptamer selection [J]. Biotechnol Appl Biochem. 2022, 69(5): 1771-1792.