Revolutionary Peptide Aptamers: Enhancing SELEX Screening Applications

Introduction of Peptide 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.

Peptide aptamers are short peptides that have a similar nature to nucleotide aptamers, which are composed of 5-20 amino acid residues. Peptide aptamers are screened from peptide libraries that contain random amino acids through the SELEX technique. The peptide aptamers are fixed to a very stable protein scaffold, such as thioredoxin, to increase their binding strength and stability to the target in a way that is easy to fold and prevents oligomerization.

The advantages of peptide aptamers are (1) High stability, low immunogenicity, high affinity, and high tissue permeability; (2) It has the characteristics of a simple structure, and there is no difference between different synthesis batches; (3) The cost is lower and the dependence on animal immunity is lower; (4) The hydrophobic amino acids can be preferably removed to maintain a higher hydrophilic ability.

Screening of Peptide Aptamers

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

Yeast two-hybrid technique

The yeast two-hybrid technique studies protein interactions in vivo by fusing protein X with the DNA binding domain (BD) of the transcription factor and candidate peptide with the transcription activation domain (AD) of the transcription factor, respectively. If the X protein binds to a candidate peptide, it activates transcription, and gene expression, and can be monitored by colorimetric enzyme assays or growth selection methods.

The technique is simple to operate, has wide applicability, and can be used for large-scale short peptide screening. However, this technique has limitations: it is only suitable for targets with large molecular weight (membrane proteins cannot be studied), is prone to false positives, and costs screening time.

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

Phage display technology

The principle of phage display technology is to fuse foreign DNA fragments encoding peptides with phage surface protein-coding genes.

The technology can preserve the activity of peptide aptamers during the whole screening process, and the competitive environment promotes the improvement of affinity for the screened peptide aptamers.

Molecular docking technology

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

Aptamer SELEX

Aptamer SELEX is a method for in vitro screening of aptamers. The target range of SELEX technology 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. 

The method of SELEX in nucleic acid aptamer development that the target range of SELEX technology has expanded from the original purified proteins and organic small molecules to almost all targets such as cells, viruses, tissue slices, and ions.

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.

The method of the principle is as follows:

(1) Nucleic acid ligand library design

Nucleic acid ligand libraries can be prepared by chemical synthesis or biosynthesis.

(2) 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.

(3) elution

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

(4) PCR amplification

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

(5) SELEX screening

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

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.

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.

Application of Peptide Aptamers

Peptide aptamers are used in the field of medicine. In the diseases, aptamers can be used as biological probes targeting cancer cells. A new peptide aptamer has been successfully designed for livestock and poultry diseases by combining the computer bioinformatics method. In the field of cancer research, aptamer-peptide conjugate is used as a targeted chemotherapy sensitizer in the treatment of breast cancer.

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 ligand library design, antibody expression and purification, affinity determination, antibody sequencing, etc., to meet customer needs.

References:

[1] Yuan Y, Li Y, Liu S, Gong P, et al. An overview of aptamer: Design strategy, prominent applications, and potential challenge in plants [J]. J Plant Physiol. 2024, 296: 154235.

[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.