Advancements in Screening and Optimization of Nucleic Acid Aptamers

Introduction of aptamers

Monoclonal antibody (mAb) 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 screening 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.

Screening and Optimization of Aptamers in Vitro

1. Rapid screening method

Rapid screening methods can improve the quick separation of complex or reduce the number of screening rounds. One of the most influential rapid screening methods is the SELEX screening based on CE, microfluidic chips, and magnetic beads.

CE-SELEX requires only 1-3 cycles to obtain aptamers and is currently mainly used for screening protein target nucleic acid aptamers.

The rapid aptamer screening method based on microfluidic technology (M-SELEX) lays a foundation for miniaturization, automation, and integration.

Magnetic bead-based SELEX (MB-SELEX) rapid screening method separates the bound sequence from the unbound sequence by chemically fixing the target on the functional magnetic sphere, which requires low efficiency.

2. Methods suitable for small molecule target aptamer screening

The in vitro screening technology based on target fixation has been developed to solve the problems of non-specific binding of aptamer libraries, exposure of target binding sites, poor affinity, and chemical modification.

Capture SELEX, a screening technique based on aptamer library fixation, overcomes the above technical problems. The aptamer library is composed of random sequences, docking sequences, and primer sequences.

Homogeneous screening technology improves the screening efficiency, and the screened nucleic acid aptamers can be converted into sensors.

3. Screening of highly stable nucleic acid aptamers in vivo

Chemical modification, from the initial phosphate skeleton, pentose, and base modification, to the introduction of artificial bases, is the most commonly used method to improve the stability of aptamers in vivo. This method can be used to screen both chemically modified aptamer libraries and libraries containing natural bases.

Mirror aptamers are L-type nucleic acids, which cannot be degraded by natural nucleases and have high stability.

Circular nucleic acids (CNAs) are resistant to exonuclease degradation and are widely used in biosensors based on roller amplification.

4. Screening methods to improve the specificity of nucleic acid aptamers

At present, there are two methods, one is through negative screening, but often subject to background interference and high cross-reaction; The other is to combine 2-3 SELEX screenings to eliminate non-specific sequences that may be introduced by one SELEX screening.

5. Screening methods to improve the aptamer affinity maturation

Conventional screening methods to improve aptamer affinity maturation include reducing the concentration of positive screening targets, using chemical modification libraries, and increasing screening pressure. With the development of technology, engineering design or in vitro screening of high-priced nucleic acid aptamers has become a technical means to improve nucleic acid aptamers.

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.

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 peptide aptamer affinity maturation.

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.

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 library construction, SELEX screening, aptamer affinity maturation, antibody expression, affinity determination, antibody sequencing, etc. to meet customer needs.

References:

[1] Gao S, Zheng X, Jiao B, Wang L. Post-SELEX optimization of aptamers [J]. Anal Bioanal Chem. 2016, 408(17): 4567-4573.

[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.