Breakthrough in Protein-Nucleic Acid Aptamer Screening Revealed

Introduction of Aptamers

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. In recent years, the aptamer development rapid, in the sensing analysis and detection of small molecules has been mainly used in food and environmental detection and biological analysis.

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.

Introduction of Protein Aptamer

Proteins are found in all biomolecules and form complex relationships with various molecular substances, from individual atoms/ions to giant macromolecules. The interaction between molecules is particular and important in life. Since small molecules bind to proteins with fewer atomic interactions, aptamer technology has emerged.

Interactions between nucleic acids and proteins play a role in basic cellular processes. Based on DNA/RNA polynucleotides can specifically target proteins in a wide range, including proteins that do not interact with nucleic acids under physiological and pathological conditions. Changing the structure of the aptamer can regulate the interaction with the protein and further affect the relevant pathways.

The first structural characterization of protein aptamer complexes was reported many years ago, but until now, progress has been slow. Van der Oost did a preliminary analysis of protein aptamer complexes in 2012. In 2016, a study characterizing the structural topic of protein aptamer recognition described only 16 protein aptamer complexes. Recently, Novoseltseva et al. investigated the structure of 45 such complexes.

In recent years, with the in-depth study of the protein aptamer, nucleic acid aptamer screening technology has been widely developed and has become a powerful tool for biomolecular detection. Targeted protein degradation is significant in chronic diseases, rare diseases, cancer, etc. Therefore, aptamers have been actively applied in the stability of targeted protein degradation. There has been only one aptamer-based drug on the market for years, pegaptanib sodium, which was approved by the FDA in 2004 to treat macular degeneration. Recently, the FDA approved a second RNA aptamer, avacincaptad pegol, for geographic atrophy secondary to age-related macular degeneration.

Nucleic Acid Aptamer Screening Techniques for Proteins

①  Aptamer sequence design

SELEX screening results are related to library design, so aptamer sequence design is important. The nucleic acid chain length is within 300 bp/nt. Because the long nucleic acid molecule will have multiple binding sites to bind the protein, some cases cannot be fitted with existing models. Therefore, short nucleic acid fragments (the core region involved in binding) for interaction studies are recorded.

②  Fixed protein or nucleic acid

In aptamer binding assay, protein and nucleic acid can be fixed separately, depending on the sample and the purpose of the analysis. If the protein sample is not pure, the protein can be fixed by the trapping method. If it is necessary to test the affinity between different kinds of proteins and nucleic acid molecules, the nucleic acid is fixed; If it is screened for aptamers with high affinity between the nucleic acid library and the protein, the protein is fixed.

Technology Development of Proteins Aptamer Screening

1. In vitro screening technology

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.

2. A screening technique based on library fixation -Capture-SELEX

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.

3. Homogeneous screening technology

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

4. Automated -SELEX

The cycle of traditional SELEX screening is too long. Automated SELEX screening improves the efficiency of aptamer screening. Studies have shown that tumor or disease-related proteins are selected for back screening, coupled with magnetic beads through chemical modification, then co-incubated with nucleic acid libraries, and the target proteins are screened through multiple rounds of washing and elution.

After years of development, aptamers have become another useful molecular recognition tool in addition to antibodies. They bind specifically to protein targets, providing researchers with resources and tools. Using protein aptamer screening technology, high-affinity aptamers can be obtained and applied in covalent drugs to overcome protein targets that are difficult to produce the drug.

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, aptamer binding assay, antibody expression, affinity determination, antibody sequencing, etc to meet customer needs.

References:

[1] Miao Y, Gao Q, Mao M, et al. Bispecific Aptamer Chimeras Enable Targeted Protein Degradation on Cell Membranes [J]. Angew Chem Int Ed Engl. 2021, 60(20): 11267-11271.

[2] Ji C, Wei J, Zhang L, et al. Aptamer-Protein Interactions: From Regulation to Biomolecular Detection [J]. Chem Rev. 2023, 123(22): 12471-12506.

[3] Troisi R, Balasco N, Autiero I, et al. Structural Insights into Protein-Aptamer Recognitions Emerged from Experimental and Computational Studies [J]. Int J Mol Sci. 2023, 24(22): 16318.