Aptamer Screening Advances: New Progress in Small Molecule Targets

Introduction of 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-aptamer development.

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.

Classification of small molecule targets

1. Toxins

The toxin molecules do not produce antibodies and are difficult to detect. The aptamer is used to screen out toxin molecules and construct sensing elements, which can effectively and quickly detect toxins. Now, aptamers of many toxin molecules have been produced, such as saxitoxin, ochratoxin, and actinotoxin.

2. Antibiotics

Antibiotics can effectively prevent and control animal diseases to promote growth and are widely used in animal husbandry. The abuse of antibiotics leads to the accumulation of antibiotics in animal-derived foods and the environment, causing harm to human health and environmental safety.

At present, nucleic acid aptamer sequences have been screened for antibiotics, and these aptamers can be used for rapid antibiotic detection. For example, rapid antibiotic detection test strips have been developed for kanamycin, with kanamycin-specific aptamers as probes and complementary oligonucleotides as signal amplification elements.

3. Hormones

Hormones are used to treat diseases, mostly in pharmacological doses for anti-inflammatory, anti-toxic, anti-shock, and immunosuppressive treatments. Aptamers for various hormones (clenbuterol, 17β-estradiol, etc.) have been obtained by SELEX screening technology, and their affinity has been determined by nano-gold colorimetry.

4. Other small molecules

Biogenic amines/bases, peptides, natural products, dyes, pesticides, important monomer small molecules, and metal ions can be used as targets for small molecule target nucleic acid aptamer screening.

Small molecule target aptamer screening

There are few binding sites for small molecule targets, is difficult to obtain small molecule target binding aptamers with high affinity and specificity. The following is the way of small molecule target aptamer screening:

1. Screening technology based on a fixed target

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. GO-SELEX

GO has the advantages of strong hydrophilicity and good dispersion, which is the best new material applied in SELEX screening technology. GO novel materials can improve screening efficiency and increase compatibility.

5. Quartz Crystal Microbalance -SELEX (QCM-SELEX)

The sensitivity of quartz crystal microbalance is extremely high. The affinity can be judged by detecting the resonance frequency caused by mass change, and the selection can be based on this.

6. Human-genome SELEX

It has been reported that RNA aptamers that bind ATP have been screened using the human genome.

7. Computer-Aided -SELEX (In Silico SELEX)

① mFold software was used to predict the action site between the target and the aptamer and optimize and truncate the effective sequence of the aptamer.

② By computer simulation of specific points of the aptamer sequence and point mutation, the nucleic acid sequence is modified and optimized to improve the affinity of the aptamer.

Screening of small molecule drug aptamers

Small-molecule drugs have contributed to medical advances and improved patients' life levels, especially in treatment for specific indications. According to the molecular structure and size, molecularly targeted drugs are mainly divided into two categories: (1) small molecule targeted drugs with fixed molecular formula and molecular weight, such as kinase, and proteasome inhibitors. (2) Macromolecular targeted drugs with complex chemical structures, such as monoclonal antibodies, antibody-drug conjugates, etc. Small molecule targeted drugs mainly play anti-tumor and anti-inflammatory roles by blocking signal transduction pathways, in different fields such as solid tumors, hematologic malignancies, and immune-mediated inflammatory diseases.

After years of development, aptamers have become another useful molecular recognition tool in addition to antibodies. They bind specifically to small molecule targets, providing researchers with resources and tools. Using small-molecule drug 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, antibody expression, affinity determination, antibody sequencing, etc to meet customer needs.

References:

[1] Kim YS, Gu MB. Advances in aptamer screening and small molecule aptasensors [J]. Adv Biochem Eng Biotechnol. 2014, 140: 29-67.

[2] Nakatsuka N. Aptamer-Field-Effect Transistors for Small-Molecule Sensing in Complex Environments [J]. Methods Mol Biol. 2023, 2570: 187-196.

[3] Li X, Chen X, Mao M, Peng C, Wang Z. Accelerated CRISPR/Cas12a-based small molecule detection using bivalent aptamer [J]. Biosens Bioelectron. 2022, 217: 114725.