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Metal Ion Nucleic Acid Aptamer Screening Service
With several years of research experience in the field of antibody discovery, TekBiotech provides customers with efficient, highly specific and high-affinity in vitro screening services for nucleic acid aptamers. It tailors solutions based on customers' specific screening targets and quickly and accurately screens out aptamers for target targets. TekBiotech can provide in vitro screening services for nucleic acid aptamers based on a variety of sample types (including proteins, peptides, amino acids, small molecules, cells and bacteria, metal ions, etc.), and the services provided to customers cover the upstream and downstream of in vitro screening of nucleic acid aptamers, from gene analysis and synthesis, nucleic acid aptamer sequence design, aptamer in vitro screening, aptamer synthesis, to affinity determination services.
█ Metal Ion Nucleic Acid Aptamer Screening Service
Nucleic acid aptamer screening refers to the principle of systematic evolution of ligands by exponential enrichment (SELEX), through incubation, washing and amplification of target molecules with nucleic acid aptamer libraries, and obtaining target oligonucleotide fragments (DNA fragments or RNA fragments) from nucleic acid aptamer libraries. Aptamers have become increasingly sophisticated in the fields of cells, fungi, proteins, peptides and most small molecules due to their high specificity, strong affinity, excellent stability and easy preparation and labeling. However, heavy metal ions and some small molecules that cannot be chemically modified are difficult to be subsequently screened for nucleic acid aptamers by self-modification. Therefore, scientists at Tianjin Tekbiotech have designed a new screening system based on conformational changes of nucleic acid aptamers, which does not require modification of the screening target and uses a small amount of target raw materials.
The nucleic acid aptamer service provided by TekBiotech for various types of samples is based on the exponential enrichment ligand system evolution technology (SELEX screening technology) to screen and obtain the corresponding DNA or RNA sequences, and to meet the screening requirements of different samples through a variety of screening methods (including cell SELEX, liquid phase SELEX, etc.). At the same time, the aptamer library capacity of TekBiotech can reach 10^13-10^14. After 8-10 rounds of screening and enrichment, it can ensure the acquisition of aptamers for target substances (binding affinity can reach nM - pM level), which has greater advantages compared with conventional aptamer screening. TekBiotech's scientific research team lays a solid foundation for customers' subsequent aptamer function verification (including affinity verification, competitive ELISA verification, in vitro targeted cell functional verification (for example: in vitro recognition and inhibition function verification of nucleic acid aptamers, in vitro flow blocking function, etc.), in vivo functional verification (for example: in vivo targeted inhibition function verification of aptamers, signal pathway blocking function verification, etc.)), and provides support for customers' subsequent scientific research work such as targeted molecular drug development.
█ Principle of Metal Ion Aptamer Screening Service
For unconventional chemical small molecules, such as metal ions, toxin small molecules, etc., TekBiotech uses conformational changes and photoelectric reporting systems to redesign the aptamer library from the original 40nt length to a 28nt saturation mutation library and redesign the homology arms at both ends of the saturation mutation region for subsequent small molecule screening. As shown in Figure 1, the schematic diagram of the screening principle of unconventional small molecules:
Figure 1 Schematic diagram of screening based on conformational changes of aptamers
█ Process of Metal Ion Aptamer Screening Service
SELEX screening technology covers four key links: binding, separation, amplification, and purification. By cyclically executing these steps, oligonucleotides with high specificity to the target are accurately identified from the library, and sequence information is obtained by sequencing. Different aptamer sequence designs will shape different three-dimensional structures and show affinity for specific metal ions. Although the chemical properties of metal ions, such as hardness and coordination structure, affect screening, their single binding site and similar structures of different metal ions will interfere with the specific recognition of metal ions in the same group by aptamers. Conventional SELEX methods may make it difficult to separate highly specific aptamers. The process of TekBiotech is shown in Figure 2. When the target molecule binds to a specific aptamer, the aptamer undergoes a conformational change and has a different surge rate from the unbound aptamer in the capillary electrophoresis system. This difference is used to separate the bound aptamers and perform multiple rounds of panning through PCR amplification technology.
Figure 2 Metal ion nucleic acid aptamer screening service process
█ Metal Ion Nucleic Acid Aptamer Screening Service Content and Cycle
Steps | Service Content | Cycle |
Step1: Nucleic acid aptamer screening | (1) The customer provides the screening target; (2) Aptamer library screening and enrichment, generally 8-10 rounds; (3) Screening products for NGS sequencing; (4) Delivery: Priority is given to providing 8-20 pre-aptamer sequences, NGS sequencing raw data, experimental report, raw data; | 10-15 Weeks |
Step2: Aptamer synthesis and affinity determination (optional) | (1) Aptamer synthesis according to sequence; (2) Aptamer and target affinity determination, BLI or SPR, KD determination; (3) Delivery: experimental report, raw data; | 2-3 Weeks |
█ Advantages of Metal Ion Aptamer Screening Service
A variety of screening targets are available: proteins, peptides, amino acids, small molecules, etc. | Large library capacity, high project success rate | Mature SELEX technology platform: the affinity of nucleic acid aptamers obtained by screening can reach nM-pM level | Rich supporting downstream verification experiments: affinity verification (including BLI and SPR affinity verification), competitive ELISA verification, flow blocking verification, etc. | Experimental records are traceable: Chinese and English experimental reports, original experimental records | One-to-one personalized solution customization to meet the scientific research project needs of various customers |
The aptamer is a small nucleic acid folded in a three-dimensional conformation to make the aptamer a specific binding target. The target has a variety of species, either proteins small molecules, or metal ions. In contrast to antibodies, aptamers enable in vitro selection with low immunogenicity. Among them, heavy metal, as the most toxic metal pollutant, can pollute the natural environment, and detecting heavy metal pollution has become an important task. At the same time, heavy metals are also classified as trace elements because of their low content. Therefore, developing an accurate and sensitive heavy metal detection method is very important to ensure human and environmental safety. Aptamers as biological probes show high binding affinity that can be directly converted to high detection sensitivity. On the other hand, high selectivity and stability enable it to detect various targets, especially metal ions. Since identifying aptamers for metal ions, aptamer-based sensors and detection methods have become possible and provide new methods for detecting metal ions, including electrochemical, colorimetric, and fluorometry.TEK Biotech has been studying in the field of nucleic aptamers for a long time, which can provide customers with metal ion aptamers with excellent performance to help customers better research follow-up projects.
Long-term exposure to toxic heavy metals may also cause cancer in both humans and animals. Therefore, the monitoring of toxic metal ions is an important issue in environmental protection as well as in disease prevention and treatment. However, the high selectivity and sensitivity detection of trace heavy metals remains a challenging area of research. Capapping of target ions from the complex matrix is a key step in detecting heavy metals. So far, researchers have designed several DNA aptamers to identify and detect heavy metal ions, mainly lead, mercury, silver, and so on. These ions can specifically interact with DNA bases to form strong and stable complexes, and some aptamer-based sensors can be used to monitor the concentration of heavy metal ions in water, providing strong support for the early warning and treatment of environmental pollution. In addition, metal ion aptamers can also be used in analytical chemistry, food science, and other fields. In analytical chemistry, aptamers can be used as efficient isolation and enrichment materials for the extraction and purification of metal ions in complex samples. In food science, aptamers can be used to detect heavy metal ion residues in food, which can ensure food safety. As novel recognition elements in biosensors, aptamers outperformed antibodies in terms of low cost, good thermal stability, simple production, ease of modification with functional groups, and conformational changes induced by binding.
The Capture-SELEX method is often used to screen metal-ion nucleic aptamers. The Capture-SELEX method uses the strong binding force between biotin and avidin, labels the DNA chain in the library using biotin, and then uses the library with avidin, where the ssDNA library is fixed on the magnetic beads. Then, using the fixed library bound to the target, we eluted the magnetic bead library bound to the target and performed PCR amplification to obtain a certain number of secondary libraries. After the whole screening process is completed, we may find that the aptamer affinity does not meet the expected requirements, or the aptamer is not specific enough. In the above situation, we can reduce the non-specificity and improve the affinity by changing the library buffer conditions during the screening process. At the same time, using Capture-SELEX can have higher affinity than using other methods to cure the target, and ePCR can be amplified to avoid the lack of library diversity. TEK Biotech can improve the efficiency of aptamer screening through the above techniques, but also by optimizing the experimental conditions, to better obtain aptamers. In addition to providing cell nucleic aptamer screening services, it can also provide protein customization and phage display services.
In the synthesis of metal ion aptamers, we will encounter errors in library design and synthesis or insufficient library diversity. The nucleic aptamer library is up to 10 ^ 10-10 ^ 14, and the number of random sequences in the library is enough to cover all possible aptamer sequences. Meanwhile, strict quality control, including sequencing verification, was performed to ensure the accuracy of the library. Too low target concentration may lead to the screening of high-affinity aptamers, while too high concentrations may increase non-specific binding. At the same time, TEK Biotech also optimized the experiment according to the properties of the target to determine the appropriate target concentration, pH value of buffer, and ionic strength and these parameters will affect the binding of the aptamer and the target. The researchers determined the appropriate screening temperature based on the thermal stability of the target and the melting temperature of the aptamer. Negative screening through blank magnetic beads, removal, and nonspecific binding of magnetic beads. Primer dimers and non-specific amplification may occur during PCR amplification. We optimized the PCR amplification conditions, adjusted the primer concentration, and annealing temperature, and used high-quality primers and enzymes for amplification. Finally, TEK Biotech can provide professional technicians to molecularly perform the sequencing data to ensure the accuracy of the final results.
The recognition of copper ions and aptamers is a process that requires highly regulated aspects, and this recognition process requires the ability of DNA to assist. There are now two possible accounts for the mechanism by which copper ions bind to aptamers, a sandwich where copper ions and 2 adjacent G form a sandwich structure and G and T binding on copper ions and different nucleotide chains. Some researchers have also found that platinum can bind to nucleotides on G and T, which can improve the stability of nucleotide DNA and prevent DNA from continuing replication, transcription, and cell division. Regarding mercury and silver ions, researchers have proposed that they interact with aptamers and lead to structural degeneration. However, the mechanism of metal ions potassium, sodium, and aptamer is related to the four-chain structure. The G-teplex is stable in the presence of monovalent cations (K +, Na +, NH4 +, etc.) because these positive ions coordinate with the negative oxygen atoms in the base to stabilize the G-teplex (G-quadraplex). The binding mechanism of metal ions to aptamers is a complex process involving a variety of interacting forces and factors. This combination has broad applications in biomedicine, materials science, and other fields.
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