Cell Nucleic Acid Aptamer Screening Service

KMD Bioscience has many years of research experience in the field of drug antibodies, and has accumulated profound experience in antibody development, aptamer screening, aptamer assay, affinity maturation, etc. KMD Bioscience can provide aptamer screening services for a variety of sample types, such as proteins, peptides, amino acids, small molecular substances, cells and bacteria, metal ions, etc. KMD Bioscience can provide customized in vitro aptamer screening services to ensure efficient, accurate and rapid screening of aptamer sequences required by customers. In addition, KMD Bioscience can provide customers with upstream and downstream services covering aptamer screening, from gene analysis and synthesis, aptamer in vitro screening, aptamer synthesis, aptamer assay, to affinity determination. KMD Bioscience can provide strong support for subsequent functional verification of aptamers (including but not limited to affinity verification, competitive ELISA verification, in vitro targeted cell functional verification (such as in vitro recognition and inhibition function verification of nucleic acid aptamers, in vitro flow cytometry blocking function verification, etc.), and in vivo functional verification (such as in vivo targeted inhibition function verification of aptamers, signal pathway blocking function verification, etc.), laying a solid foundation for subsequent work of customers, such as downstream research and development of targeted specific molecular drugs.

The nucleic acid aptamer screening service based on SELEX technology provided by KMD Bioscience not only includes a variety of types of samples, but also has a variety of aptamer screening methods (magnetic bead SELEX, aptamer cell SELEX, capture SELEX, etc.) to meet the screening needs of different samples. Among them, in the cell targeting aptamers screening, the screening method with whole cells as the target is the most commonly used, that is, aptamer cell selex. KMD Bioscience's huge aptamer library capacity (10^13-10^14) guarantees the breadth of screening and is sufficient to obtain nucleic acid aptamers for customer targets. After multiple rounds of screening (generally 6-10 rounds), it ensures that high-specific nucleic acid aptamers are obtained, and the binding affinity of aptamers can reach the nM- pM level, far exceeding the standard of conventional aptamers. According to the different needs of each project, KMD Bioscience's team of scientists can plan the best screening scheme to ensure that the requirements of customers are met.

Cellular Aptamer Screening Service

As a functional nucleic acid, aptamer has many advantages. Based on its high specificity, it can accurately find the target molecule, thus avoiding the interference caused by nonspecific binding. Through in vitro selection technology (SELEX) screening, aptamers can be synthesized rapidly and in large quantities without complex biological preparation process. Aptamers have good chemical stability, can keep their structure and function unaffected under a variety of environmental conditions, and have higher thermal stability and lower immunogenicity. The molecular weight of aptamers is relatively small, making it easier for them to penetrate cell and tissue barriers and reach the site of action quickly. The core of aptamer screening is SELEX technology, which can accurately screen nucleic acid molecules that can achieve highly specific binding with specific targets from a large number of nucleic acid libraries with diverse sequences after multiple rounds of selection and amplification cycles. DNA or RNA aptamers obtained by in vitro screening technology have a unique three-dimensional structure, which can accurately identify and tightly bind to the corresponding target molecules. Compared with traditional antibodies, aptamers exhibit a variety of advantages, such as thermal stability, chemical synthesis and modification ability, and low immunogenicity. These characteristics together determine the great application potential of aptamers in a wide range of fields, such as bioanalysis, biomedical research, sensor technology innovation, and so on.

For the samples used for aptamer screening, some requirements need to be met.

Cellular Aptamer Screening Service Process

The specific screening process and sample requirements will vary according to the target characteristics and experimental requirements. The process of cell targeting aptamers screening is mainly based on SELEX technology and optimized using the aptamer cell selex method to meet the screening needs at the cellular level. A aptamer library containing a large number of random nucleic acid sequences (DNA or RNA, typically between 20 and 60 nucleotides in length) is constructed. Specific molecules on target cells or cell surfaces are used as targets (cell culture, purification, or labeling). The target cells are mixed with nucleic acid sequences from the aptamer library, and after incubation, the nucleic acid sequences bind to molecules on the surface of the target cells. The nucleic acid sequence that binds to the target is separated through filtration, centrifugation, magnetic bead separation, and other methods. The combined nucleic acid sequence is eluted and collected for the next round of screening. The secondary library is generated by amplifying the eluted nucleic acid sequence using PCR technology. The steps of binding, separation, elution, and amplification are repeated to gradually enrich high affinity aptamers. Through sequencing and sequence analysis, candidate aptamers were identified from the final enriched library. Simply put, SELEX technology is mainly to synthesize a single stranded oligonucleotide Library in vitro, and obtain nucleic acid aptamers with high affinity with target substances through repeated screening and amplification. Cell SELEX is to screen target substances such as cells and bacteria, separate and remove unbound suitable ligands by centrifugation and precipitation, alternate positive screening with negative screening of cells that do not express target proteins, and finally obtain aptamers. The specific process is shown in Figure 1.

Figure 1 cell targeting aptamers screening service process based on SELEX technology

Cellular Aptamer Screening Service Workflow

Advantages of Cellular Aptamer Screening Service

FAQ-Cell Nucleic Aptamer

1. What is the cell aptamer screening service, and what are the advantages of cell aptamers?

A: Our common aptamers include RNA aptamers and DNA aptamers, which targets have high affinity and specificity and are capable of high binding to the target. These aptamers have multiple functions in disease treatment and scientific research. First, it acts as a cell agonist to activate cell receptors and promote the cell to exert its effects. Secondly, it has the effect of antagonist to block the mutual binding and interaction between various structures. Furthermore, the aptamer can bind to the target so that the drug can be accurately transmitted to the therapeutic target. During the cell-SELEX screening, intact live cells were screened as a target, while the associated cell lines were used as negative controls for negative screening to exclude non-specific binding. The Cell-SELEX technology targets multiple cellular targets to generate nucleic acid aptamers, thus providing an advantage in identifying cells as molecules. In disease diagnosis, we can use aptamers for labeling, or use aptamers as a fixative to depurify the cells. These properties of aptamers provide new ideas for novel drug development. One of the highlights of the Cell-SELEX technology is the ability to retain the original conformation of the cellular receptor, which is different from screening using recombinant proteins as targets. KMD Bioscience not only provides the screening service of cell nucleic acid aptamers but also covers protein customization and phage display services, providing customers with a full range of one-stop solutions.

Figure 2 A Schematic representation of the Cell-SELEX step. (Reference documentation: Rahimizadeh K, AlShamaileh H, Fratini M, Chakravarthy M, Stephen M, Shigdar S, Veedu RN. Development of Cell-Specific Aptamers: Recent Advances and Insight into the Selection Procedures. Molecules. 2017 Nov 27;22(12):2070.)

2. Comparison of serum stability of DNA aptamer and RNA aptamer? How do we improve the aptamer stability?

A: In the case of aptamers, the stability of aptamers in serum has been limiting its application, evaluating the stability of RNA and DNA aptamers. Because RNA aptamers are less stable in serum and RNA aptamers are cheaper than DNA aptamers, researchers tend to screen DNA aptamers in serum. In terms of the experimental data, no modified RNA aptamers have an extremely short half-life in plasma and are rapidly degraded after co-incubation with rat and human serum, whereas DNA aptamers have a half-life of up to 30-60 minutes, showing that DNA aptamers are significantly more stable than RNA aptamers in serum. Therefore, in cases where high serum stability is required, most researchers prefer the DNA aptamers, although the RNA aptamers and DNA aptamers show higher affinity. In response to the aptamer in serum degradation of the disadvantage, scientists use chemical modification to improve stability, 2'-F modified nucleotides, or ribonucleotide 2' -hydroxyl for methoxy, or the use of methoxyethyl replacement ribonucleotide 2'-hydroxyl hydrogen, these modifications can improve the aptamer and target binding, increase oligonucleotide for nuclease resistance, prolong the half-life of the aptamer. The ability of the aptamer to enhance the aptamer to bind DNA or RNA. In addition, improving the serum stability of the aptamer can also be achieved by lipid modification, PEG modification, and optimization of the aptamer sequence.

3. How do we guarantee the specificity of the selected aptamer when screening the cellular nucleic acid aptamer? How to ensure that the development of aptamers can play the desired role?

A: KMD Bioscience biological in the process of nucleic acid aptamer screening, through several rounds of screening and enrichment process, step by step to improve the affinity of nucleotides in the library, the previous screening products as the library of the next round of screening, nucleotide enrichment, and screening, at the same time, in the negative control using other cell lines, other cell lines, and target cell structure, can be used as a negative screen target, can further elute the non-specific binding sequence, to improve the specificity of the final selected aptamer. After multiple rounds of selection, we performed the sequencing analysis of the final resulting nucleotide library. Through bioinformatics methods, the optimal aptamer sequence can be determined for the next step of optimization and validation. We often use fluorescence validation, SPR, and cell experiments to verify the specificity of the selected nucleic acid aptamers. Based on the practical experience of Teker tech, we recommend functional validation of aptamer using flow cytometry. Unlike other methods, flow cytometry enables the analysis and sorting of individual cells. Experiments can also be designed based on the desired application of the aptamer, for example, to verify whether the therapeutic aptamer can block tumor cell signaling or apoptosis.

4. What is the fitness system for internalization? How to help cell aptamers?

A: Aptamers are generated by the SELEX screening process. Compared with techniques such as antibodies, aptamers synthesized are less expensive and have higher specificity and affinity. Despite the outstanding advantages of aptamers demonstrated in in vitro experiments, most aptamers cannot autonomously enter the cell and require the assistance of external mechanisms. Scientific studies have shown that only a few aptamers have successfully achieved cell internalization. Aptamer internalization refers to the entire entry of the aptamer into the interior of the cell. Aptamers are essentially single-stranded oligonucleotide fragments that cannot cross the cell membrane directly into the cell interior without help. The internalization of aptamers is important in in vivo applications. Through internalization, aptamers can carry drugs or other therapeutic molecules into the cell interior and target binding, realizing the precise treatment of the target. To make aptamers with the ability to internalize, we can optimize the design and screening steps of the whole process and adopt the most cutting-edge technology to select aptamers with higher affinity and specificity, thus strengthening their binding force to cell surface receptors. At the same time, the gamete can also be chemically modified, such as adding fluorine, amino, methoxy, and other functional groups to adjust the physical and chemical properties of the aptamer, enhance stability, promote the interaction between the gamete and the cell membrane, and then promote the internalization process of the gamete.

5. What factors should we pay attention to in the cell nucleic aptamer screening? How to improve the affinity of the aptamer screening?

A: The same cell culture conditions are crucial for the success of Cell-SELEX. It is best to match the cell cycle, passage times and growth conditions, and the confluence of cells. Both prolonged cell growth and changes in culture conditions affect cell morphology and cells indicate protein expression, resulting in reduced efficiency of the Cell-SELEX screen. Furthermore, to reduce the nonspecific binding of oligonucleotides, we can decal target cells using reagents such as tRNA, salmon sperm DNA, or an endogenous biotin-blocking kit.Cell-SELEX allows screening of adherent and suspended cultured cells, first cleaned using PBS to reduce the effect of medium and serum on specific screening before subsequent aptamer screening. To be careful, treatment with trypsin causes degradation or damage of the markers indicated by the cells, so the trypsin-treated cells need to be recovered by incubating with the medium for at least 2 hours. Moreover, if we use adherent cells as cell fluid for aptamer screening, we shake the flask every time to prevent cell adhesion.Cell-SELEX screening was performed in the range of 4-37℃, and both appropriate temperature and prolonged incubation time would increase the affinity of the aptamer and increase the possibility of raptor incorporation into cells. Aptamer affinity can be improved by optimizing cell screening conditions, chemical modification of aptamers, and combining high-throughput sequencing and bioinformatics.

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