Nucleic acids play a central role in human biology, making them suitable and attractive tools for therapeutic applications. Conventional drugs often target proteins to induce transient therapeutic effects, while nucleic acid drugs can achieve lasting or curative effects by targeting the genetic basis of the disease. However, natural oligonucleotides are characterized by low in vivo stability due to nuclease sensitivity and unfavorable physicochemical properties due to their polyanionic properties, which represent obstacles to their therapeutic applications. Countless synthetic oligonucleotides have been prepared over the past decades, and it has been shown that appropriate chemical modifications to nuclear nucleases, nuclefuranose units, or phosphate backbones protect nucleic acids from degradation, enabling efficient cellular uptake and target targeting, thereby ensuring the effectiveness of oligonucleotide-based therapies. The initial aptamer generated using SELEX consists of a random region consisting of approximately 30 – 50 nucleotides and two fixed primer parts. The constant primer region is mainly used for PCR amplification, and the randomly selected regions of SELEX have different roles in interacting with the target molecules. Aptamers are oligonucleotides with high affinity and specificity for their targets, sometimes referred to as "chemical antibodies" because of their similarity to biological receptors. Suppose that these aptamers form secondary / tertiary structures that provide a binding pocket for the target. Compared with antibodies, these aptamers have unique characteristics, so the high attention to these aptamers is also due to their unique characteristics, including low production cost without living systems, easy chemical modification during chemical DNA synthesis, custom characteristics, low levels of immunogenicity and toxicity, do not interfere with the selection of aptamers, and high chemical stability, binding affinity, reproducibility and reusable.

Process of oligonucleotide synthesis. During biosynthesis, the enzyme accumulates nucleic acids from the 5′end monomer to the 3′end. The chemical synthesis of oligonucleotides and artificial nucleic acids (usually performed by automated solid-phase oligonucleotide synthesis (SPOS)) occurs in the opposite directions. In this case, the first nucleoside is connected to the solid phase by its 3′-OH group, which is usually a controlled pore glass (CPG), while the 5′-hydroxyl group is protected in the form of a 4,4′-dimethoxytriphenyl (DMTr) ether. Then, a synthetic cycle consisting of four reactions is started. First, the DMTr, the group is trichloroacetic acid (TCA) and cleaved to release the 5′ -OH group. In the next step, the 3‘-O- (N, N-diisopropyl) phosphoramide derivative of the 5′-O-DMTr-nucleoside is added with the activator (acidic azole catalyst, such as 1H-tetrazole or 4,5-dicyanogenic imidazole). In this coupling step, an interphosphotriester nucleotide bond is formed. Subsequently, requiring capping due to the incompleteness of the coupling implies protecting the remaining 5′-hydroxyl group by acetylation with Ac 2 O in order to exclude unreacted monomers from further synthetic cycles. In the final step of the cycle, the obtained phosphate derivatives are oxidized to iodine-containing phosphate. The above steps must be repeated n-2 times to obtain oligomers of length n nucleosides. Finally, the protecting group was removed and the intact oligomer was cleaved from the solid phase. Benzoyl group is used to protect the amino group of nuclear bases in cytidine and adenosine, and isobutylyl group is used to protect the amino group of guanosine.

Bioinformatics tool used to examine the contribution of various regions of more than 2,000 aptamers to target recognition, consistent primer sequences contribute little to aptamer-target binding. Certain non-primed domains are not required for the aptamers to recognize their targets. Unwanted nucleotides in the aptamer may increase the free energy and may hinder the way in which the aptamer and the target molecules interact. Indeed, eliminating the primer-binding site and further reducing the aptamer sequence often yields simpler structures with the same or even higher affinity as the target. Thus, to maximize the performance of the aptamers, many studies have shortened those unnecessary nucleotides. On the other hand, the addition of nonspecific nucleotides may enhance the ability of the aptamer to bind the target, maintain thermostability, and be resistant to nuclease degradation. Furthermore, the aptamer is engineered by containing short hairpin DNA fragments, which improves thermostability and stabilizes its resistance to the nuclease. The insertion of the additional G-C pairing enhances the stem region of the aptamer. Sequence length affects signaling in aptamer-based device determination and the intrinsic characteristics of the aptamer. Using the aptamer-coated gold nanoparticles (AuNPs), an aptamer sensor was created by Tian et al. Furthermore, they show that aptamers with flanking nucleotides can adhere very closely to AuNPs, resulting in separation from, but not completely, AuNPs. Therefore, the length needs to be optimized to reduce the synthesis costs and improve the performance of the preselected aptamers.

Nuclear bases can be modified by introducing a variety of functional groups, designed to enhance the binding strength between the aptamer and the protein. To further enhance the binding efficiency of the modified aptamers, additional base designs were developed. In addition to these strategies, they also used SELEX post-modification techniques to successfully integrate amino acids into the TBA nuclear base structure. The experimental results showed that these chemical modifications not only strengthened the binding affinity, but also improved the anticoagulant properties. In addition, the adjustment of nucleic acid ribose fragments, such as the inclusion of 2 ′ -fluorine (2 ′ -F) or 2 ′ -fluorobarabinoic acid, is also helpful to enhance the binding force. In general, unmodified apgametes have relatively limited binding capacity to target molecules, while the apgametes optimized with 2 ′ -fluorine (2 ′ -F) modification show excellent binding and tolerance to nucleases. The β -D-furan ribose unit of the native NA can be replaced by either α or L stereoisomers or other sugars. L-nucleotides are enantiomers of type D, so their oligomers are called "spiegelmer". Spiegelmer Is resistant to nucleases, and is not immunogenic. They are not used for gene silencing, but have great potential as aptamers.

We can use the appropriate aperture ultrafiltration tube (such as 10 kd millipore ultrafiltration tube) for ultrafiltration recovery, but pay attention to the adsorption of nucleic acid by ultrafiltration tube, as long as the loss is not too much can be accepted. This method yields higher quality but also higher cost. It can also be performed with high efficiency, but the obtained single-stranded mass may be unstable.It is recommended that the nucleic acid aptamer be scanned by UV spectrum after purification to identify the quality.Use a kit suitable for the recovery of short acids, such as the Oligonucleotide Purification kit for Shanghai Biotech. Using the long and short chain method combined with denaturing PAGE electrophoresis, the resulting single chain mass is stable, but many operation steps. In aptamer synthesis we will encounter the formation of primer dimer, TEK Biotech by optimizing PCR conditions, such as adjusting the annealing temperature, primer concentration, etc. Avoid aerosol contamination and keep the experimental environment clean. High-quality primers and raw materials are used to avoid dimer formation.TEK Biotech is deeply engaged in a variety of nucleic aptamer screening platforms, which can provide a range of services including screening to synthesis, and is committed to better customer service.