Academic: A Brief Analysis of Nucleic Acid Aptamer Screening

Previous words

An Aptamer is a single-stranded oligonucleotide that can specifically bind to target molecules. It undergoes self-adaptive folding through various interaction forces such as nucleotide base complementary pairing, hydrogen bonds, π-π stacking, and electrostatic forces to form a specific three-dimensional structure. This three-dimensional structure specifically binds to the target molecule through intermolecular forces.

Its binding and dissociation constants can reach nanomolar and picomolar levels, which are comparable to those of monoclonal antibodies. It mainly involves numerous fields such as proteomics, biological detection, molecular detection, targeted therapy, drug delivery, biosensors, food safety, molecular imaging, disease diagnosis and treatment, gene regulation, drug development, nanomaterials and ecological environment. It has high affinity and strong specificity, stable aptamer properties and structure, is not easily disturbed by the external environment, can be reused, will not have an immune effect on the experimental subjects, has a small particle size, can recognize multiple targets, is easy to label and modify, and has a low synthesis cost.

Random libraries generally select ssDNA rather than RNA. First of all, DNA is more stable chemically and biologically, which makes its selection and application easier. This is especially true when microarray platforms are applied to high-throughput SELEX; Secondly, DNa-based SELEX is more cost-effective and time-efficient because it does not require the additional reverse transcription steps necessary for RNA SELEX. Thirdly, from a commercial perspective, in terms of shelf life, DNA is easier to synthesize and more robust than RNA (Lakhin et al.,2013).

When designing a random library, it should be as short as possible, but not too short either. Because the synthesis of short random sequences is simple and the cost is relatively low; Secondly, the possibility of forming PCR by-products is reduced; And promote subsequent sequence truncation and application. To be compatible with common PCR polymerases, the random region is generally designed to be 30-50 nt(Cowperthwaite and Ellington et al.,2008).

The most commonly used method for preparing dsDNA is through polymerase chain reaction (PCR). However, there is a base bias phenomenon during PCR. SELEX reflects the law of "survival of the fittest", that is, only library sequences that show strong binding to the target of interest are enriched and amplified through polymerase chain reaction.

Ideally, in order to effectively select the best conjugate from the initial library, after PCR amplification, the proportion of different sequence species in the PCR amplicon should faithfully reflect the composition of the original template. However, it has long been observed that PCR amplification of complex oligonucleotide libraries with high diversity is very complex, unlike the amplification of homogeneous templates (Polz and Cavanaugh et al.,1998). Because each sequence in the initial library has different structural characteristics, such as GC content, secondary structure and melting temperature, they show adaptability to DNA polymerase, primers and other PCR conditions.

In the past few years, the rapid development of NGS technology has led to the invention of a novel PCR variant called emulsion PCR(ePCR) (Kanagal-Shamanna et al.,2016), by encapsulating each oligonucleotide sequence into a single PCR droplet surrounded by a hydrophobic organic phase. ePCR can significantly reduce PCR deviations and the formation of by-products to undetectable levels.