Introduction to Nucleic Acid Aptamer Library Screening Technology-Molecular Biology Service Topics-Tekbiotech-Yeast Display Service,Phage display technology

Introduction to Nucleic Acid Aptamer Library Screening Technology

Aptamers are oligonucleotide sequences that are screened by an in vitro method called systematic evolution of ligand exponential enrichment (SELEX). Aptamers are known as "chemical antibodies" that can achieve specific recognition of targets through high-affinity conformational changes. They have attractive characteristics such as simple chemical synthesis, low cost, relative stability, low batch-to-batch variability, easy modification and signal amplification, and low immunogenicity. At present, aptamers have shown great application potential and economic benefits in disease diagnosis, environmental testing, food safety, drug delivery and discovery, and have attracted the attention of researchers from more than many disciplines.

I.Aptamer Screening

The library contains a random region with two fixed primer binding sequences at both ends to amplify the enriched sequence by PCR. Ideally, the developed aptamer sequences should bind to their targets through a tertiary structure formed only by nucleotides in the random region. However, in the SELEX process, the interaction between the fixed primer binding site and the random region sequence is inevitable. Studies have shown that the primer binding site is involved in the formation of the functional secondary structure of the aptamer. Functional motifs that structurally exploit constant primer binding regions may be more prevalent in enriched libraries and dominate in genetics.

As an essential component of SELEX, PCR amplification begins with the binding of primers to fixed primer binding sites. Sequences with random regions exhibit strong interactions with fixed primer binding sites (competing for primer binding to primer binding sites) and may exhibit a selective disadvantage in primer binding and subsequent amplification. After multiple rounds of selection and amplification, this selective disadvantage is likely to outweigh any selective advantage of functional motifs formed by random regions. In contrast, sequences with random regions do not show interactions with fixed primer binding sites.

Like proteins, aptamers require a secondary structure to bind to their targets. It has been reported that there are 4n (equal to the number of bases) species of oligonucleotides, and a classic NA library contains up to 10^15 sequences, constituting a huge, highly diverse library for aptamer selection. With such a library, one can expect that aptamers can be targeted to any target you can think of. Unfortunately, the success rate of SELEX is only 30%, which is confounded by the many sequences in the library showing simple structural motifs and unstable pillars, both of which are unlikely to form high-quality aptamers. By introducing a designed pattern, the emergence of functional secondary structures in structurally random regions that are conducive to information-rich structures can be significantly increased, which is a practical method for designing efficient SELEX libraries.

Nucleic acid aptamer screening process-tekbiotech.png

Figure 1 Nucleic acid aptamer screening process

II.Summary of Aptamer Screening Methods

Methods	Advantages	Disadvantages
Cultivation of targets and libraries under nitrocellulose membrane filtration in matrix-free conditions	-- Easy to operate	-- Applicable only to proteins (cannot bind small peptides due to membrane size); -- Low efficiency due to nonspecific binding of nucleic acids to the membrane;
Electrophoretic mobility shift assay	-- Simple to handle; -- Accessible to routine laboratories; -- Enables real-time monitoring of the selection process; -- Minimizes nonspecific binding;	-- Applicable only to protein targets; -- Low resolution due to native gel electrophoresis; -- Difficult to maintain stable selection conditions such as temperature, salt concentration;
Capillary electrophoresis system	-- High separation resolution, supports single round of selection; -- Applicable to a wide range of targets, especially small molecules; -- Develop smart aptamers with predefined properties (Kd, koff, kon) by manipulating separation parameters; -- Minimizes nonspecific binding;	-- Relatively limited library input; -- Difficult to optimize separation conditions for some proteins;
Solid-phase immobilized targets	-- Simple handling; -- Applicable to a wide range of targets; -- Accessible to routine laboratories; -- Enables real-time monitoring of the selection process;	-- Non-specific binding to the immobilization matrix; -- The method used to immobilize the target can affect the native structure of the target;
Sequences with preferred properties are eluted with salts, DNases, etc.	-- Simple and inexpensive; -- Develop aptamers with preferred binding properties;	Its reliability needs further study
Immobilized library sequence SELEX on solid-phase capture	-- Suitable for developing aptamers for small molecules that cannot be immobilized -- Minimize non-specific binding and selection rounds -- Select aptamers that undergo structural transformation -- Minimize non-specific binding -- Single round of aptamer selection -- Candidate aptamers can be physically identified and collected by atomic force microscopy (AFM) and AFM tip	-- Only aptamers that have undergone structural transformation can be developed -- Introducing additional changes through docking sequences

III.Obtaining Short Aptamer Sequences Through Truncation and Site-directed Mutagenesis

The length of aptamers generated by the SELEX process is usually 60-100nt, containing a random region of 25-50nt and a fixed primer binding region at each end. The fixed primer binding sequence does not contribute to the binding properties of the aptamer. Therefore, when analyzing sequence data, the primer binding site is usually deleted, leaving only the random region for binding studies. The random sequence region of the aptamer can be further shortened by structural analysis of the developed aptamer. The binding properties of aptamers to targets are mainly contributed by more complex and information-rich structures, such as G-quadruplexes, three/five-way structures, and hairpin structures. In many cases, the truncated sequence does maintain the same or better binding affinity and target specificity than the parent aptamer sequence.

The aptamer in vitro screening service provided by TekBiotech can quickly and accurately screen out aptamers with high affinity and high specificity for customers, saving costs for customers' experimental projects. We provide a one-stop in vitro aptamer screening service by flexibly utilizing the natural evolution of ligand systems through the SELEX method (for example, the SELEX method can identify different cells or detect the molecular level differences between two types of cells).

Previous:Overview of Aptamer Synthesis Technology Next:Issues Related to Aptamer Library Screening