In vitro Selection of Aptamers

In vitro selection, also referred to as "test-tube evolution"

In vitro selection, or also referred to as SELEX (systematic evolution of ligands by exponential enrichment) is a test tube-based experiment that mimics the evolutionary process found in Nature. A population, organism or even molecule that can adapt to their environmental pressure will thrive while those that fail to derive key traits to survive will wither away. The purpose of an in vitro selection experiment is to identify DNA or RNA molecules that have unique molecular functions. Every in vitro selection begins with a large, randomized library of 10^12 - 10^16 DNA (or RNA) molecules that are subjected to successive rounds of screening. Nucleic acid sequences that express the desired function are enriched while inactive sequences are removed. Through this test tube evolution experiment, synthetic molecules that function as molecular receptors known as aptamers.

The process

In vitro selection is an iterative process where every cycle aims to enrich sequences with a desired function. Each cycle can be broken down into three separate components: Selection (negative and positive), Enrichment, and Regeneration.

Negative Selection: This is a crucial yet often overlooked step of in vitro selection. To obtain functional sequences of high specificity, DNA species that can cross-react or have the capabilities to bind multiple targets are removed. This is a fine-tuning that allows the user to determine what kind of binding properties is desired.

Positive Selection: This is where the magic happens. The DNA sequences within the library must be able to carry out the function of interest or it will be eliminated. For example, if you are identifying aptamers, then the addition of your target ligand will force the DNA sequences to compete to bind to the target. Sequences that fail to do this will be discarded while sequences that were successful are isolated and prepared for enrichment.

Enrichment: Sequences that survived the selection step will be allowed to multiply. This is done using polymerase chain reaction (PCR). However, something special also happens during PCR. As the DNA is being synthesized by the polymerase, minor mutations are introduced and may provide the sequence with a fantastic advantage in the upcoming cycle!

Regeneration: The result from PCR will produce a double stranded library that needs to be separated to regenerate the single stranded DNA pool.

The evolution

Since we are mimicking the evolutionary process, the condition for selection needs to be more stringent in the later cycles. After the first few cycles, the DNA library should contain many different sequences that can carry out the function of interest. We often categorize these sequences into classes, which are grouped by sequence similarity. Although each class now possess the desired function, their effectiveness will vary to a large degree. By increasing the selection condition, you will force the remaining classes to compete with one another. The end result will yield a functional DNA sequence that is highly efficient at performing the task of interest.

What are aptamers

Aptamers are short single-stranded DNA or RNA molecules capable of binding a wide range of molecules. They have been shown to possess immense potential in replacing antibodies for the development of new biosensors, diagnostics, and therapeutics. The discovery of aptamers is a rewarding process as its identification will lead to cost-effective production and scalability that can easily service the needs of any industry.

Advantages of aptamers

Thermal Stability - DNA aptamers are highly resistant to thermal degradation. They able to revert back to their native structure without loss of activity. This extends their shelf-life, making storage and shipping at room temperature a viable option.

Batch Consistency - Aptamers are chemically synthesized in controlled environments. This results in little to no batch-to-batch variation, no contamination from biological sources, and rapid scale-up capabilities.

In vitro Screening - Unlike antibodies, aptamers are isolated in vitro. This significantly reduces the economic burden as the use of animals and their associated costs for maintenance and ethics requirements. 

Production Scalability - Cost of aptamer production is predictable and cost-effective. This features makes them an attractive option for use in assay and therapeutic development.

Immunogenicity - Unmodified aptamers do not elicit a negative immune response and are generally safe for handling. 

Handling and Processing - Aptamers are highly soluble in aqueous solutions and will not aggregate and form insoluble precipitates as seen with proteins. 

Range of Selection Targets - Antibodies are limited by the choice of target as it requires a strong immune response for production. In contrast, aptamers have been developed for a broad range of targets from small molecules to whole cells.

Chemical Modification - Aptamers are highly amenable to chemical modifications allow them to change their physical and chemical attributes. This also extends their utility for conjugation to various organic and inorganic molecules.