Oligonucleotide synthesis is the process of producing short, single-stranded DNA molecules with precise sequences. The ability to synthesize custom DNA oligos with specific sequences has expanded the range of applications for life sciences from basic research to therapies.

Custom oligos are frequently used as probes to detect specific DNA sequences or as primers for PCR (polymerase chain reaction) to amplify specific regions of DNA. Incorporating modified bases into oligonucleotides has led to new advancements in gene therapy and other applications.

About Oligonucleotides

Synthetic (modified) nucleotides used in oligo synthesis include dATP, dCTP, dGTP and dTTP, which are the deoxyribonucleoside triphosphates for the bases adenine, cytosine, guanine and thymine, respectively. dUTP is a modified nucleotide used in oligonucleotide synthesis to replace dTTP and create RNA oligos that contain uridine instead of thymine. This allows the creation of RNA probes and the inhibition of specific genes through antisense therapy and RNA interference. These nucleotides are used in PCR and DNA sequencing applications. Other modified nucleotides are used for stabilizing oligonucleotides and reducing degradation due to nucleases.

Oligonucleotides vary in length from 5 to 120 or more nucleotides depending on applications. For example, PCR oligonucleotide primers vary from 18 to 25 nucleotides in length.

Long oligonucleotides are challenging to make because the process is prone to errors, such as base misincorporation and truncation, which become more frequent as the length of the oligonucleotide increases. Additionally, the synthesis yield decreases as the length increases, making it harder to obtain enough of the desired product.

Phosphoramidite Method of Oligonucleotide Synthesis

The phosphoramidite method is widely used due to its sequence accuracy when producing high-quality single-stranded DNA or RNA oligonucleotides. It is widely used in molecular biology, genetic engineering and drug development applications. Highly efficient, this method is accurate and cost-effective and can produce high-quality oligonucleotides with precise sequences. Additionally, this method allows for greater flexibility in modifying oligonucleotides.

Step 1 - Oligonucleotide Detritylation

Phosphoramidites are protected nucleosides that contain a phosphate group and an amine group. These are assembled on a controlled pore glass (CPG) support via a continuous flow process (polystyrene is another support option). The first step in the synthesis is to deprotect the 5′-hydroxyl group (5’ DMT) of the CPG support.

Oligonucleotide detritylation refers to the process of removing the protective groups (also known as "protectors" or "capping groups") from the termini (ends) of the synthesized oligonucleotide. These protective groups are added during synthesis to prevent the oligonucleotide from being degraded or modified by unwanted reactions.

Detritylation typically involves using chemical reagents such as acid or base solutions to cleave the protective groups from the oligonucleotide. For example, if the protective group is a dimethoxytrityl (DMT) group, it can be removed by treatment with a strong acid solution, such as trifluoroacetic acid (TFA). The acid solution cleaves the DMT group from the oligonucleotide, leaving the unprotected ends available for further reactions or applications.

Detritylation is an important step in oligosynthesis because it allows the oligonucleotide to be used in downstream applications, such as PCR, sequencing, or hybridization assays, without interference from the protective groups. Additionally, detritylation may also be used to purify the oligonucleotide by removing any impurities or by-products that may have been generated during the synthesis process.

Step 2 - Oligonucleotide Coupling

Oligonucleotide coupling joins two or more individual oligonucleotides together using phosphoramidites to form a covalent bond between the 5' phosphate group of one oligonucleotide and the 3' hydroxyl group of another. This bond formation is typically carried out in the presence of a catalyst, such as a metal ion, and under specific temperature and pH conditions. The process is generally automated and carried out in a stepwise manner, with each successive coupling reaction building on the previous one to create a longer oligonucleotide.

Step 3 - Oligonucleotide Capping

Oligonucleotide capping methods are techniques used to add a modified base or chemical group to the ends of the oligonucleotide. These modifications can serve various purposes, such as increasing the stability or specificity or preventing oligonucleotides with deletion mutations spurred by unreacted 5’ hydroxyl groups.

Step 4 - Oligonucleotide Oxidation

After the capping phase, the oxidizing agent is added to the oligonucleotide synthesis reaction. The reaction is typically monitored by gel or capillary electrophoresis, mass spectrometry or chromatography to ensure that the desired oxidation level has been achieved.

This process involves using an oxidizing agent, such as iodine and water, which oxidizes phosphite into phosphate and leads to a phosphodiester linkage between the oligonucleotide and the added nucleotide. The oligonucleotide is then cleaved from the CPG support using a concentrated ammonia solution.

The phosphoramidite method allows the repetitive addition of nucleotides to the growing oligonucleotide chain, with each nucleotide being added one at a time in a stepwise manner. This process results in synthesizing a single-stranded DNA or RNA oligonucleotide with the desired sequence.

Oligonucleotide Chemical Modifications

Chemical modifications to the backbone or base structure can enhance the properties of oligonucleotides and expand their range of applications. The ability to control the sequence and modifications of oligonucleotides provides researchers with a versatile platform for designing probes, primers and therapeutic agents that can be tailored to specific needs.

One common modification is the introduction of non-nucleotide linkages, such as phosphorothioate or methylphosphonate. These modifications increase the resistance of the oligonucleotide to nuclease degradation and improve its binding affinity to target sequences, making it a useful tool for in vitro and in vivo studies.

Another type of modification is the introduction of modified nucleotides, such as 2'-O-methyl or 5-methylcytosine, into the base structure of the oligonucleotide. These modifications can increase the specificity of the oligonucleotide and reduce the risk of non-specific binding to off-target sequences, making them useful for targeted therapies.

In addition, covalent modifications, such as conjugation with a fluorescent or radioactive label, can be used to track the oligonucleotide in vivo and monitor its activity. These modifications also allow the designing of imaging and therapeutic agents for disease diagnosis and treatment.

Future Applications of Oligonucleotide Synthesis

The ability to synthesize small, synthetic pieces of DNA with defined sequences has opened new possibilities for studying gene function and creating new therapeutic approaches. From personalized medicine and gene editing to gene regulation, the potential for oligonucleotide synthesis will continue to expand, resulting in new and useful applications.

Innovation and future applications for oligonucleotides may include a greater focus on personalized medicine if combined with CRISPR-Cas9 to introduce changes in target specific genes or RNA transcripts involved in various disease processes. By designing oligonucleotides that can target specific mutations or genomic regions, it may be possible to develop therapies that are tailored to each patient’s individual needs.

DNA oligonucleotides also have the potential to play a key role in the continuing development of new tools. They will continue to be used to research gene function and regulation. By synthesizing oligonucleotides that mimic specific regulatory elements, such as promoter or enhancer regions, researchers can gain a deeper understanding of how genes are regulated and how they contribute to disease.

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