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Oligonucleotide Synthesis: The Future of Evolutionary Biology and Medicine

Opemipo Oduntan
22 min readOct 23, 2023

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The innovative prospect of genomics and its application to modern biology and medicine offers a new opportunity that has never been available to anyone else before. Throughout all of human existence, never before have we been able to have a greater impact on the life on Earth than during the 21st century. Never before have we been given the ability to influence entire ecosystems as we are able to now.

Before, scientists held the belief that the limit to our ability to turn the Earth into a sandbox was limited only by our creative ability to edit the genome of various organisms. The idea always was to take what was already existing, change it, and then reproduce/replicate. What is new? A new peak of biological creativity has been developed and is being commercialized — the ability to create things that have never been seen before in any living creature; the ability to synthesize DNA.

The following article will focus on giving an indepth explanation of one of the many process that makes up DNA synthesis — oligonucleotide synthesis.

Table of Contents

– Background
– Basic Principles of Oligonucleotide Synthesis
– Chemistry of Oligonucleotide Synthesis
– Solid-Phase Synthesis Techniques
– Purification of Oligonucleotides
– Applications of Synthesized Oligonucleotides
– Case Study
– Conclusion
– References

Background

To begin, let us take a second to understand a major piece of vocabulary that will be present throughout the remainder of this article — oligonucleotide. The word oligonucleotide is derived from a combination of the suffix oligo, which is commonly used in biology, and the term nucleotide, which we’ll discuss later in this article. The suffix oligo comes from the Greek word oligos (ολίγος). It means few or little. When combined with the word nucleotide, it means “few neucleotides” or “partial nucleotides”. This is key to our understanding of oligonucleotide synthesis because the main principle that makes up the chemical synthesis of oligonucleotides is this idea of the synthesis of parts of an entire gene sequence — oligonucleotides — and combining them to create nucleic acids. In summary, oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic accids with defined chemical structures (sequence).

Importance of Oligonucleotide Synthesis

Throughout this article, you will come to understand the efficiency of the process of oligonucleotide synthesis. You will also learn where some of the strengths and weaknesess of this technique for synthesising DNA. Because oligonucleotides are a lot easier to work with than entire nucleic acids, this method makes for a really compact way of creating new DNA. Here are some of the main reasons why oligonucleotide synthesis is important:

  1. Genetic Research Advancement: Oligonucleotide synthesis is crucial for deciphering genetic information, enabling researchers to manipulate and study DNA and RNA sequences. This capability is foundational for understanding the genetic basis of diseases, evolution, and the intricacies of cellular functions.
  2. Biomedical Applications and Therepeutics: Oligonucleotides serve as key components in various biomedical applications, including gene therapy and targeted drug delivery. Their programmable nature allows for the development of precision medicines, contributing to advancements in personalized treatments and therapeutic interventions.
  3. Biotechnology and Industrial Impact: Oligonucleotide synthesis plays a pivotal role in biotechnological industries, impacting sectors like pharmaceuticals, agriculture, and bioproduct development. It facilitates the production of diagnostic tools, genetically modified organisms, and novel biomaterials, driving innovation and progress in these fields.

Basic Principles of Oligonucleotide Synthesis

Before diving into the chemistry of oligonucleotide synthesis, let us first take a quick look at some of the basic principles that form the foundation of our understanding and application of oligonucleotide synthesis.

“If DNA is the building block of life, then the nucleotides are the building blocks of DNA.” — (Cabral, 2019)

Nucleotide Structure

Nucleotides are the building blocks of nucleic acids, which can include both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleotides are essential molecules for the storage and transmission of genetic information in living organisms. Generally, to fulfill this purpose, every nucleotide consists of up to three main components:

  1. Phosphate Group — made up of one phosphorus and four oxygens, two of which, in some cases, hold a negative charge.
  2. Sugars — made up of a collection of hydrogen and oxygen atoms which connect to the phosphate group through a methelene molecule (CH₂).
  3. Nitrogenous Base — made up of a collection of nitrogen which form aromatic rings that connect to hydrazine (NH₂) which can refer to a number of chemical compounds.

When diagrammed, they usually look something like Fig. 1.

Fig. 1 | Phosphate Group (orange); Sugars (blue); (A) Adenine — Nitrogenous Base (yellow)

The phosphate group and the sugars form the structural backbone of a DNA sequence and allow for any given portion of our DNA to kind of act like a lego piece. In short, they allow for DNA nucleotides to concatinate and form larger, more complex structures of DNA.

The nitrogenous base, indicated by it is yellow color, that was included in Fig. 1 was an organic compound known as Adenine (A). In total, there are four nitrogenous bases that are present in DNA:

  1. Cytosine (C)
  2. Guanine (G)
  3. Adenine (A)
  4. Thymine (T)

There is also one nitrogenous base that is present in RNA — Uracil (U).

Nucleotides link together through phosphodiester bonds, forming long chains known as polynucleotide strands. These strands serve as the foundation for the double helix structure of DNA and the single-stranded structure of RNA (Note: RNA includes the three same nitrogenous bases as DNA but with Uracil instead of Thymine).

The most important thing to remember is that the nitrogenous bases determine the DNA sequence. Any combination of these bases can be transcripted and translated by an RNA producing enzyme, which can unlock a plethora of complexity and diversity in all organisms.

For a more indepth explanation of DNA nucleotides in the context in genome sequencing, you can check out my previous article, Decoding Life’s Blueprint: An Introduction to Genomics.

Solid-Phase Synthesis

Now with an understanding of what nucleotides are, let us begin to take a look at what oligonucleotides are and some of the techniques for synthesising them.

Solid-phase synthesis is a technique used in organic and bioorganic chemistry for the synthesis of molecules, particularly oligonucleotides (DNA, RNA), peptides, and small organic compounds. This method involves attaching the starting material (e.g., a resin-bound molecule) to a solid support, such as a polymer or a resin bead. The key feature of solid-phase synthesis is that the reaction takes place while the molecule is attached to the solid support, simplifying purification steps and allowing for the synthesis of complex molecules.

“Solid phase synthesis generally involves the addition of one nucleotide residue at a time (e.g. 121a in the phosphotriester approach) to an immobilized protected nucleoside or oligonucleotide.” — Tetrahedron, 2002

To gain a better understanding, here is a brief overview of the general process of solid-phase synthesis:

  1. Attachment to Solid Support: The first building block (e.g., the first nucleotide in oligonucleotide synthesis or the first amino acid in peptide synthesis) is covalently attached to a solid support. This support is typically a resin bead with functional groups that facilitate the attachment.
  2. Chemical Reactions: Subsequent building blocks are added in a stepwise fashion. Each addition involves a chemical reaction, such as coupling, and these reactions occur while the growing molecule is still attached to the solid support.
  3. Washing and Purification: Unreacted reagents and byproducts are washed away after each step, simplifying the purification process. Since the growing molecule is attached to a solid support, it is easier to separate it from impurities.
  4. Deprotection: If protecting groups are used to prevent unwanted reactions during synthesis, they are selectiely removed to expose reactive functional groups for the next coupling step.
  5. Final Cleavage and Release: After the desired sequence is synthesized, the molecule is cleaved from the solid support. This final step typically involves treatment with appropriate reagents to release the fully synthesized molecule.

Solid-phase synthesis is widely used in the synthesis of oligonucleotides, peptides, and other biopolymers due to its efficiency, ease of purification, and ability to automate the synthesis process. Automated synthesizers are often employed for high-throughput and reproducible synthesis of these molecules in research and industrial settings.

Chemistry of Oligonucleotide Synthesis

Organic chemistry forms the foundation of oligonucleotide synthesis. Scientists use a process similar to the process of natural DNA synthesis done by enzymes during cell replication within our bodies and the bodies of other multicellular or unicellular organisms.

The process through which we replicate this subconscious effect of nature is by combining compounds together using synthesising technology. One of the prevailing methods that has been developed over the last few decades for synthesising nucleotides is the use of phosphoramidite chemistry for oligonucleotide synthesis. The following section of this article will be covering, in depth how that process works.

Phosphoramidite Chemistry

Phosphoramidite chemistry is a method used in oligonucleotide synthesis to create DNA and RNA sequences with defined chemical structure or sequence. The method was first described in 1981 and has since become the gold standard for DNA synthesis.

The phosphoramidite chemistry method for oligonucleotide synthesis is a four-step cycle that is repeated for each nucleotide that is added to the growing oligonucleotide chain. Here are the four main steps:

  1. Detritylation— remove the trityl protecting group from the 5'-hydroxyl group of the nucleotide that is attached to the solid support. In DNA synthesis, protection refers to the process of masking certain functional groups or nucleotide monomers to prevent them from interfering with the desired 3' to 5' sequential condensation of monomers to the growing oligonucleotide. Detritylation, a step that removes this protection, is necessary to expose the 5'-hydroxyl group for the next step.
  2. Coupling — add the next nucleotide to the growing oligonucleotide chain. This is done by coupling the 3'-hydroxyl group of the incoming nucleotide to the 5'-hydroxyl group of the nucleotide that is already attached to the solid support.
  3. Capping — cap any unreacted 5'-hydroxyl groups on the solid support to prevent them from reacting with the next incoming nucleotide. Any reactions outside of the intended reactions that occur at the same time as the main reaction, but produce different products, are generally referred to as side reactions.
  4. Oxidation — oxidize the phosphite group of the newly added nucleotide to a phosphate group. Oxidation is a chemical process that involves the loss of electrons or the gain of oxygen by a molecule, atom, or ion. This is done to stabilize the newly formed phosphodiester bond — occurence resulting from the formation of an ester bond. An ester bond is a covalent bond between a carbon atom that is double bonded to an oxygen atom and another oxygen atom that is attached to an alkyl or aryl group.

The following diagram asserts that step three of this process is optional.

Fig. 2 | | The dominating four-step, phosphoramidite chemistry widely applied in… | Download Scientific Diagram (researchgate.net)

The reason being the general lack of efficiency associated with capping. Capping is not always 100% efficient, and some support-bound oligonucleotides may not couple with the added nucleotide, leaving a free 5′ hydroxyl group. If not blocked, this reactive group can couple to the nucleotide added in the next cycle, producing an oligo with a deletion in its sequence.

However, it is important to take into consideration the fact that capping is a critical process in the synthesis cycle to reduce the accumulation of deletion mutations that are difficult to purify and could render the oligonucleotide ineffective for subsequent applications.

One of the things that can be noted about the process of phosphoramidite chemistry for oligonucleotide synthesis utilizes phosphoramidite building blocks instead of phosphates because they are more stable and easier to work with. Phosphoramidites are, in summary, modified nucleosides and they are a key component in solid-phase synthesis when synthesising oligonucleotide chains. They are the compound found inside nucleotides used in oligonucleotide synthesis. Phosphoramidites contain a phosphite instead of the typical phosphate in nucleotides, as well as four specific protecting groups — Dimethoxytrityl (DMT), 2-Cyanoethyl (CE), Benzoyl (Bz), Isobutyryl (iBu). These groups provide stable protection but can be easily removed when necessary.

Phosphates, on the other hand, are more reactive and less stable than phosphoramidites, which can lead to unwanted side reactions during oligonucleotide synthesis. Additionally, phosphates require harsher conditions for deprotection, which can damage the oligonucleotide chain.

Protecting Groups

Likely, up until this point in this article, you’ve seen the use use of the word protection or protecting group. Luckily, protecting groups are one of the few concepts in biology that means what it looks like.

Protecting groups are used to protect reactive functional groups during the synthesis of oligonucleotide. Because of the reactivity of the chemical bases used in the chemical synthesis of oligonucleotides, side reactions tend to be a pretty common occurence. When protecting groups are introduced, they can prevent side reactions and kind of act like a seal on a can of soda, preventing the carbon dioxide from exiting the drink.

There are many different types of protecting groups used in oligonucleotide chemistry and the choice of protecting group depends on the specific functional group that needs to be protected and the conditions of the synthesis. Below are several protecting groups utilized in oligonucleotide synthesis, encompassing the four mentioned earlier. Each comes with a short description explaining its purpose.:

  1. Dimethoxytrityl (DMT) — This group is used to protect the 5'-hydroxyl group of the nucleotide that is attached to the solid support. It is removed during detritylation.
  2. 2-Cyanoethyl (CE) — This group is used to protect the 3'-hydroxyl group of the incoming nucleotide during coupling. It is removed during oxidation.
  3. Benzoyl (Bz) — This group is used to protect the exocyclic amino group of cytosine during coupling. It is removed during detritylation.
  4. Isobutyryl (iBu) — Has the same function as Bz.
  5. Tert-butyldimethylsilyl (TBS) — This group is used to protect the 2'-hydroxyl group of ribonucleotides. It is removed from the oligonucleotide after the synthesis and deprotection of the oligoribonucleotide — oligonucleotide consisting of ribonucleotides.

An important note about protecting groups is their removal process, which has been alluded to numerous times throughout this section of this article. The removal of protecting groups is typically achieved by exposing the oligonucleotide to a specific reagent or set of conditions that selectively removes the protecting group without damaging the rest of the molecule.

An example of a common removal process in oligonucleotide synthesis is the removal process of DMT. The removal process for DMT protecting groups in oligonucleotide synthesis involves exposing the oligonucleotide to a specific reagent or set of conditions that selectively removes the DMT protecting group without damaging the rest of the molecule. It is typically achieved by exposing the oligonucleotide to a solution of ammonium hydroxide or ammonium hydroxide/methylamine (AMA) for a specific amount of time.

Coupling Reactions

One of the most important parts of oligonucleotide synthesis is coupling. It is the process that adds nucleotides to the growing olignucleotide chain, creating the actual tangible smaller DNA pieces that we look to as the end goal of oligonucleotide synthesis.

The diagram below shows the coupling of bases in order to form a larger chain of nucleic acids.

Fig. 3 | Nucléotide : définition, rôle, structure et plus (aquaportail.com)

This process is synthetically replicated through the process of coupling in oligonucleotide synthesis, enabling the creation of short chains of DNA.

Coupling usually happens right after Detritylation where the DMT protecting group is removed. When the DMT protecting group is removed during Detritylation, the 5'-hydroxyl group is exposed and the addition of the synthetic nucleotide can take place.

Coupling agents are used to activate the phosphoramidite building block and facilitate the coupling reaction. They are typically added to the reaction mixture along with the phosphoramidite building block and the activator. Some common coupling agents include 1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthiotetrazole, 4,5-dicyanoimidazole, and others. The choice of the coupling agent entirely depends on the specific conditions of the synthesis.

After the completion of a coupling reaction, a small percentage of the suppor-bound 5'-OH groups remains unreacted and needs to be permanently blocked from further chain elongation to prevent the formation of oligonucleootides with an internal base deletion commonly referred to as (n-1) shortmers. The unreacted 5'-hydroxy groups are, to a large extent, acetylated by the capping mixture which is to say that an acetyl group is introduced to the compound.

Another important note about coupling reactions in oligonucleotide synthesis is that they are highly chemoselective, meaning that they will only react with a desired set of molecules and will not react with other functional groups in a given molecule.

Before diving into the next section of this article, let us take a second to acknowledge what we’ve covered throughout this article thus far. So far, this article has been focused on oligonucleotide synthesis in the context of only half of the entire process, phosphoramidite chemistry. We’ve focused on how the chemistry of this process works, as well as some of the chemical compounds involved in the process.

This next section will focus on a sort of macro-perspective on what we’ve just covered, by looking into solid-phase synthesis.

Solid-Phase Synthesis Techniques

One clear destinction needs to be made before diving deeper into this topic. Please note that solid-phase synthesis and phosphoramidite chemistry are two important aspects of oligonucleotide synthesis, and that they are not different from each other. In fact, solid-phase synthesis is implemented using phosphoramidite chemistry, which involves the use of phosphoramidite building blocks to add nucleotides to the growing oligonucleotide chain. Solid-phase synthesis can be identified as an overarching term to describe the main chemical processes that govern oligonucleotide synthesis.

Support Materials

Solid-phase synthesis is a widely used method in oligonucleotide synthesis, and it involves the use of a solid support, typically controlled pore glass (CPG) or polystyrene, to which the oligonucleotide is bound during synthesis.

CPG is the most commonly used solid support material in oligonucleotide synthesis. It is a high-silica glass that contains pores with a specific size distribution. CPG is rigid, non-swelling, and compatible with any solvent and it is also relatively stable under high pressure or temperature scenarios. It is also stable to corrosive solvents, making it ideal for oligonucleotide synthesis.

Solid support materials are used to immobilize the growing oligonucleotide chain and facilitate purification. Here’s the purpose broken down into its basic components:

  1. Solid support materials: These are typically resin beads or similar structures with functional groups. They provide a physical foundation for the synthesis reactions.
  2. Immobilize the growing oligonucleotide chain: As the oligonucleotide chain (DNA or RNA sequence) is synthesized, each nucleotide is added to the growing chain while attached to the solid support. This attachment allows for ease of handling and simplifies subsequent purification steps.
  3. Facilitate purification: Because the oligonucleotide is attached to a solid support, it becomes simpler to separate the desired product from impurities. After each addition of a nucleotide, unreacted reagents, and byproducts can be washed away efficiently, streamlining the purification process.

The following diagram depicts the process of synthesizing DNA through the solid phase approach. The solid support materail is indicated by the white circular base.

Fig.3 | Slide depicting the process of synthesising DNA through the solid phase… | Download Scientific Diagram (researchgate.net)

As new bases are added on to the growing nucleotide chains, protecting agents are removed and they link to the T-base in the diagram via the phosphate group.

For perspective on the size of solid supports, they are typically 50–200 μm in diameter. Smaller particles will not permit rapid flow of solvents and regents through the synthesis column and can block filters.

Solid support materials used in oligonucleotide synthesis are typically prepared by functionalizing the surface of the support with a linker molecule that can covalently bind to the first nucleotide of the oligonucleotide chain.

Automated Synthesis vs. Manual Synthesis

Generally, there are two main approaches to solid-phase synthesis. The first is automated synthesis, and the second is manual synthesis. The process I’ve described thus far uses automated synthesis. Here’s a quick breakdown of a comparison of the two:

Automated Synthesis:

  • Faster, more efficient, and less prone to errors than manual synthesis.
  • Achieves the same goals as manual synthesis at a faster, more accurate rate.
  • More expensive than manual synthesis, but more cost-effective for large-scale synthesis.
  • Amenable to automation on computer-controlled solid-phase synthesizers.
  • Widely used in peptide synthesis, oligonucleotide synthesis, oligosaccharide synthesis, and combinatorial chemistry.

Manual Synthesis:

  • More time-consuming and labor-intensive than automated synthesis.
  • Requires more human intervention and is more prone to human error.
  • Less expensive than automated synthesis, but less cost-effective for large-scale synthesis.
  • Performed by anchoring the first nucleotide to a resin and coupling each successive nucleotide to the 3'-terminus.
  • Solid-phase synthesis of peptides is performed by anchoring the first amino acid to a resin and coupling each successive amino acid to the N-terminus.

In summary, automated synthesis is faster, more efficient, and less prone to errors than manual synthesis, but it is more expensive. Manual synthesis is more time-consuming and labor-intensive than automated synthesis, but it is less expensive. Automated synthesis is amenable to automation on computer-controlled solid-phase synthesizers, while manual synthesis requires more human intervention and is more prone to human error.

As a result of some of the points that were previously brought up in the comparison between automated and manual synthesis, automated synthesis is the most common approach to synthesizing oligonucleotides.

Purification of Oligonucleotide Synthesis

Another important part of oligonucleotide synthesis is purification. Oligonucleotide synthesis requires purification to remove impurities such as truncated oligonucleotides and sequences containing chemically-modified bases.

This next section will cover some of the most common methods for purifying oligonucleotide chains and some of the peculiarities of each of them.

High-Performance Liquid Chromatography (HPLC)

High-Performance Liquid Chromatography (HPLC) is a highly efficient method for both the analysis and purification of synthetic oligonucleotides. Operating based on principles of hydrophobicity or charge, HPLC separates oligonucleotides in the 5 to 50 bases length range, effectively eliminating failure sequences. This method is particularly favored for large-scale oligonucleotide synthesis required in therapeutic or diagnostic applications, with the choice of HPLC or ultra-performance liquid chromatography (UPLC) depending on the specific oligonucleotide being synthesized.

Polyacrylamide Gel Electrophoresis (PAGE)

Polyacrylamide Gel Electrophoresis (PAGE) is a routinely employed method for synthetic oligonucleotide analysis and purification. It efficiently separates full-length products from shorter species, notably enhancing purity even though there is a reduction in the overall mass of the final oligo product.

PAGE stands out as the preferred technique for achieving base-level resolution across short and long oligonucleotides, routinely achieving purity levels exceeding 90% of the full-length product. It is especially recommended for longer oligos, around 100 bases and beyond, where other purification methods encounter challenges.

Capillary Electrophoresis

Capillary Electrophoresis is a powerful tool for the analysis and purification of oligonucleotides, leveraging the separation of molecules based on electrical charge and hydrodynamic properties. In comparison to standard HPLC on a reversed-phase C18 column, capillary electrophoresis using a polyvinylalcohol (PVA)-coated capillary demonstrates enhanced precision.

The effectiveness of purification shines through in capillary electrophoresis using a PVA-coated capillary, making clear distinctions in the quality of purified and unpurified oligoribonucleotides. This method is particularly valuable for the precise purification of oligonucleotides and offers advantages in specific scenarios compared to traditional HPLC methods.

Another important important aspect of the purification process is characterization. Characterization in oligonucleotide synthesis refers to the process of analyzing and identifying the properties of synthetic oligonucleotides to ensure their quality and purity. Some parts of this topic were previously covered in this article, as HPLC and PAGE are efficient methods for analysis or/and purification of oligonucleotides, however, this article will not be going in depth into how this process works and some of the existing methods for carrying it out.

Applications of Synthesized Oligonucleotides

Now with an understanding of how oligonucleotide synthesis works, why is this important, and where is this technology being applied? This section will take a deep dive into two of those applications.

Polymerase Chain Reaction (PCR)

Oligonucleotides are used as primers in polymerase chain reaction (PCR) to amplify specific regions of DNA. A PCR primer is a short, single-stranded DNA molecule, usually around 20 nucleotides in length, that is used in PCR to amplify a specific region of DNA.

Two primers are used in each PCR reaction, and they are designed so that they flank the target region (region that should be copied). The primers bind to the template by complementary base pairing. PCR primers are also designed to be complementatry to the 5' end of the sequence targeted for amplification.

The goal of PCR is to make enough of the target DNA region that can be analyzed or used in some other way, such as sequencing, visualization by gel electrophoresis, or cloning into a plasmid for further experiments.

Through oligonucleotide synthesis, PCR primers have become more accessible, which has revolutionized the study of gene expression and disease processes.

Therapeutic Applications

Oligonucleotide therapeutics represent a promising category of drugs that utilize short DNA and/or RNA molecules to influence the activity of specific RNA targets, offering a new approach for treating various diseases. These therapeutic molecules come in different types, such as antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs) that participate in RNA interference (RNAi), small activating RNAs (saRNAs), aptamers, and guide RNAs (gRNAs) employed in CRISPR gene editing technology. Essentially, these oligonucleotide therapeutics work by either blocking or enhancing the expression of specific RNA sequences to address various medical conditions.

Moreover, oligonucleotide conjugates, which are combinations of oligonucleotides with other molecules, find applications beyond therapeutics. They are widely used as probes in quantitative polymerase chain reaction (qPCR), for sequencing and hybridization of nucleic acids, and in microscopy. In these contexts, oligonucleotide conjugates serve as versatile tools for understanding and manipulating genetic material, playing crucial roles in research, diagnostics, and imaging technologies.

Case Study

Oligonucleotide synthesis has emerged as a transformative force in molecular and clinical research. It has fast tracked our understanding of genetic corelations and is pioneering the development of targeted therapeutics. This case study explores the tangible real-world impacts of oligonucleotide synthesis, focusing on groundbreaking applications in the revolutionary field of Antisense Technology and the treatment of Spinal Muscular Atrophy (SMA).

Antisense Technology

Background:
Antisense Technology represents a frontier in oligonucleotide synthesis, offering a unique approach to drug discovery. Antisense oligonucleotides (ASOs) are short, synthetic, chemically modified chains of nucleotides that have the potential to target any gene product of interest. ASOs are single-stranded RNA molecules that offer new opportunities for therapeutic intervention because they act inside the cell to influence protein production.

ASOs are designed to bind to a specific RNA sequence and modulate gene expression by inhibiting or promoting RNA splicing, blocking translation, or inducing RNA degradation. The ASO gapmer design is a common approach to increase the stability and specificity of ASOs.

Fig.4 | Antisense oligonucleotide (ASO) ‘gapmer’ design. Example of a 20-base… | Download Scientific Diagram (researchgate.net)

The diagram shown above displays the the conventional gapmer desin of ASOs, where there is a central unmodified block of DNA nucleotides that is surrounded by chains of modified 2' molecules.

Impact:
ASOs, designed to complement specific RNA sequences, hold promise in treating a diverse range of diseases such as orphan genetic alterations and cancer. They’ve also been used to treat deadly viral infections. Chemical modifications have further enhanced the efficacy of ASOs, illustrating how oligonucleotide synthesis is revolutionizing current drug discovery and treatment methods.

Spinal Muscular Atrophy (SMA)

Background:
SMA is a devastating genetic disorder affecting motor neurons, causing muscle weakness, and atrophy. It is caused by a deficiency of a motor neuron protein called SMN2, for “survival of motor neuron.” This protein, as its name implies, is necessary for normal motor neuron function.

Fig. 5| Spinal muscular atrophy (a2zmedicalnote.in)

The disease, as shown in the diagram above, can damage and kill specialized nerve cells in the brain and spinal cord (motor neurons). Motor neurons are essential for control of the arms, legs, face, chest, throat, and tongue, which enable things like walking, speaking, swallowing, and breathing.

Now, the way our cells normally make proteins involves a process called splicing. It is like putting together a puzzle. However, in SMA, this splicing process does not work quite right, and not enough of the important protein is made.

A drug known as Nusinersen steps in like a helpful guide. It tells the cell how to do the splicing correctly. It is like giving instructions to fix the puzzle pieces so that more of the needed protein is produced. In more technical terms, Nusinersen inhibits some factors that were causing the splicing to go wrong, making sure the cells create the right protein that muscles need.

In 2016, Nusinersen was approved by the FDA, enabling clinical use in North America. This marked a pivotal moment in genetic disorder therapeutics.

Impact:
Nusinersen’s approval and subsequent use have showcased the rapid and effective translation of oligonucleotide synthesis into tangible treatments. The drug has demonstrated substantial improvements in motor function and increased survival rates among infants with SMA, underscoring the potential of oligonucleotide synthesis to address previously incurable genetic disorders in a remarkably short timeframe.

Case Study Conclusion

Oligonucleotide synthesis has propelled the field of molecular medicine into uncharted territories. The SMA case study exemplifies how oligonucleotide synthesis can swiftly translate into life-changing treatments. Antisense Technology opens avenues for treating targets that were not previously treatable (ex. Viral Infections). Innovative delivery methods promise to make oligonucleotide-based therapeutics a cornerstone of precision medicine.

In conclusion, the impact of oligonucleotide synthesis in the real world is evident in the lives changed and diseases targeted. As technological advancements continue, the horizon of possibilities in medicine expands, offering hope to patients and practitioners alike.

Conclusion

With the science of gene editing becoming more and more of a field that is being pumped full of money, people’s hopes, and dreams, it is vital for us to have a basic understanding of the underlying process that governs this industry. The science of oligonucleotide synthesis, though daunting, is currently an industry standard that is pioneering developments and innovations in this space, making way for a new level of creativity and hope that humanity has never had before. I hope this article has been helpful in some way to aid your understanding of the topic of oligonucleotide synthesis, and I hope you, like myself, will come to appreciate the intricacies of this process which turns the tables on nature itself. Through science, we have been gifted the true ability to create what was not, before. The true creative nature of the world is being unfolded before our eyes, and we humans get to be a part of it. Truly a privilege worth marvelling at.

References

Genetic Engineering
Genetics
Science
Medicine
Healthcare

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Opemipo Oduntan

Written by Opemipo Oduntan

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