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Oligonucleotides, short chains of nucleic acids, are essential tools in modern molecular biology and biotechnology. From DNA sequencing and PCR to gene therapy and drug discovery, these molecules play a pivotal role. These short DNA or RNA sequences are pivotal in biotechnology, diagnostics, and pharmaceuticals, forming the backbone of numerous scientific advancements. From genetic research to personalized medicine, oligonucleotides have become indispensable tools, driving innovation and enabling groundbreaking discoveries. This article delves into the fascinating process of oligonucleotide synthesis.
What are Oligonucleotides?
Oligonucleotides are synthetically produced, short sequences of DNA or RNA. They are typically 10-100 nucleotides long and can be customized to match any desired sequence. This versatility makes them invaluable for various applications.
One of the most important tools in modern molecular biology is the oligonucleotide (oligos for short). Without oligos, today’s biotechnology, diagnostic, and pharmaceutical industries simply couldn’t exist. The word oligonucleotide is derived from the Greek word olígoi, meaning “few” or “small,” and nucleotide, which are the building blocks of nucleic acids, such as those in DNA. Oligos are chemically synthesized and are used in many different areas of scientific research and development.
Oligonucleotides are short single- or double-stranded segments of nucleic acids that are linked together to form single-chain biological polymers. The individual nucleotide bases can be thought of as equivalent to the monomers that make up classical chemical polymers. A nucleotide consists of three parts: a nitrogen-containing base, a sugar molecule with five carbons (these two parts form the nucleoside), and one or more phosphate groups. Alternatively, nucleotides can consist of non-natural or non-canonical bases. Common examples include locked nucleic acids (LNA), morpholino, and structurally modified bases or backbones.
The complex linking of nucleotides forms the biologically critical RNA and DNA biomolecules. In RNA, which is a single-strand biopolymer, the sugar is ribose. In DNA, the double-strand molecule contains the sugar deoxyribose. It is the short sequences of deoxyribonucleic acids and ribonucleic acids that are the building blocks of important oligonucleotides. Specific linkages of these nucleotides create the target biomolecules used in biological, medical, forensic, and clinical applications.
How are Oligonucleotides Used?
Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids can be manufactured as single-stranded molecules with any user-specified sequence, and so are vital for artificial gene synthesis, polymerase chain reaction (PCR), DNA sequencing, molecular cloning and as molecular probes. In nature, oligonucleotides are usually found as small RNA molecules that function in the regulation of gene expression (e.g. microRNA), or are degradation intermediates derived from the breakdown of larger nucleic acid molecules.
Oligonucleotides are powering the next generation of synthetic biology. Synthetic biology, the design and construction of biological devices and systems, promises to enhance biological life to produce outcomes we dictate. This field is used for designing and constructing new biological parts, devices, and systems as well as for redesigning existing natural biological systems for useful purposes. Synthetic biology represents an intersection of biology and engineering that focuses on the modification or creation of novel biological systems. Therefore, it combines the knowledge of genomics and the chemical synthesis of DNA for the rapid production of cataloged DNA sequences.
What is Oligonucleotide Synthesis?
Oligonucleotide synthesis is the chemical process of creating short sequences of nucleotides—the building blocks of DNA and RNA. These sequences can be meticulously designed to match specific genetic codes, making them essential for various applications, including gene editing, diagnostic assays, and therapeutic development.
The Process of Oligonucleotide Synthesis
The synthesis of oligonucleotides involves several precise steps:
- Monomer Preparation: Nucleoside phosphoramidites, the basic units of oligonucleotides, are prepared and protected to prevent unwanted reactions during synthesis.
- Chain Assembly: Using automated synthesizers, the oligonucleotide chain is assembled one nucleotide at a time through a series of chemical reactions: deprotection, coupling, capping, and oxidation.
- Deprotection and Purification: Once the desired sequence is assembled, protecting groups are removed, and the oligonucleotide is purified to eliminate incomplete or erroneous sequences.
The most common method for synthesizing oligonucleotides is solid-phase phosphoramidite chemistry. This process involves the stepwise addition of nucleotides to a growing chain attached to a solid support. Here’s a simplified overview of the steps:
- Solid Support: The first nucleotide is attached to a solid support, often a resin bead.
- Detritylation: The protecting group on the 5′ end of the attached nucleotide is removed, exposing the hydroxyl group for the next reaction.
- Coupling: A new activated nucleotide (phosphoramidite) is added, forming a phosphodiester bond with the growing chain.
- Capping: Unreacted hydroxyl groups are capped to prevent side reactions.
- Oxidation: The unstable phosphite triester is converted to a stable phosphate triester.
These steps are repeated for each nucleotide in the desired sequence, building the oligonucleotide one base at a time.
How Are Oligonucleotides Synthesized? A Focus on DNA Oligos
Oligonucleotide synthesis is a precise chemical process that involves linking nucleotides to create specific sequences. The most commonly used method is solid-phase synthesis, which typically employs a packed-bed column for the production of oligonucleotides, though batch or slurry reactions are sometimes used. The process consists of several cyclical steps: first, a protecting group (4,4’-dimethoxytrityl) on the solid-phase nucleotide is removed to expose the hydroxyl group. Next, an activated phosphoramidite monomer is coupled to this free hydroxyl group, forming a dinucleoside linked by a phosphite triester. This triester is then oxidized to stabilize the backbone, and any unreacted nucleotides are capped to prevent side reactions.
To enhance product yield and quality, controlled pore glass (CPG) is commonly utilized, as it offers a significant increase in surface area for the synthesis. The cyclic nature of the synthesis process necessitates physical separation of columns, with chemical waste being flushed away to reduce cross-contamination. Automation allows for the synthesis of up to 1,536 oligos simultaneously. Additionally, microfluidic technologies provide a cost-effective method for high-throughput oligo synthesis, enabling the production of tens of thousands of oligos per chip. Innovations include microreactor chips that integrate fixed silica beads to greatly enhance surface area and yield, coupled with inkjet printers to precisely deliver chemical reagents.
Modern synthesis techniques, exemplified by companies like Twist Bioscience, have revolutionized the field by enabling the simultaneous production of approximately 1 million unique oligonucleotides. These advanced methods use silicon surfaces for synthesis, allowing for unparalleled scale and customization. The combination of these technological advancements ensures that oligonucleotides can be produced efficiently, at high yields, and tailored for specific applications, supporting both academic research and commercial needs.
Research, Diagnostic, Forensic and Therapeutic Applications
Oligonucleotide Primers and Probes
Synthetic oligonucleotides, typically up to 30 nucleotides in length, are crucial tools in molecular biology, serving as primers and probes. Primers are essential for amplifying target DNA sequences through techniques like polymerase chain reaction (PCR). In PCR, short oligos complementary to target DNA sequences initiate the copying process, enabling the exponential amplification of specific DNA segments for analysis. This technology underpins various applications, including DNA sequencing, gene expression analysis, cloning, and molecular diagnostics.
Oligonucleotide probes are designed to bind to specific DNA or RNA sequences, confirming their presence in samples. They are used in blotting procedures, microarrays, and molecular diagnostics to detect specific genetic markers or pathogens. Probes play a vital role in identifying changes in gene expression, screening for genetic diseases, and diagnosing infections. Both primers and probes leverage the precision and specificity of synthetic oligonucleotides to facilitate accurate and targeted molecular biology applications.
Recent advancements have improved oligonucleotide synthesis by enhancing coupling efficiency, refining purification techniques, and increasing automation. These improvements have made the synthesis process more efficient and cost-effective, expanding the applications of oligonucleotides. Long oligonucleotides, now producible with current synthesis methods, are used in advanced applications such as drug development, DNA-based nanostructures, and digital data archiving. These advancements continue to drive the progress of molecular biology and biotechnology, enabling more sophisticated and diverse applications.
Oligonucleotide Primers and Probes
Synthetic oligonucleotides, often up to 30 nucleotides in length, are essential tools in a variety of molecular biology applications, primarily as primers and probes. Primers are used to initiate enzymatic reactions to create millions to billions of copies of target DNA or RNA sequences. Prominent examples of these processes include the polymerase chain reaction (PCR) and Sanger sequencing. PCR involves synthesizing short, single-stranded oligos that are complementary to the edges of the target DNA sequence. By adjusting the temperature, the double-stranded DNA helix is separated, allowing the oligonucleotides to bind to their complementary sequences, enabling DNA polymerase to start the replication process. This technique is fundamental in DNA sequencing, gene expression analysis, cloning, and molecular diagnostics.
PCR can be likened to photocopying a page of a book, where the enzyme DNA polymerase acts as the copier, making numerous copies of the target DNA. Through specific temperature cycles, the target DNA strand is repeatedly amplified, resulting in billions of identical copies. This technology is indispensable in diagnosing unknown pathogens in biological samples and conducting genetic fingerprinting in forensic science. Modern DNA synthesis methods can produce oligos longer than 200 bases, which are being utilized to expedite drug development, create DNA-based nanostructures for targeted drug delivery, and archive digital data for thousands of years.
Oligonucleotide probes are designed to bind to specific DNA or RNA sequences, confirming their presence in samples. These probes are used in blotting procedures like northern blotting (for RNA) and southern blotting (for DNA), as well as in microarrays with fluorophore-conjugated sequences to detect changes in gene expression. Probes are also crucial in screening for genetic diseases and identifying specific pathogens in molecular diagnostics. The precision and specificity of synthetic oligonucleotides as primers and probes enable accurate and targeted applications in molecular biology, facilitating advancements in medical research and diagnostics.
Oligo Therapeutics/Gene Therapy
In therapeutic applications, antisense oligonucleotides (ASOs), typically 20 to 30 nucleotides in length, exploit natural biological processes to inhibit or silence undesirable RNA sequences, thereby blocking the expression of harmful or overactive proteins associated with various diseases. This innovative approach to gene therapy has seen significant research advancements, leading to the approval of several oligonucleotide-based drugs in recent years, highlighting their potential in treating a range of genetic disorders and other medical conditions.
Future Use of Synthetic Nucleotides: Exploring DNA and RNA Vaccine Modalities
The future use of synthetic nucleotides is particularly promising in the realm of DNA and RNA vaccine modalities, which, although not oligonucleotides by strict definition, leverage synthetic nucleotide technologies. DNA- or RNA-based vaccines, such as mRNA or plasmid/vector-based nucleic acids, contain hundreds or thousands of bases and represent cutting-edge advancements in vaccine development. These vaccines focus on encoding only the necessary parts of a pathogen’s genetic material, instructing the patient’s body to produce specific antigens or fragments that trigger an immune response. This approach eliminates the need for including whole pathogens, making the vaccines potentially safer and more targeted.
Modern computing and in silico modeling have revolutionized the development of these nucleic acid-based vaccines, enabling rapid design and production within days or weeks once a target sequence is identified. As platform technologies, DNA and RNA vaccines utilize standard building blocks, allowing for versatile and relatively inexpensive production compared to traditional vaccines. Despite these advantages, this approach is still evolving, with ongoing research addressing unique challenges associated with oligo and long nucleic acid products. These challenges include ensuring stability, efficient delivery, and robust immune responses, as well as overcoming issues shared with other biotherapeutic modalities.
Storing Digital data
One of the most groundbreaking applications of oligonucleotides extends beyond biological systems—it’s in digital data storage. Researchers from Microsoft, Twist Bioscience, and the University of Washington have pioneered a method to store digital information within DNA sequences. Unlike traditional binary data storage, which relies on ones and zeros, this technique encodes data into quaternary sequences using the four nucleotides of DNA: adenine (A), thymine (T), cytosine (C), and guanine (G).
DNA offers an extraordinary capacity for data storage, with a single gram potentially holding up to 1 zettabyte of information—equivalent to one trillion gigabytes. This immense density, combined with DNA’s physical compactness and remarkable stability over millennia, makes it an ideal medium for long-term data preservation. To retrieve the stored information, DNA sequences are analyzed using sequencers, which translate the nucleotide sequences back into binary code. While the encoding and decoding processes are already established, ongoing research aims to refine these technologies to make them more cost-effective and scalable. As the field progresses, we can anticipate further innovations in using DNA for high-density, long-term data storage.
Advances in Oligonucleotide Synthesis
Researchers use oligonucleotides to power more efficient, greener chemical synthesis. By assembling oligonucleotides into longer fragments that encode synthetic genes, they can instruct bacteria or yeast cells to make specific enzymes (a type of protein). All cells make enzymes to catalyze chemical reactions. However, many of the chemical reactions required for life can be repurposed to produce useful compounds that would be very difficult or expensive to obtain using traditional chemical synthesis methods.
Instead of traditional chemical synthesis, researchers string together enzyme reactions. The genes encoding these enzymes are stitched together into entirely new genetic pathways that bacteria or yeast can utilize. These organisms can be considered miniature factories. Billions of cells, each containing the introduced enzyme pathway, are used to convert low-value precursors like plastic waste into high-value chemical products, without the need for harsh reaction conditions or environmentally dangerous chemicals. Using oligonucleotide-based technologies, new and novel therapeutics, commodity chemicals like perfumes, and even textiles for durable clothes are being developed.
Recent technological advancements have significantly enhanced the efficiency and accuracy of oligonucleotide synthesis:
- High-Throughput Synthesis: Automation and miniaturization have enabled high-throughput synthesis, allowing the simultaneous creation of large libraries of oligonucleotides for screening and research.
- Enhanced Purity: Improved purification techniques, such as high-performance liquid chromatography (HPLC), ensure high purity of synthetic oligonucleotides, essential for sensitive applications like therapeutics.
- Modified Oligonucleotides: Chemical modifications, such as locked nucleic acids (LNAs) and peptide nucleic acids (PNAs), enhance the stability and binding affinity of oligonucleotides, expanding their potential applications.
Oligonucleotide Synthesis Market: A Growing Force in Biotechnology
Introduction
Oligonucleotide synthesis has become a cornerstone of modern biotechnology, revolutionizing scientific research and medical advancements with its ability to create custom-designed DNA and RNA sequences. This article explores the oligonucleotide synthesis market, analyzing its growth drivers, key segments, and competitive landscape.
Market Overview
The oligonucleotide synthesis market has experienced significant growth in recent years, driven by the expanding adoption of biotechnology, advancements in genomics and proteomics, and the rising demand for personalized medicine. The global oligonucleotide synthesis market size is estimated to be worth $ 4.8 billion in 2024 and is expected to grow at compounded annual growth rate (CAGR) of 9.9% during the forecast period 2024-2035
Oligonucleotides are usually manufactured using chemical synthesis approaches, such as phosphoramidite synthesis and solid-phase
synthesis (used for manufacturing small molecules). However, these approaches for production of synthetic oligonucleotides are fraught
with various challenges, such as long development timelines, lack of purification techniques for oligonucleotides synthesized, regulatory
and compliance-related issues, and lack of expertise among the developers. Therefore, several researchers and therapy / diagnostic
developers in the oligonucleotide synthesis market prefer to outsource their oligonucleotide manufacturing operations to contract
service providers, which possess the required manufacturing capabilities and expertise in the oligonucleotide synthesis market.
Key Market Drivers
- Advancements in Genomics and Proteomics: The growing understanding of the human genome and proteome has increased the demand for oligonucleotides as essential tools for research and development.
- Rising Prevalence of Chronic Diseases: The increasing burden of chronic diseases such as cancer, diabetes, and autoimmune disorders is driving the development of oligonucleotide-based therapeutics.
- Growing Adoption of Personalized Medicine: The trend towards tailoring medical treatments to individual patients is boosting the need for custom-designed oligonucleotides.
- Technological Advancements: Innovations in oligonucleotide synthesis technologies, such as solid-phase synthesis and microfluidic-based methods, have improved efficiency and reduced costs.
Market Segmentation
Market Share by Type of Product Synthesized
The global oligonucleotide synthesis market is segmented into active pharmaceutical ingredients and finished dosage forms, based on
the type of product synthesized. The oligonucleotide finished dosage occupies the highest share in 2024 and is expected to remain
dominant during the forecast period. This can be attributed to the rising approval of oligonucleotide therapies in the coming years and
higher profit margins for finished dosage forms.
Market Share by Type of Oligonucleotide Synthesized
Based on the type of oligonucleotide synthesized, the global oligonucleotide synthesis market is segmented into antisense, DNA, siRNA
and other oligonucleotides. The antisense oligonucleotide segment occupies the highest share in 2024 and is expected to be dominant
during the forecast period. With the rising number of approved siRNA and antisense oligonucleotides-based therapies for various rare
diseases, genetic disorders and other disorders, these segments are likely to grow at a higher CAGR as compared to other types of
oligonucleotides in the coming years.
Market Share by Scale of Operation
This section segments the global oligonucleotide synthesis market across different scales of operation, such as clinical and commercial
scale. Presently, the market is dominated by revenues generated from commercial scale synthesis of oligonucleotides and this trend (in
terms of market share) is likely to remain same during the forecast period.
Market Share by Therapeutic Area
The global oligonucleotide synthesis market is segmented into cardiovascular disorders, genetic disorders, liver disorders, rare
diseases and other disorders. The rare diseases segment occupies the highest share in 2024. However, cardiovascular disorders
market segment is expected to witness substantial market growth in the coming years.
Market Share by Company Size
This section segments the global oligonucleotide synthesis market based on the company size into small, mid-sized, and large and very
large companies. Presently, the market is dominated by large and very large players. This can be attributed to the fact that large and
very large players have well-established facilities and capabilities to improve operational efficiencies and reduce the manufacturing cost
of novel therapeutics. It is worth highlighting that mid-sized companies are likely to grow at a relatively higher pace in the coming years,
as compared to small, and large and very large companies in the oligonucleotide synthesis market.
Market Share by Key Geographical Regions
This segment highlights the distribution of oligonucleotide synthesis market across various geographies, such as North America,
Europe, Asia-Pacific and rest of the world. According to our projections, the market in North America is likely to capture majority (45%)
of the share, and this trend is unlikely to change in the future. It is worth highlighting that the market in Asia-Pacific is likely to grow at a
relatively higher CAGR (10.7%), during the period 2024-2035.
North America and Europe currently dominate the oligonucleotide synthesis market due to established biotechnology and pharmaceutical industries and significant investments in research and development. However, the Asia Pacific region is emerging as a major growth market, driven by increasing government support for life sciences research and a growing number of pharmaceutical and biotechnology companies.
Competitive Landscape
The oligonucleotide synthesis market features a mix of established players and emerging companies. Key players include [insert major market players, such as Thermo Fisher Scientific, Agilent Technologies, Integrated DNA Technologies, Merck KGaA]. These companies are focusing on expanding their product portfolios, investing in research and development, and forming strategic partnerships to strengthen their market positions.
Future Outlook
The oligonucleotide synthesis market is set for continued growth, fueled by ongoing advancements in biotechnology and expanding applications of oligonucleotides. The development of novel oligonucleotide-based therapeutics, the increasing use of oligonucleotides in diagnostics, and the growing adoption of personalized medicine are expected to create significant opportunities for market players.
Challenges and Future Directions
While oligonucleotide synthesis is a well-established process, challenges remain. These include:
- Coupling Efficiency: Achieving high coupling efficiency at each step is crucial for obtaining high-quality products.
- Purification: Removing impurities and side products is essential for downstream applications.
- Cost: Synthesizing long oligonucleotides can be expensive.
Recent advancements have addressed some of these challenges:
- Improved Coupling Efficiency: Optimized reagents and reaction conditions have led to higher coupling efficiencies, ensuring more consistent and high-quality oligonucleotide products.
- Advanced Purification Techniques: Techniques such as high-performance liquid chromatography (HPLC) and mass spectrometry have improved the purification process, yielding products with greater purity and fewer impurities.
- Automation: The development of automated synthesizers has significantly increased throughput and reduced costs, making the synthesis of oligonucleotides more efficient and economically feasible.
While oligonucleotide synthesis has made remarkable strides, several challenges remain:
- Cost and Scalability: The cost of synthesis, especially for long and modified oligonucleotides, can be high. Advances in synthesis chemistry and automation are needed to reduce costs and improve scalability.
- Delivery: Efficient delivery of therapeutic oligonucleotides to target cells and tissues remains a significant hurdle. Developing novel delivery systems, such as nanoparticles and conjugates, is crucial for the success of oligonucleotide-based therapies.
- Regulatory Approval: As more oligonucleotide-based drugs enter clinical trials, navigating the regulatory landscape to ensure safety and efficacy is essential for bringing these therapies to market.
Conclusion
Oligonucleotide synthesis is a cornerstone of modern biotechnology and medicine, driving innovations in genetic research, diagnostics, and therapeutics. As synthesis technologies continue to advance, the potential applications of synthetic oligonucleotides are expanding, promising new solutions for some of the most challenging problems in health and disease. The future of oligonucleotide synthesis looks bright, with ongoing research and development poised to unlock even greater possibilities.
References and Resources also include:
https://www.twistbioscience.com/blog/perspectives/how-oligos-changed-world
https://www.rootsanalysis.com/reports/oligonucleotide-synthesis/304.html
