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Oligonucleotide synthesis

The synthetic biology, the design and construction of biological devices and systems, promises  to augment biological life, in order to have it producing outcomes which we dictate. It is used for designing and constructing new biological parts, devices, and systems as well as for re-designing of 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.


Oligonucleotides (oligos for short) are one of the most important tools in modern-day molecular biology. 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, or oligos, 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 are the nucleoside), and one or more phosphate groups. Alternatively, nucleotides can consist of non-natural or non-canonical bases. Common examples include LNA (locked nucleic acids), Morpholino, and structurally modified bases or backbones.


The complex linking of nucleotides form the biologically-critical RNA and DNA biomolecules. In the case of 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 deoxyriobonucleic acids and ribonucleic acids that are the building blocks of important oligonucleotides. Specific linkages of these nucleotides create the target biomolecules that are used in biological, medical, forensic and clinical applications.


Oligonucleotides are Powering the Next Generation of Synthetic Biology. Researchers also use oligonucleotides to power more efficient, greener chemical synthesis. By assembling oligonucleotides together 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 a 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.


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.

Research, Diagnostic, Forensic and Therapeutic Applications

Oligonucleotide Primers and Probes

The most common use of synthetic oligonucleotides is as relatively short probes and primers (up to 30-mer) in a wide variety of applications. This involves synthesizing a nucleotide sequence that is paired or ‘reverse-complimentary’ to a larger, target DNA or RNA strand (target sequence). As primers, oligos are typically used to initiate enzymatic reactions to e.g. create millions to billions of copies of a short or long target sequence. Well-known examples are the polymerase chain reaction (PCR) or the Sanger sequencing method. Applications for oligo primers include DNA sequencing, gene expression, cloning and molecular diagnostics.


The PCR is like photocopying a page of a book for DNA. However, unlike normal photocopying, in PCR the photocopier (an enzyme called DNA polymerase) has to be brought to the paper (the DNA) to make copies. Researchers use short, single-stranded oligos (20-30 nucleotides) that are synthesized to have a nucleotide sequence complimentary to the outside edges of the DNA sequence that is the target for copying. By using specific temperatures, the double stranded helix of the target DNA is opened, the oligonucleotides bind their complimentary sequence on the target, and DNA polymerase starts copying from the oligo-DNA duplex. Repeating the temperature cycles amplifies a single, specific DNA strand into billions of identical strands.


This technology powers many applications, including diagnosis of unknown pathogens in biological samples and genetic fingerprinting in forensic science. While PCR uses short oligos, current DNA synthesis methods can make oligos of 200 or more bases long. Long oligos are being used to speed up drug development processes, to generate DNA-based nanostructures for applications like targeted drug delivery, and to archive digital data for tens of thousands of years.


As probes, oligos serve to identify and bind to a specific DNA or RNA target sequence in order to confirm presence of this sequence in a given material. Applications using oligo probes include blotting procedures such as northern blotting (for RNA) or southern blotting (for DNA), as fluorophore-conjugated sequences in microarrays that detect changes in genes expression or used in screening for genetic diseases or to identify specific pathogens (molecular diagnostics).


Oligo Therapeutics/Gene Therapy

In therapeutic applications, antisense oligonucleotides (ASO), generally 20 to 30-mer species, take advantage of natural biology and facilitate gene inhibition or gene silencing (destruction) of undesirable or over-active RNA sequences, this in turn blocks expression of certain damaged or overactive proteins which may be causing or facilitating disease. Research on oligonucleotide based therapeutics has intensified tremendously and several drugs have been approved in recent years.


Future Use of Synthetic Nucleotides: Exploring DNA and RNA Vaccine Modalities

Although not an oligonucleotide by strict definition, DNA- or RNA-based vaccine products, such as mRNA or plasmid or vector-based nucleic acids, of many hundreds or thousands of bases in length, represent the frontlines of evolving synthetic nucleotide technologies.


In concept, DNA or RNA vaccines would dispense with all unnecessary or harmful parts of a pathogenic bacterium or virus. Instead, such a nucleic acid-based vaccine would contain code for just a few parts of the pathogen’s DNA or RNA. These DNA or RNA strands instruct the patient’s own body to produce individual antigens or fragments of the pathogen, and then promote an immune response to the antigen. With modern computing and in silico modeling, these oligonucleotide vaccine modalities are capable of being created in a matter of days or weeks given an appropriate target sequence to design against. As a platform technology, nucleic acid-based vaccines rely on standard sets of building blocks or raw materials enabling myriad of combinations almost at will. As such, they are also relatively cheap and easy to produce compared to traditional vaccine modalities. However, this is still a maturing paradigm for the biopharmaceutical industry and new challenges are constantly being addressed, some unique to oligo and long nucleic acid products and some shared with other biotherapeutic modalities.


Storing Digital data

Possibly the most innovative application for oligonucleotides has nothing to do with living organisms — they can be used to store digital data. A collaboration between researchers at Microsoft, Twist Bioscience, and the University of Washington has developed a way to store digital data as sequences of DNA. Instead of the binary data (ones and zeros) being stored on tapes, data can be encoded into quaternary data and be stored in DNA as A, T, C and G nucleotides.

DNA is a truly amazing resource for storing data. The upper-limit for storage in one gram of DNA is around 1 zettabyte. That’s 10 exp(21) bytes, or one trillion gigabytes! DNA oligos also take up very little physical space and are stable for thousands of years, making them ideal candidates for long-term data storage. To extract data stored in this way, the oligos would be run on a DNA sequencer. The sequence can then be decoded back to the binary digital data. Both the encoding (writing millions of oligos) and decoding (DNA sequencing) steps already exist, now it is up to researchers to further develop these technologies into an affordable data storage solution. We expect many more exciting advances in this field in the future.



How are Oligonucleotides Synthesized?

Example: DNA Oligos

Oligonucleotide synthesis is the chemical process by which nucleotides are specifically linked to form the desired sequenced product. Continuous solid phase synthesis using a packed-bed column is commonly used for producing oligonucleotides. In some cases, a batch, slurry reaction is employed.


The process of producing the target oligomer sequence takes a number of cycles consisting of specific synthetic steps. In the first step, a 4,4’ dimethoxytrityl protecting group on the solid phase supported nucleotide is removed. Next, a phosphoroamidite monomer is activated and rapidly coupled through the free hydroxyl group on the solid phase bound nucleotide to create a dinucleoside that contains a phosphite triester link. In the third step, the phosphite triester is oxidized. In the final step (of the first cycle), any unreacted supported nucleosides are “capped” via acetylation to prevent unwanted side products forming in the next cycle.


To achieve high product yield, controlled pore glass (CPG) is widely employed due to the significant enhancement of surface area. As the oligo synthesis is a cyclic process, the CPG is maintained in physical separated columns while the chemical waste between adjacent reactions is flushed away. The nature of this process minimizes the cross contamination between columns and leads to high quality products. This column-based process can typically automate 96–1536 oligos synthesis in parallel.


Microfluidics provides opportunities to achieve high throughput oligo synthesis at low cost. Microarray-based oligo synthesis is well known for the extremely high throughput that can be as high as tens of thousands of oligos per chip


An oligonucleotide synthesizer based on a microreactor chip and an inkjet printer

For applications requiring a large number of oligos with high concentration, it is critical to perform high throughput oligo synthesis and achieve high yield of each oligo.


Researchers have developed microreactor chip for oligo synthesis that integrates fixed silica beads in the microreactors. The microreactors are fabricated on a silicon wafer using an anisotropic etching process. Silica beads are self-assembled in the microreactors to increase the surface area. The surface area of each microreactor with beads is 71 times more than an empty microreactor. As oligo synthesis occurs on the surface of the beads, the surface area is critical to the product yield. An inkjet printer is utilized to deliver chemical reagents in the microreactors.


There are 100 micro-reactors for synthesizing different oligonucleotides on each chip with a size of 20mm×20mm. Cartridges of the inkjet printer are loaded with chemical reagents which can be independently ejected by 90×6 nozzles. The introduction of chemical reagents into corresponding micro-reactors is realized by the PC-controlled printer, which can control the types, volume and location of the reagents through inputting target sequences to the PC. Each synthetic process subsequently starts after introducing reagents into the micro-reactors. While the waste in the micro-reactors is collected by vacuum pump.


Today, many companies, including Twist Bioscience, synthesize oligonucleotides en masse for academic and commercial use. While the early methods focused on building oligonucleotides one at a time, current synthesis technology developed at Twist Bioscience can write approximately 1 million unique synthetic oligonucleotides simultaneously. Each oligo’s sequence is bespoke — designed for their specific application after synthesis.  Instead of the traditional plastic or glass used as the surface on which to chemically “write” DNA sequences, Twist Bioscience harnesses the properties of silicon to enable synthesis of oligos on a world-leading scale.


Oligonucleotide Synthesis Market

According to the Transparency Market Research (TMR) report, the global a market was valued at US$ 1,966.2 Mn in 2018 and is projected to expand at a CAGR of 9.5% from 2019 to 2027.


There are various lengths of oligonucleotides, and sequences and design modifications available. Thanks to the increasingly developing biotechnology and life sciences markets, there has been a n ever-rising need for oligonucleotides on the global market, as well as rising acceptance and use of oligonucleotides in clinical and diagnostic fields.


Synthesized oligonucleotides Segment to Dominate Market

  • Based on product type, the global oligonucleotide synthesis market has been divided into reagents & consumables, equipment, and synthesized oligonucleotides. The synthesized oligonucleotides segment has been bifurcated into DNA oligonucleotides, RNA oligonucleotides, and others.
  • The synthesized oligonucleotides segment dominated the global oligonucleotide synthesis market in 2018 and the trend is projected to continue during the forecast period. Synthesized oligonucleotides are widely used in research as well as in therapeutics and diagnostics applications. Synthesized oligonucleotides are ready to use oligonucleotides. These are also available as customized oligos prepared as per need. There are two types of commercially used synthesized oligonucleotide: DNA oligonucleotides and RNA oligonucleotides. Bridged Nucleic Acid (BNA) and Locked Nucleic acid (LNA) are the other types of synthesized oligonucleotides used in research.


Research Applications to be Highly Lucrative Segment

  • In terms of application, the global oligonucleotide synthesis market has been classified into research, diagnostic and therapeutic. Among the applications, the research applications segment dominated the global oligonucleotide synthesis market in 2018. Rising R&D focus of pharmaceutical & biotechnology companies, along with technological advances in the techniques such as polymerase chain reaction, and next generation sequencing, are some of the major factors driving the growth of the research segment in 2018. However, rising applications in therapeutics, and growing R&D in RNAi therapeutic drugs is expected to drive the growth of the therapeutic segment during 2019-2027


North America to Dominate Global Market

  • In terms of region, the global oligonucleotide synthesis market has been segmented into North America, Europe, Asia Pacific, Latin America, and Middle East & Africa. North America dominated the global Oligonucleotide Synthesis market in 2018, followed by Europe.
  • North America accounted for major share of the global oligonucleotide synthesis market in 2018, According to the data obtained from the ongoing clinical trials in the oligonucleotides segment, there were more than 70 oligonucleotide ongoing or recently completed trials in the U.S. alone. Fast growing life science industry, rising focus on genetics and genomics in the research and diagnostics applications in North America, and growing focus of pharmaceutical organizations on oligonucleotides as therapeutic agents comprising RNAi and antisense therapeutics are driving demand for oligonucleotides in North America.
  • However, rising focus on personalized medicine and growing biotechnology and life science research activities in Asia Pacific, along with rising healthcare spending on research & development in India, China, and Japan are factors expected to boost the growth of the market for oligonucleotide synthesis in Asia Pacific from 2019 to 2027.



The advanced synthesis of oligonucleotides provides longer oligos with better sequence fidelity and higher purity that can be used in a number of molecular biology applications. Various pharmaceutical and biotechnology companies are continuously working on developing newer and advanced methods in synthetic chemistry. For instance, Integrated DNA Technologies, leader of synthesized oligonucleotides has developed various pioneered methods to synthesize oligonucleotides of extreme lengths. The company has developed oligonucleotides up to 200 base length which is used with mass spectrometry for the manufacture of mini-genes.


The global oligonucleotide synthesis market is fragmented in terms of number of players. Key players in the global market include GE Healthcare, Thermo Fischer Scientific, Agilent Technologies, Inc., Merck KGaA, Integrated DNA Technologies, Inc., Kaneka Eurogentec S.A., BioAutomation, Gene Design, Inc., Eurofins Genomics, and ATDBio, Ltd. among others.




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