Synthesis of oligonucleotides (2023)

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What is oligonucleotide synthesis?

Oligonucleotides can be generated by enzymatic cleavage of larger biomolecules or in a targeted mannerchemical compositionIn the latter case, organic nucleotide synthesis is a chemical process for the synthesis of nucleoside phosphoramidites, the basic monomeric building blocks of oligonucleotides. Nucleoside phosphoramidites (also known as nucleosides or amidites) are themselves derivatives of natural or synthetic nucleosides.

Nucleotides are therefore complex molecules composed of covalently linked nucleosides and phosphate groups. Nucleotides combine in a specific order to form the desired product. Thus, oligonucleotides consist of small fragments of nucleic acids linked together to form single-stranded biopolymers ("-mers"). Since most oligonucleotides (oligonucleotides) typically consist of up to 20 linked nucleotides (although there may be more), they can be considered small biopharmaceutical molecules.

Like other drug-related small molecules, oligonucleotide synthesis requires careful control of temperature, pH, substrate quality, etc. Due to the standard methods used in oligonucleotide synthesis, the longer and more complex the nucleotide sequence, the more difficult the synthesis. to achieve performance, purity and cost objectives. Process Analysis Technology (PAT) is very useful in achieving these goals.

What are oligonucleotides?

Create biomolecules for biotechnological, medical, forensic and clinical applications

Oligonucleotides or oligonucleotides are small single-stranded or double-stranded nucleic acid fragments linked together to form single-stranded biopolymers. The individual nucleotide bases can be considered equivalent to the monomers that make up the polymers in classical chemistry. Nucleotides consist of three parts: a nitrogenous base, a sugar molecule with five carbon atoms (both parts are nucleosides), and one or more phosphate groups. Alternatively, the nucleotides may consist of unnatural or non-canonical bases. Typical examples include LNAs (encapsulated nucleic acid), morpholinos and structurally modified bases or backbones.

Complex nucleotide bonds form biologically important RNA and DNA biomolecules. In the case of RNA, a single-stranded biopolymer, the sugar is ribose. In DNA, the double-stranded molecule contains the sugar deoxyribose. Short sequences of deoxyribonucleic acid and ribonucleic acid are building blocks of important oligonucleotides. The specific combination of these nucleotides allows obtaining biomolecules useful in biological, medical, forensic and clinical applications.

How are oligonucleotides used?

Research, diagnostic, forensic and therapeutic applications

Oligonucleotide primers and probes

The most common use of synthetic oligonucleotides is relatively small probes and primers (up to 30-mer) in a variety of applications. This involves the synthesis of a nucleotide sequence that pairs with, or is "reversibly complementary to" a larger target DNA or RNA strand (target sequence). Oligonucleotides are often used as primers to initiate, for example, enzymatic reactions. Make millions to billions of copies of short or long target sequences. Well-known examples are polymerase chain reaction (PCR) or Sanger sequencing methods. Applications of oligonucleotide primers include DNA sequencing, gene expression, cloning and molecular diagnostics.

As probes, oligonucleotides are used to recognize and bind to a specific DNA or RNA target sequence to confirm the presence of that sequence in a given material. Applications using oligonucleotide probes include blotting procedures such as Northern blotting (for RNA) or Southern blotting (for DNA) as conjugated to fluorophore sequences on microarrays to detect changes in gene expression or to screen for genetic disorders or to identify specific pathogens (molecular diagnosis) .

Oligonucleotide/gene therapy

In therapeutic applications, antisense oligonucleotides (ASOs), typically 20-30 mer, exploit the natural biology and promote gene repression or gene silencing (destruction) of unwanted or hyperactive RNA sequences, which in turn prevents the expression of certain damaged of RNA sequences. or hyperactive proteins that may cause or promote disease. Research on oligonucleotide-based drugs has intensified significantly in recent years, and several drugs have been approved.

Future applications of synthetic nucleotides: exploring the modality of DNA and RNA vaccines

Although not strictly oligonucleotides, DNA- or RNA-based vaccine products such as mRNAs of hundreds or thousands of bases or plasmid- or vector-based nucleic acids represent the evolving frontier of synthetic nucleotide technology.

Conceptually, a DNA or RNA vaccine would eliminate all unnecessary or harmful parts of a pathogenic bacterium or virus. In contrast, such nucleic acid-based vaccines would encode only some parts of the pathogen's DNA or RNA. These DNA or RNA strands instruct the patient's body to produce a single antigen or pathogen fragment, which then promotes an immune response against the antigen. Thanks to modern computing and computer modeling, these oligonucleotide vaccine templates can be generated in days or weeks, provided the appropriate target sequence is designed. As a platform technology, nucleic acid-based vaccines are based on standard sets of building blocks, that is, raw materials that can be freely combined in almost unlimited combinations. As such, they are also relatively cheap and easy to produce compared to traditional vaccines. However, it remains an established paradigm in the biopharmaceutical industry, and new challenges are constantly being faced, some specific to oligonucleotide products and long nucleic acids, and others common to other biotherapeutic approaches.

Synthesis of oligonucleotides (1)

How to synthesize oligonucleotides

Example: Chemical synthesis of DNA oligonucleotides

Oligonucleotide synthesis is a chemical process in which nucleotides are specifically linked to form the desired sequence product. Continuous solid-phase synthesis using packed columns is commonly used to produce oligonucleotides. In some cases suspension batch reactions were used.

The process of producing an interesting oligomer sequence requires multiple cycles consisting of specific synthesis steps. In the first step, the 4,4'dimethoxytrityl protecting group is removed from the solid-supported nucleotide. The phosphoramidine monomers are then activated and rapidly conjugated via free hydroxyl groups to the solid-phase bound nucleotides to produce dinucleosides containing phosphite triester linkages. In the third step, the phosphorous triester is oxidized. In the last step (first cycle), any unreacted charged nucleosides are "blocked" by acetylation to prevent the formation of unwanted byproducts in the next cycle.

Synthesis of oligonucleotides (2)

How long does an oligonucleotide last?

Oligonucleotide length and effect on yield

The length of the oligonucleotide is determined by the number of nucleotides that make up the sequence. For nomenclature purposes, sequences of 20 nucleotides are designated as "20-mers". The length of the oligonucleotide is critical for several applications. For example, short oligonucleotides are commonly used in PCR, while very long sequences (200 bases or more) are used in demanding applications such as cloning or CRISPR editing. The synthesis of long chain specific oligonucleotides is challenging and requires efficient synthesis controls and purification methods to ensure pure sequences. On average, most oligonucleotides are less than 60 meters in length. Since the oligonucleotide synthesis method requires repeated reaction cycles to add additional nucleotides, the longer the desired sequence, the greater the risk of unwanted byproducts, which may include deletions or truncated sequences.

Depending on the qualitative, quantitative and cost objectives, different processing methods are used. For example, unsalted oligonucleotides remove byproducts of chemical synthesis, but the sequence remains incorrect. In terms of cost and efficiency, these desalted oligonucleotides are very useful in more common applications such as PCR. For more demanding applications, reverse-phase chromatography can remove unwanted sequence from the product, but is not as efficient as gel electrophoresis, which provides a highly purified sequence product, but usually in limited quantities. Particularly for clinical reasons, maintaining yield is a constant concern during synthesis and downstream processing.

Solid phase oligonucleotide synthesis

Production of customized peptides and oligonucleotides

Classical solid-phase chemical synthesis usually involves the use of polymers or specialized glass beads that are independent of reaction conditions. These beads can be placed in columns through which reactants, reagents and solvents flow. The purpose of the beads is to act as a platform for the covalent attachment of substrate molecules, which then act through chemical reactions. Protection and deprotection of certain functional groups on the substrate molecule is key to the synthesis and delivery of the desired product. The composite is removed by chemically breaking the bond between the composite and the underlying polymer sphere.

Solid-phase synthesis is efficient and rapid in producing purified products because impurities and unreacted substances are washed away at each step of the synthesis. In addition, the entire synthesis process lends itself to computer control and automation. Solid-phase synthesis is the most common method for producing custom peptides and oligonucleotides. In the latter case, many repeat cycles are required to add successive nucleotides to form the target sequence.

Synthesis of oligonucleotides (3)

Alternative forms of oligonucleotide synthesis

liquid or enzyme phase

Solid-phase oligonucleotide synthesis faces many challenges, including decreasing yield as chain length increases. It is often assumed that steric hindrance is a low-efficiency mechanism where one or more surface-immobilized chains can interfere with each successive extension of its neighbors.

To address these and other challenges, several methods exist for the synthesis of oligonucleotides in the liquid phase.

Many of the basic steps of liquid phase synthesis are similar to solid phase synthesis. The main difference is that the synthesis takes place in a solution or liquid phase (ie it is not immobilized on any surface). Since the process is still cyclic, there are several random methods for removing unreacted reagents while retaining the extended oligonucleotide products.

The technique of oligonucleotide synthesis by PCR was developed in 1985. In 1986, Taq polymerase was used for the first time. However, the industrial synthesis of oligonucleotides has been difficult to adopt the enzymatic synthesis of oligonucleotides for three reasons:

  1. Enzymatic synthesis requires a template on which DNA polymerases can act to extend the sequence. Therefore, the enzymatic synthesis of deoligonucleotides cannot proceed without a template.
  2. Many ASO oligoprimers, probes, or targets that can be used as templates are inherently too short to effectively prime and activate polymerase synthesis.
  3. Many artificial therapeutic oligonucleotides have been chemically modified to increase the half-life and enhance the circulatory function of body fluids, and these modifications make it almost or completely impossible for them to interact with evolutionary polymerases (mainly larger oligonucleotides, e.g., mRNA).

Continued research and development of engineered polymerases capable of synthesizing ASOs from chemically modified nucleotide stocks

Oligonucleotide Synthesis Monitoring and Control Technology

complete approach

The complexity of oligonucleotide synthesis can be beneficial through a greater degree of process observation, understanding of reaction mechanisms and errors, and hands-on control. Real-time analysis using PAT can minimize or eliminate the need for time-consuming or slow offline analysis.

  • reactive infraredIReactive RamanIt can be used to monitor the kinetics of oligonucleotide synthesis reactions and compare assays between assays.
  • Precise temperature, mixing and dispensing controlAutomatic EasyMax Chemical Synthesis ReactorThe inclusion of trends and response mechanisms facilitates data collection and simplifies management with iC software

Suggested publication: Real-time PAT prediction for oligonucleotide synthesis process

ReactIR and chemometric modeling reduce risk

Rydzak, J. W., White, D. E., Airiau, C. Y., Sterbenz, J. T., York, B. D., Clancy, D. J. and Dai, Q. (2014). Providing real-time process analytical technology for oligonucleotide process synthesis.Organic Process Research and Development, 19(1)203-214.

The authors note that while automation protocols for solid-phase oligonucleotide synthesis are well established, the chemistry involved remains difficult and expensive. The main risks identified during production include:

  • Connecting the wrong chemical solution to the specified input port of the synthesizer
  • The solution was quantitatively prepared incorrectly
  • Errors that may come from synthesizer machines or other unknown sources

Failure to detect and address any of these threats can result in complete process failure. Therefore, the application of real-time spectral monitoring was evaluated using discriminative, quantitative and MSPC modeling based on mid-infrared data. ReactIR has been found to provide confidence and real-time synthesis control and enhance automated oligonucleotide synthesis. In particular, the sensitivity, depth of information, broad spectrum and empirical availability of this method are believed to allow the method to be easily applied to many of the critical production challenges associated with these compounds, the most important of which is the confirmation of sequence and online purity.

Featured Publication: Online Phosphamidite Identification and Oligonucleotide Sequence Confirmation

Real-time process control with ReactIR

McElderry, J.-D., Hill, D., Schmitt, E., Su, X., and Stolee, J. (2021). Online FTIR identification of phosphoramidites for real-time oligonucleotide sequence confirmation.Organic Process Research and Development, 25(2),262–270.

The authors note that the sequence of antisense oligonucleotides (ASOs) is a critical quality attribute that must be confirmed as part of the waiver suitability testing. However, confirming the identity of the full-length product (FLP) can be difficult because there are many potentially closely related structures. Related structures can arise from chemical reaction failures, random coupling to solid-phase templates, starting material preparation, dosing, and reagent identification errors. Any of these events can lead to incorrect sequencing or other unacceptable quality characteristics of the oligonucleotide polymer.

ReactIR was used to model and differentiate phosphoramidites and solvents in flux identification and synthesis. Spectral differences between modified phosphites allow for reliable real-time process validation, allowing material assignment errors to be captured in real-time before they lead to sequencing failure. ReactIR provides the ability to take corrective action that would otherwise not be possible.

Using this method, eight different phosphoramidites were identified in acetonitrile solution by processing infrared spectra collected in real time. The resulting models showed high sensitivity and specificity, with no misclassifications. The model also shows robustness to changes in flow rate, device-to-device variability, detector variability, changes in amidite solution concentration, and wave number.

Synthesis of oligonucleotides (4)

Synthesis of oligonucleotides and PAT

real-time response monitoring

Real-time monitoring capabilityIn situ Fourier transform infrared spectroscopyIRaman spectroscopyChemical flow reactions are well documented for key steps in oligonucleotide synthesis.

  • Synthetically modified phosphoramidites and natural nucleomonomers for structure and purity
  • Reduce risk due to synthesizer mechanical problems or incorrect solution delivery
  • Monitoring the coupling reaction of phosphoramidine monomers with free hydroxyls on solid-phase bound nucleotides
  • Monitor denaturation and hybridization of complementary oligonucleotides during double-strand synthesis.
  • Determinekinetics of chemical reactionsDuring annealing - detect and monitor intermediates or unknowns
  • Relate the spectral changes to key points in the process
  • Suitable for SPS, bulk peptide and polymer synthesis (SPS + solution phase)
Synthesis of oligonucleotides (5)

Automated oligonucleotide synthesis

A well-controlled synthetic environment is essential to ensure reaction results. At the same time, the automated reactor platform ensures certainty and consistency of all critical process parameters.

  • A repetitive environment is the foundation of good learning
  • Understand the impact of process parameters on critical quality attributes by combining sensor and PAT data online
  • Protocols and automation reduce the risk of manual or inconsistency
  • Make it easy to search and organize your data
  • Rely on decades of quotes and evidence
  • Suitable for SPS, bulk peptide and polymer synthesis (SPS + solution phase)

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