Solid-phase oligonucleotide synthesis is designed to create short strands of nucleic acids with predetermined sequences. It forms the backbone of gene editing by driving the production of custom DNA/RNA oligo sequences for various applications, including CRISPR, antisense oligonucleotides, and gene-based drug and vaccine development. Custom oligonucleotide sequences can be used in precision medicine to treat rare genetic diseases and cancer, where the patients carry varying target sequences and may not respond to conventional treatments.

Background and evolution of solid-phase oligonucleotide synthesis

History of oligonucleotide synthesis: Basic Nucleic Acid Chemistry

The foundations of oligonucleotide synthesis date back to the 1950s, when the structure of nucleic acids and the chemical composition of DNA and RNA had been revealed.

Nucleotides, the building blocks of nucleic acids, are composed of:

1. Sugar (deoxyribose for DNA and ribose for RNA)
2. A nitrogenous base that may be one of the following: Purines - Adenine (A), Guanine (G) and pyrimidines - Cytosine (C), Thymine (T) in DNA, Uracil (U) in RNA.
3. Phosphate group to link to other nucleotides.
4. The sugar + nitrogenous base (without the phosphate group) complex is called a nucleoside.

This information is essential for synthesizing oligonucleotides with high stability and specificity.

The first dinucleotide, synthesized by Michelson and Todd, was a thymidine nucleotide containing two thymidine nucleosides with a phosphate link.¹ Khorana synthesized more stable oligonucleotides by devising a sequential oligonucleotide synthesis protocol that joins nucleotides via phosphodiester bonds.²

Foundation of Modified Oligonucleotides, Solid-phase Support, and Automated Oligonucleotide Synthesis

Letsinger laid the foundations of the solid-phase synthesis technology, which allowed a more controlled step-by-step synthesis of oligonucleotide sequences.³ He also developed phosphotriester and phosphite-triester coupling methods to stabilize phosphate linkages, prevent nucleotide branching, and expedite coupling reactions in a semi-automated way. His research introduced an oxidation step to stabilize linkages and marked the beginning of modified oligonucleotides.⁴

These advancements were limited to only a few nucleotides until Caruthers and Beaucage invented the phosphoramidite approach, using phosphoramidite derivatives of the nucleotides to produce scalable and storable oligonucleotides.⁵ These modified oligonucleotides harbored protective groups to prevent unwanted reactions, increasing the purity and quality of the product. More importantly, the method made oligonucleotide synthesis amenable to automation, producing longer sequences in short timeframes.

Advancements in Oligonucleotide Synthesis

Further modifications, from fluorescent tags to bioconjugation^6,7, were tested to integrate oligonucleotides into high-throughput research and combinatorial drug development applications.

The advent of automated oligo synthesizers gave rise to oligonucleotide libraries consisting of large sets of synthetic oligonucleotides.⁸ These libraries can be screened for their ability to bind target genes and alter cellular functions. Therefore, they lend themselves to gene therapies, such as antisense oligonucleotide (ASO) therapy, RNA interference (RNAi), aptamers, and CRISPR-based systems.

Meanwhile, alternatives to solid-phase oligonucleotide synthesis were invented with flow chemistry, which enabled better control over reaction conditions, enhanced yield, and streamlined purification. Nevertheless, solid-phase oligonucleotide synthesis remains widely used due to reaction times, ease of purification, and automation.

The synthesis cycle: A stepwise process

Preparation of solid support

Initially invented by Merrifield, solid-phase synthesis is used to synthesize oligonucleotides, oligosaccharides, and peptides. A solid support is a material that provides a platform for sequentially adding nucleotides.

Different types of solid-phase supports are as follows:

• Polymer-based supports, such as polystyrene and polyethylene glycol (PEG)
• Glass-based supports, such as silica gels
• Controlled-Pore Glass
• Bead-based supports
• Resin-based supports

Each solid-phase support type has distinct advantages for robust nucleotide anchoring, minimum background reactions, stability, and convenient washing. Furthermore, an ideal solid support must comprise functional groups to facilitate covalent bonding between nucleotides.

Additionally, a universal support system can overcome the limitations of standard solid-phase supports. This system does not require a pre-attached base; instead, this base is added to the support in the first coupling reaction. It features a single column for synthesizing any oligonucleotide base instead of requiring a separate column for each standard and modified base.

Steps in the synthesis cycle

The phosphoramidite method is the gold standard for solid-phase oligonucleotide synthesis. It proceeds in cycles, with one nucleotide added in each cycle, in the 3'—to 5' direction.

• The first nucleoside is pre-attached to the solid support and contains a 5'-DMT protecting group that prevents the nucleotide from participating in unwanted side reactions.
• The protecting group is removed in a process called detritylation, which opens the 5' OH group of the pre-attached nucleoside for adding the next base.
• During coupling, a nucleoside phosphoramidite monomer is added to the first nucleoside in the presence of an optimum solvent and a catalyser. A phosphite triester bond is formed.
• Unreacted 5'- OH groups on the first nucleoside are blocked with a capping reagent to prevent it from partaking in future cycles. This step also mitigates the risk of deletion errors in the oligonucleotide sequence.
• The newly formed phosphite triester bond is stabilized with an oxidation step.
• The steps are repeated for adding each nucleotide, starting with removing the DMT group at the 5'- end of the oligonucleotide chain.
• After the desired sequence is assembled completely, the oligonucleotide chain is cleaved off the solid support. Finally, the remaining protecting groups on the bases and the backbone are removed. The resulting oligonucleotide is ready for purification.

Coupling efficiency and challenges

High coupling efficiency is critical for producing high-quality pure oligonucleotides with precise sequences. Key factors include:

• Purity of the phosphoramidites, coupling activators, and solvents
• The optimum concentration of the phosphoramidites and the activator
• Optimum coupling time
• A strong coupling activator must be chosen, such as tetrazole and its derivatives.
• Temperature
• Solid support features, such as pore size and surface area, which significantly affect proper reagent diffusion

Coupling efficiency can be assessed by measuring the detritylation color absorbance. More specifically, a cleaved DMT group should produce an orange color with a visible-range absorbance at 495 nm.⁹

Depending on the source of the problem, coupling efficiency can be improved in several ways. For example, introducing the coupling reagent 5-(ethylthio)-1H-tetrazole into the protocol increased coupling efficiency.¹⁰ Another protocol involves an isothermal biocatalytic process with polymerases and endonucleases to streamline coupling.¹¹

Purification and quality control

Techniques for oligonucleotide purification

Commonly practiced oligonucleotide purification methods include:

1. Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC): This technique is used to separate truncated oligonucleotides without the 5'-DMT from full-length oligonucleotides with the 5'-DMT based on the difference in their hydrophobicity.
2. Polyacrylamide Gel Electrophoresis (PAGE): Its principle is size-based separation, where the migration distance is affected by the size of oligonucleotides. Thus, truncated sequences that migrate faster can be easily sequestered from the full-length ones.
3. Ion-Exchange Chromatography (IEX): This separation method is based on the differences between complete and truncated oligonucleotides in charge density.
4. Reversed-phase cartridge purification: The oligonucleotide mixture is passed through a column cartridge, where failed sequences or short oligonucleotide products are immediately washed away.

Quality assurance

Despite the purification and separation methods, truncated sequences, failure sequences, byproducts, and protecting groups can still copurify with the final product. Furthermore, premature strand cleavage may occur due to suboptimal experiment conditions, mechanical stress, and prolonged reactions.

Quality assurance is essential to mitigate the issues that diminish product quality in oligonucleotides. Efficiency must be carefully monitored during coupling, capping, and purification to detect byproducts or incomplete chains timely. The solid support must be handled carefully, while detritylation and oxidation must be timed thoroughly under controlled environmental conditions.

Comparison with peptide synthesis

Oligonucleotide synthesis is similar to peptide synthesis in many ways. Both start with a molecule anchored to a solid support, followed by the stepwise addition of monomers (amino acids in case of peptide synthesis). The workflow for the stepwise addition involves protective groups, coupling, deprotection, and the necessary purification post-synthesis.

On the other hand, peptide synthesis is unique in the protecting groups used, the appropriate coupling reagents for peptide bond formation, and the cleavage reagents.

References

1. Michelson A, Todd AR. Nucleotides part XXXII. Synthesis of a dithymidine dinucleotide containing a 3′: 5′-internucleotidic linkage. J Chem Soc (Resumed) 1955:2632-2638.
2. Gilham PT, Khorana HG. Studies on polynucleotides. I. A new and general method for the chemical synthesis of the C5 ″-C3 ″internucleotidic linkage. Syntheses of deoxyribo-dinucleotides1. J Am Chem Soc 1958;80(23):6212-6222.
3. Letsinger RL, Mahadevan V. Oligonucleotide synthesis on a polymer support1, 2. J Am Chem Soc 1965;87(15):3526-3527.
4. Letsinger RL, Ogilvie KK, Miller PS. Nucleotide chemistry. XV. Developments in syntheses of oligodeoxyribonucleotides and their organic derivatives. J Am Chem Soc 1969;91(12):3360-3365.
5. Beaucage SL, Iyer RP. Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tetrahedron 1992;48(12):2223-2311.
6. Benizri S, Gissot A, Martin A, Vialet B, Grinstaff MW, Barthélémy P. Bioconjugated oligonucleotides: recent developments and therapeutic applications. Bioconjugate chem 2019;30(2):366-383.
7. Hennessy J, McGorman B, Molphy Z, Farrell NP, Singleton D, Brown T, et al. A Click Chemistry Approach to Targeted DNA Crosslinking with cis‐Platinum (II)‐Modified Triplex‐Forming Oligonucleotides. Angew Chem 2022;134(3):e202110455.
8. Sabary O, Orlev Y, Shafir R, Anavy L, Yaakobi E, Yakhini Z. SOLQC: Synthetic oligo library quality control tool. Bioinformatics 2021;37(5):720-722.
9. Ferrazzano L, Corbisiero D, Tolomelli A, Cabri W. From green innovations in oligopeptide to oligonucleotide sustainable synthesis: differences and synergies in TIDES chemistry. Green Chem 2023;25(4):1217-1236.
10. Kundu J, Ghosh A, Ghosh U, Das A, Nagar D, Pattanayak S, et al. Synthesis of phosphorodiamidate morpholino oligonucleotides using trityl and fmoc chemistry in an automated oligo synthesizer. J Org Chem 2022;87(15):9466-9478.
11. Moody E, Obexer R, Nickl F, Spiess R, Lovelock S. An enzyme cascade enables production of therapeutic oligonucleotides in a single operation. Science 2023;380(6650):1150-1154.

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