What Is Oligonucleotide Synthesis? Enzymatic synthesis
Learn how scientists create custom DNA and RNA strands for use in research, diagnostics, and drug development.
This first article will cover method 4. Enzymatic synthesis
Oligonucleotide synthesis represents a foundational technique in molecular biology, biotechnology, and genomics, enabling the creation of custom-designed short sequences of nucleotides—DNA or RNA fragments—crucial for numerous applications such as genetic testing, diagnostics, gene editing, synthetic biology, and therapeutic development. The ability to synthesize these molecules with high specificity and precision has revolutionized the study of nucleic acids and facilitated a broad range of innovations across multiple scientific disciplines. Typically ranging between 5 and 100 nucleotides in length, oligonucleotides serve as primers for PCR, molecular probes, antisense therapies, and components in gene assembly, underscoring their indispensable role in both experimental and applied molecular sciences.
As this article series unfolds, we’ll explore each method in detail—covering workflows, reagents, advantages, limitations, and real-world applications across biotech, pharma, and academic research. This first article will cover method 4.
Enzymatic synthesis
Enzymatic DNA synthesis is an emerging and exciting alternative to traditional chemical DNA synthesis methods like the phosphoramidite approach. This technique leverages template-independent DNA polymerases to directly synthesize DNA without requiring a pre-existing template strand. By mimicking the natural enzymatic processes used by living organisms to build DNA, this method offers several advantages over chemical synthesis, including the potential to produce longer, more complex sequences with higher accuracy and fewer errors.
Enzymatic DNA synthesis is still a developing technology, but it promises to overcome many limitations of traditional methods, particularly for large-scale applications in synthetic biology, genomics, and biopharmaceutical development.
Let’s dive deep into the mechanisms, advantages, and challenges of enzymatic DNA synthesis.
Key Components of Enzymatic DNA Synthesis
The central idea of enzymatic DNA synthesis is to use specialized DNA polymerases that do not require a template strand for DNA synthesis. These polymerases are capable of adding nucleotides to a growing DNA strand in a controlled manner, directed either by sequence-specific signals or by random addition, depending on the application.
Key components involved in this process include:
Template-Independent DNA Polymerases: Unlike traditional DNA polymerases that require a complementary template strand to guide DNA synthesis (like those used in PCR), template-independent polymerases such as Terminal deoxynucleotidyl transferase (TdT) or engineered polymerases are used in enzymatic DNA synthesis. These enzymes add nucleotides to the 3’-OH end of a DNA molecule without the need for base pairing with a template.
Nucleotide Triphosphates (dNTPs): As with all DNA synthesis, dNTPs (deoxynucleotide triphosphates: dATP, dCTP, dGTP, dTTP) are required as the building blocks of the new DNA strand. These nucleotides are added to the 3’-OH end of the growing DNA chain by the polymerase.
Controlled Nucleotide Addition: In order to synthesize specific DNA sequences rather than random sequences, methods have been developed to control the addition of specific nucleotides at each step. This can be achieved by controlling the availability of individual nucleotides or by using modified nucleotides that prevent uncontrolled elongation.
Modified Nucleotides: In many enzymatic synthesis approaches, chemically modified nucleotides are used to regulate the synthesis process. These modified nucleotides often have protecting groups that block further elongation until they are removed. This ensures that only one nucleotide is added at a time, allowing for precise control over the sequence.
Surface Attachment (Optional): Similar to chemical synthesis, enzymatic DNA synthesis can be performed on a solid surface, where the growing DNA strand is attached to a surface (e.g., a bead or microarray). This allows for the synthesis of many different DNA sequences in parallel.
Mechanism of Enzymatic DNA Synthesis
Enzymatic DNA synthesis can be broken down into two primary categories: random polymerization and sequence-controlled polymerization.
Random Polymerization (Using TdT)
One of the most well-characterized enzymes for template-independent DNA synthesis is Terminal deoxynucleotidyl transferase (TdT). This enzyme, which is naturally found in vertebrate immune systems, adds nucleotides to the 3' end of a single-stranded DNA (ssDNA) molecule without the need for a template. TdT plays a key role in generating diversity in the immune system by randomly adding nucleotides during the recombination of antibody genes.
In random polymerization:
No template is required: TdT can add any available nucleotide to the 3’-OH end of a growing DNA strand.
TdT has low sequence specificity, meaning that it adds nucleotides in a random order unless additional controls are imposed.
This randomness is useful in certain applications where random DNA sequences are needed, such as in the generation of DNA libraries for directed evolution or combinatorial chemistry. However, for more controlled synthesis of specific DNA sequences, other methods must be used to control the nucleotide addition process.
Sequence-Controlled Enzymatic DNA Synthesis
The primary challenge of enzymatic DNA synthesis is achieving sequence control—ensuring that nucleotides are added in the correct order to build a specific DNA sequence. Several approaches have been developed to address this, including:
Stepwise Controlled Synthesis (using TdT + Modified Nucleotides)
To gain precise control over the sequence of the DNA being synthesized, a technique known as stepwise synthesis can be used, which mimics the stepwise nature of phosphoramidite chemistry. Here’s how it works:
Initiation: The synthesis begins with a short DNA primer that has a free 3'-OH group. This primer serves as the starting point for DNA elongation.
Addition of a Modified Nucleotide: In each cycle, a single, chemically modified nucleotide (e.g., dA*, dT*, dC*, dG*) is introduced into the reaction. These nucleotides carry protecting groups on the 3’-OH that prevent further nucleotide addition. TdT will add this modified nucleotide to the 3’-OH end of the growing DNA chain.
Blocking Further Extension: After the modified nucleotide is added, the polymerase cannot add additional nucleotides because the 3’-OH group is chemically protected by the blocking group.
Deprotection Step: The blocking group is then chemically removed (typically using mild chemical or photolytic conditions), restoring the reactive 3’-OH group and allowing the next nucleotide to be added.
Repeat the Process: The process is repeated for each nucleotide, with one modified nucleotide being added in each cycle. By controlling which nucleotide is introduced in each cycle, a specific DNA sequence can be synthesized.
This approach is analogous to solid-phase chemical synthesis but uses enzymatic catalysis instead of chemical coupling. The advantage of using enzymes is that they can potentially reduce the error rate, allow for the synthesis of longer DNA sequences, and avoid some of the harsh chemical conditions used in phosphoramidite synthesis.
Enzymatic Oligonucleotide Assembly
Another approach to achieve sequence-specific synthesis is through the assembly of short oligonucleotides into longer DNA sequences using enzymes. This technique combines the precision of oligonucleotide synthesis with the efficiency of enzymatic assembly:
Short Oligonucleotide Synthesis: Short oligonucleotides (typically 10-50 nucleotides in length) are synthesized using conventional phosphoramidite chemistry.
Enzymatic Assembly: These oligonucleotides are then enzymatically ligated or extended to create longer DNA molecules. For instance, an enzyme like T4 DNA ligase can be used to ligate two adjacent oligonucleotides that have complementary overhangs, or a polymerase can fill in gaps between oligos to form continuous strands.
This method is often used in gene synthesis or genome assembly and is particularly advantageous for assembling very long sequences of DNA, such as entire genes or even synthetic chromosomes.
Advantages of Enzymatic DNA Synthesis
Enzymatic DNA synthesis offers several significant advantages over traditional chemical methods:
Potential for Longer Sequences:
In chemical DNA synthesis (e.g., phosphoramidite method), the yield and fidelity decrease as the sequence length increases. Errors accumulate with each nucleotide addition, making it challenging to synthesize DNA sequences longer than ~200 nucleotides with high accuracy.
Enzymatic synthesis, in contrast, mimics the natural processes used by cells to synthesize DNA. These processes are capable of producing very long DNA sequences, such as entire genomes, with much lower error rates.
Therefore, enzymatic methods could potentially allow the synthesis of DNA sequences that are thousands or even millions of base pairs long, surpassing the limitations of chemical synthesis.
Higher Fidelity and Fewer Errors:
DNA polymerases, especially those with proofreading abilities, are highly accurate enzymes. Enzymatic DNA synthesis could leverage high-fidelity enzymes to reduce the number of errors introduced during the synthesis process.
By using enzymes with proofreading activity, it may be possible to achieve significantly lower error rates compared to chemical methods, where the error rate is typically around 1 in 1000 bases.
Milder Conditions:
Traditional chemical synthesis requires harsh reagents and solvents, such as acetonitrile and trichloroacetic acid, which can be damaging to the environment and require specialized handling.
Enzymatic synthesis, on the other hand, typically occurs under milder conditions, using aqueous solutions at physiological pH. This reduces the environmental impact and may enable synthesis in more sensitive systems, such as inside living cells.
Scalability and Cost:
Enzymatic DNA synthesis is inherently more scalable because it does not rely on expensive chemical reagents and solid supports. Instead, it can be performed in solution, which reduces the cost of scaling up the synthesis.
The reduced need for purification and chemical handling also contributes to lowering the overall cost, making it a promising approach for large-scale applications like synthetic biology and industrial DNA production.
Challenges in Enzymatic DNA Synthesis
Despite its potential, there are several challenges that need to be addressed before enzymatic DNA synthesis can replace or complement traditional methods at scale:
Control of Nucleotide Addition:
One of the biggest challenges in enzymatic DNA synthesis is controlling the sequence specificity of nucleotide addition. Enzymes like TdT naturally add nucleotides in a non-template-directed manner, which makes it difficult to synthesize specific sequences without additional steps like protecting group chemistry.
Research is ongoing to develop engineered polymerases that can be programmed to add specific nucleotides in the correct order without the need for protecting groups.
Synthesis Fidelity:
While enzymatic DNA synthesis has the potential for high fidelity, the accuracy of nucleotide addition in the absence of a template is still a concern. Random errors or incorporation of the wrong nucleotide can occur, especially over longer sequences.
Ensuring fidelity in long, sequence-specific synthesis will require further refinement of enzyme engineering and reaction conditions.
Commercialization and Standardization:
Enzymatic DNA synthesis is still in its early stages of commercialization, and the technology is not as mature as traditional chemical synthesis methods. Developing robust, standardized platforms for high-throughput enzymatic DNA synthesis is a key challenge that must be addressed for broader adoption.
Applications of Enzymatic DNA Synthesis
The potential applications of enzymatic DNA synthesis span many fields, including:
Synthetic Biology:
Synthetic biology requires the ability to design and construct custom DNA sequences, such as synthetic genes, regulatory elements, and metabolic pathways. Enzymatic synthesis could enable the rapid and accurate synthesis of these elements, facilitating the design of complex biological systems.
The ability to synthesize very long DNA sequences without the limitations of traditional methods could enable the construction of synthetic organisms or artificial chromosomes.
Genome Editing:
Enzymatic DNA synthesis could be used to generate precise DNA sequences for use in genome editing techniques like CRISPR/Cas9. This would allow the rapid synthesis of guide RNAs (gRNAs) or donor templates for homology-directed repair (HDR).
Personalized Medicine:
In personalized medicine, where treatments are tailored to the genetic makeup of individual patients, enzymatic DNA synthesis could enable the rapid production of custom oligonucleotides, such as antisense oligonucleotides (ASOs) or gene therapy vectors.
Diagnostics:
Enzymatic synthesis could streamline the production of DNA probes and primers used in diagnostic assays, such as PCR or next-generation sequencing (NGS), making it faster and cheaper to produce the reagents needed for large-scale diagnostic testing.
Enzymatic DNA synthesis is an exciting and innovative technology that offers the potential for synthesizing longer, more complex DNA sequences with higher accuracy and fewer errors compared to traditional chemical methods. By harnessing template-independent polymerases and controlling nucleotide addition in a stepwise manner, enzymatic synthesis could overcome many of the limitations of current methods, paving the way for new applications in synthetic biology, genomics, personalized medicine, and beyond.
However, there are still significant challenges to overcome, particularly in controlling sequence fidelity and achieving efficient, scalable synthesis. As research progresses and the technology matures, enzymatic DNA synthesis could play a transformative role in the future of molecular biology and biotechnology.