DNA serves as the carrier of genetic information, playing an indispensable role in basic biology, biomaterials science, and synthetic biology. Modern biological research and engineering heavily rely on the synthesis of customized DNA sequences, encompassing oligonucleotides, synthetic genes, and even entire chromosomes. Breakthroughs in large-scale, cost-effective, and efficient construction of DNA sequences propel advancements in biological research and applications. Today, the complete reconstruction of viral and bacterial genomes validates our synthetic capabilities. While scalable methods exist for double-stranded DNA (dsDNA) production, equally effective single-stranded DNA (ssDNA) synthesis is crucial for specific applications. ssDNA synthesis has become a driving force in DNA-based biomaterials, widely applied in DNA nanotechnology, CRISPR systems, drug delivery, and more. Various synthesis methods based on chemical, enzymatic, and bacteria-based principles have been developed to meet diverse application needs, emphasizing the practical significance of choosing the appropriate synthesis method for different directions.
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Chemical Synthesis Methods of ssDNA
Chemical synthesis is a key method for producing ssDNA fragments, particularly those under 200 nucleotides (nt). Two main approaches are column-based oligo synthesis and array-based oligo synthesis.
Column-Based Oligo Synthesis
Initiated in the 1950s, de-novo synthesis of oligos using phosphodiesters advanced significantly in the early 1980s with the advent of solid-phase phosphoramidite approaches. This method, executed in cyclical steps, employs standard phosphoramidite chemistry involving deprotection, coupling, optional capping, and oxidation. Column-based synthesis is well-suited for automated synthesizers, producing oligos at scales from 10 to 100 nmol. However, efficiency decreases beyond a certain length due to yield reduction and error accumulation.
Fig. 1 Phosphoramidite-based oligonucleotide synthesis (Hao M., et al. 2020).
Array-Based Oligo Synthesis
Affymetrix introduced microarray oligonucleotide synthesis in 1990, pioneering spatially located synthesis using photoactivation-based chemical methods. Array-based platforms, a cost-effective alternative, utilize inkjet printing or chip synthesis technology for massively parallel synthesis. While cost-efficient, array-based synthesis faces challenges, such as lower product yields compared to column-based synthesis.
Enzymatic Synthesis Methods of ssDNA
Enzymatic synthesis stands out as a cost-effective, rapid, and stable approach for generating ssDNA, offering specificity and mild conditions conducive to the direct synthesis of longer oligos. With numerous biotechnology applications requiring efficient synthesis of ssDNA fragments ranging from hundreds to over 10kb, several enzymatic methods have been developed, addressing specific challenges in constructing long ssDNA fragments of individual genes.
Terminal Deoxynucleotidyl Transferase (TdT)
One method employs TdT, a polymerase that adds deoxynucleotide triphosphates (dNTPs) to the 3' end of ssDNA. TdT exhibits low substrate specificity, allowing compatibility with various modified nucleotides and easy purification. Challenges include controlling the addition of single bases, limiting the overall length, and finding suitable solid supports. Despite these challenges, TdT has shown promise in signal amplification, single-nucleotide modifications, polymerization of building blocks, and DNA information storage.
Transcription and Reverse Transcription (ivTRT)
In vitro ivTRT involves three steps: preparing dsDNA templates, transcribing RNA from dsDNA, and preparing ssDNA from RNA. Although labor-intensive and expensive, ivTRT can synthesize ssDNAs of various lengths (approximately 0.5~2kb), suitable for gene editing. Challenges include nuclease limitations and the requirement for high-quality DNA.
Asymmetric Polymerase Chain Reaction (aPCR)
aPCR provides an effective method for on-demand labeling and ssDNA production. Involving unequal concentrations of two amplification primers, aPCR produces ssDNA through exponential and linear amplification phases. Challenges include nonspecific amplification, requiring extensive optimization for desired ssDNA yield. Despite challenges, aPCR has been applied in the synthesis of short ssDNAs and DNA origami scaffolds, demonstrating potential for various applications.
Isothermal Amplification of ssDNA
Isothermal amplification techniques circumvent thermal denaturation, relying on enzyme activity or designed primers. Primer exchange reaction (PER) and rolling circle amplification (RCA) are noteworthy methods. PER offers programmable synthesis but is limited to 60nt, while RCA synthesizes long stretches of repeating ssDNA sequences. Although RCA is simple and robust, challenges include low efficiency and a relatively long reaction time.
Separation of ssDNA from dsDNA
Enzymatic or chemical approaches for denaturing dsDNA to form ssDNA provide an alternative strategy, often requiring purification steps. Separation methods include biotin-streptavidin-based interaction, lambda exonuclease digestion, and denaturing urea polyacrylamide gel electrophoresis. Each method has advantages and challenges, influencing their efficiency in generating high-quality ssDNA.
Bacteria-Based ssDNA Synthesis
Bacteria-based platforms for ssDNA synthesis yield milligrams in shake flasks, scalable through bioreactors. Utilizing bacteriophages with fast-growing E. coli, ssDNA is assembled into virions without host cell lysis. Phagemids, accommodating custom inserts, enhance production but present limitations. Phagemid libraries ease workability, producing purer ssDNA for nanotechnology. Despite mature techniques, challenges include helper phage control and infection timing. Retrons, unique genomic sequences, employ reverse transcriptase for ssDNA/RNA hybrid production, enabling precise mutations. Additionally, a Gram-positive bacteria replicating plasmid, pC194, offers a novel platform for circular ssDNA synthesis in E. coli, showcasing versatility in length and sequence.
In conclusion, the synthesis of ssDNA is pivotal for advancing genetic research and engineering applications. Chemical methods, such as column-based and array-based synthesis, offer distinct advantages and challenges. Enzymatic approaches, including Terminal Deoxynucleotidyl Transferase, transcription, reverse transcription, asymmetric polymerase chain reaction, and isothermal amplification, address the need for efficient and specific ssDNA production. Bacteria-based platforms, leveraging phagemids, retrons, and replicating plasmids, demonstrate versatility, scalability, and unique capabilities in ssDNA synthesis. Despite challenges, ongoing innovations in these methodologies hold promise for shaping the future of synthetic biology, nanotechnology, and biological research.
References
- Hao M., et al. Current and emerging methods for the synthesis of single-stranded DNA. Genes. 2020, 11(2): 116.
- Minev D., et al. Rapid in vitro production of single-stranded DNA. Nucleic Acids Research. 2019, 47(22): 11956-11962.
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