Oligos for Novel Molecular Research Beyond PCR

DNA was discovered over 150 years ago in 1869. However, the idea that DNA was responsible for inheritance of traits took a while to develop. By the mid-20^th century, scientists understood that DNA is the genetic material that encodes for protein and soon after, it was shown that DNA consists of four bases (A, T, C, and G) at 1:1 ratios. Rosalind Franklin found that the two strands of DNA form a helix and James Watson and Francis Crick found that the A-T and G-C base pairing are responsible for the annealing between strands. Advancements in understanding DNA and its role in genomes form the basis of early molecular biology. Scientists immediately recognized that the ability to synthesize DNA would be beneficial in studying basic biology.

Synthetic DNA generation became routine in the early 1980s with the combination of breakthrough discoveries [1]. First, phosphoramidite chemistry allowed for stepwise addition of nucleotides to generate a DNA strand. Secondly, solid phase chemistry allowed for automated DNA synthesis. These techniques are responsible for the routine availability of custom synthesized DNA in day-to-day research.

DNA is at the core of genetics for all organisms. The genomic DNA sequence defines normal biology and disease. Synthetic DNA oligonucleotides are essential to many experimental techniques that allow scientists to address biological questions and challenges.

Polymerase chain reaction (PCR) was commercialized in the late 80s and has since become a commonly used technique in molecular biology, both on its own and also within the broader workflow of many applications and experimental approaches. PCR utilizes synthetic DNA primers and a DNA polymerase enzyme to amplify DNA templates. Amplification occurs by temperature cycling, which causes a double-stranded DNA template to disassociate into single-strands that can anneal to the primers that are then substrates for the polymerase and subsequent elongation. PCR, and variations of it, can be used to detect and quantify DNA and RNA sequences of interest. In addition, PCR can also be used to amplify DNA for use in subsequent applications such as cloning, next generation sequencing, and epigenetics. PCR continues to be a workhorse for scientists and in recent decades more advanced applications of PCR have been developed with real-time and digital PCR technologies [2].

PCR showcases the utility of DNA’s physical attributes. DNA complementarity through Watson-Crick base pairing allows for sequence-specific binding and discrimination. Annealing and denaturing at different temperatures permit control of application-specific assay steps. DNA binding to enzymes (e.g., polymerases, ligases, transcriptase etc.) enables DNA extension, replication, recombination, gene editing, and more. Sequence-specific binding of oligos to other molecules enable many applications beyond PCR. DNA synthesis capabilities facilitate rational design of nucleic acid base modifications that allow selection of characteristics to control DNA-DNA or DNA-protein binding in order to develop new molecular techniques and cellular applications.

Reverse transcription (RT) is a method used to generate a DNA copy from an RNA template, such as mRNA or non-coding RNA. Within this method oligo dT or random primers are often used to enable first strand synthesis and ultimately produce a complementary DNA (cDNA) library. This cDNA library can then serve as input for RT-PCR, Sanger sequencing, or gene expression profiling applications.

Sanger sequencing is used in all types of experiments to determine the sequence of a specific DNA template. It uses a short DNA primer that anneals to the template adjacent to the region being studied. The process generates a mixture of DNA fragments which is run on an instrument that provides the specific sequence [3].

Determining the sequence of an RNA or DNA in a cell, tissue, cDNA clone, or clinical sample s fundamental to all molecular biology research. Sanger sequencing provides researchers with same-day determination of the exact sequence within their sample to inform their cloning, gene editing, or genotyping experiments. Success of the experiment depends on the sequence design and the quality of the DNA sequencing primer.

Next Generation Sequencing (NGS) is a high-throughput experimental approach that uses massively parallel sequencing [4]. Library preparation is the step where DNA or RNA is isolated from a biological or synthetic sample and is prepared for sequencing. The main goal is to maintain the complexity of the DNA and RNA sequence to make sure that we understand the intricate composition of the sample. Library preparation is performed routinely for targeted panels, whole genome sequencing (WGS), whole transcriptome analysis (WTA), total RNA, microRNA or small RNA sequencing.

NGS allows us to investigate the genome of an organism or the expression pattern of particular genes. This is commonly used to determine normal biological states as well as in evaluation of biological changes after a treatment. NGS can also be used to determine epigenetic methylation profiles of the genome. Because the NGS detection is so sensitive, primer sequence design and oligo purity are critical in NGS experimental design.

Genotyping is the investigation of differences in DNA sequence within a population. This genetic variation can take the form of single-nucleotide polymorphisms (SNP), insertion and deletion polymorphisms (indels), and copy number variants (CNV) [5]. These differences can be associated with disease and can become biomarkers for diagnostics. Sequencing approaches, such as Sanger and NGS described above, are primarily used for discovery of new targets or known variants for studying disease. Diagnostics leverage the specificity of primer and probe binding, often enhanced with chemical modifications, to detect these variants in experimental or clinical samples.

Gene editing in mammalian cells was revolutionized with the discovery of CRISPR systems. Many different CRISPR systems have been identified [6] but the most commonly utilized system is CRISPR-Cas9 from Streptococcus pyogenes[7]. Previously, genome engineering required extensive expertise, with CRISPR editing, any scientist can easily perform a gene knockout or knock-in experiment. This makes a wide range of applications accessible including generation of genetically engineered cell lines or organisms where genes are knocked out, mutated, or even new domains are introduced. Furthermore, changing genes in a cell has also demonstrated its tremendous potential in therapeutics [8].

Oligos feature prominently in the entire CRISPR workflow. Guide RNAs are needed to make double-stranded DNA breaks in the correct genomic location. Knock-in experiments require high-quality DNA donor oligos or longer custom DNA to ensure correct genomic alteration. Whether designing a research cell line for studying biology or manufacturing a cell line for gene therapy, precise analysis of genomic changes is essential [8]. The success of a genome engineering program relies on Sanger or NGS primers similar to what is applied in genotyping to detect specific genetic changes and unwanted off-targets resulting from CRISPR-mediated knockout and knock-in.

While biology is controlled by the primary DNA sequence, other genomic factors, including genome methylation and noncoding RNA, also play a role; this field of study is called epigenetics. Epigenetic changes in organisms’ genomes come about as a result of many environmental factors such as diet, stress-levels, and smoking. Interestingly, these changes can be passed on to progeny even though they do not alter the genomic sequence itself. This emerging field is describing novel mechanisms that influence organism biology. The two primary epigenetic mechanisms are DNA methylation, which silences transcription of particular genes, and non-coding RNA, which regulates gene expression [9]. Various epigenetic sequencing methods, including methylated DNA immunoprecipitation and bisulfite sequencing, have been identified to study methylation state of the genome. The same NGS primer requirements identified above also apply here for successful experimentation.

Oligos delivered into cells can influence a cell’s biological function. Antisense oligos (ASOs) can affect the expression of a target messenger RNA or non-coding RNA. Not only are ASOs useful for studying disease but can also be developed into therapeutics [10]. Another type of functional oligo is an aptamer; a single-stranded modified RNA that can inhibit proteins by binding to them [11] providing another mechanism for biological study and therapeutics.

ASOs and aptamers need to have improved binding and nuclease resistance characteristics to remain biologically active in the cells. To achieve this, these types of oligos can have mixed DNA and RNA bases, phosphate backbone modifications (phosphorothioates, LNA), ribose modifications (2’Ome, 2’F) in addition to 5’ and 3’ modifications.

Access to synthetic DNA oligonucleotides, including capabilities for reagent design through sequence control and the addition of specific chemical modifications, facilitates early research and development. In addition, Thermo Fisher Scientific oligonucleotide manufacturing provides options for custom post-synthetic processing and purification for specific application needs.

As research programs mature, different oligo purity levels can be matched to experimental approaches depending on the diagnostic or therapeutic need. Specifications can be controlled by the removal of contaminants such as salts or truncated products to ensure full-length DNA for fidelity in amplification or sequencing applications.

Thermo Fisher Scientific provides oligonucleotides in either tube or plate formats to facilitate efficiency of both standard and high-throughput experiments:

• Standard tube oligos are provided for lower throughput approaches where oligonucleotides in tubes enable flexible and complex experimental design.
• Oligonucleotides in 96- or 384-well plates are provided for high-throughput screening approaches such as sequence optimization and target discovery.
• Complex and large-scale oligos are provided to support diagnostic and therapeutic research programs where larger yields, custom purification, and formulation may be necessary.
• Thermo Fisher Scientific manufacturing adheres to standards described in ISO 9001 and ISO 13485 certification.
• Thermo Fisher Scientific engages through an allyship model to provide tailored service and support for unique and complex needs, including raw and oligo materials for commercial kits, nucleic acid therapeutics research, and clinical molecular diagnostics research.

Synthetic oligonucleotides are fundamental components used in research, commercialization of molecular tools, diagnostics, and therapeutics. DNA primers and probes have demonstrated tremendous utility in PCR applications, but also serve an underlying element within NGS, epigenetic, CRISPR, and yet-to-be-discovered applications. Functional oligos provide powerful research tools and cutting-edge therapeutics.

Thermo Fisher Scientific provides resources and tools to support customer needs from research through development by enabling efficient ordering and delivery of small-scale, custom DNA to facilitate rapid optimization and experimentation. In addition, dedicated support is available to assist with commercial oligonucleotide requirements for kits, therapeutics, and clinical diagnostics. These collaborations with researchers can tailor service and support to assist with customization, including large-scale synthesis and manufacturing with appropriate quality certification. Thermo Fisher Scientific oligonucleotide synthesis capabilities facilitate rapid completion of projects from research to commercialization.

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