Oligonucleotides are short chains of nucleic acids, typically 3 to 20 nucleotide units, which can be single- or double-stranded. They are built from the same components—nucleobases, sugars, and phosphate groups—as longer DNA and RNA molecules. While natural oligonucleotides exist, they are often synthetically created. These synthetic versions can be precisely designed and chemically altered to enhance their properties or give them new functions, making them versatile tools for biological research and medical applications.
Purpose of Chemical Modifications
Chemical modifications are introduced to oligonucleotides to overcome limitations within biological systems. A primary goal is to increase their stability, as unmodified oligonucleotides are rapidly degraded by enzymes called nucleases. By altering their chemical structure, modified oligonucleotides resist this enzymatic breakdown, allowing them to remain active for longer periods in a therapeutic setting. This enhanced stability translates into a longer half-life, making them more effective.
Modifications also aim to improve the oligonucleotide’s binding affinity and specificity to its target genetic sequence. Tighter, more precise binding ensures the oligonucleotide interacts only with its intended DNA or RNA, reducing unwanted off-target effects. This enhanced binding is achieved through structural changes or by introducing chemical groups that strengthen interactions with the target.
A further purpose involves enhancing cellular uptake. Oligonucleotides are relatively large and negatively charged, making it challenging for them to cross cell membranes. Certain modifications can increase hydrophobicity or facilitate interactions with cell surface proteins, helping them enter cells more efficiently. This improved delivery is a significant step towards their successful use in diagnostics and therapeutics.
Types of Structural Modifications
Modifying the core structure of an oligonucleotide involves alterations to its backbone, sugar, or nucleobase components.
Backbone Modifications
Backbone modifications commonly involve replacing an oxygen atom in the phosphate linkage with a sulfur atom, creating a phosphorothioate (PS) linkage. This imparts resistance to nuclease degradation, extending the oligonucleotide’s half-life. While enhancing stability and cellular uptake, PS linkages can introduce stereoisomers and may slightly reduce binding affinity.
Sugar Modifications
Sugar modifications focus on the ribose sugar. A common alteration is 2′-O-methylation (2′-O-Me), where a methyl group is added to the 2′ hydroxyl group. This stabilizes RNA structure, protects it from nuclease attacks, and increases the thermodynamic stability of RNA duplexes.
Another widely used sugar modification is the 2′-fluoro (2′-F) substitution, which increases binding affinity and nuclease resistance. Locked Nucleic Acids (LNAs) represent a distinct class where the ribose ring is “locked” into an RNA-mimicking conformation by an extra bridge. This significantly increases binding affinity towards complementary RNA, and offers improved specificity and resistance to enzymatic degradation.
Nucleobase Modifications
Changes to nucleobases can impact the base-pairing properties, potentially enhancing or modulating binding specificity. For example, a C-5 methyl substitution on pyrimidine nucleobases improves duplex stability and nuclease resistance. Such modifications can also mitigate unwanted immunostimulatory properties, improving the oligonucleotide’s drug-like characteristics.
Functional Group Attachments
Beyond altering the core structure of oligonucleotides, scientists can attach various functional groups to provide them with new capabilities.
Labels for Detection
One common type of attachment involves labels for detection, such as fluorescent dyes. These dyes, like FAM or Cy3, are covalently linked to the oligonucleotide, allowing it to emit light. This enables researchers to track the oligonucleotide’s location, detect its presence, or quantify target DNA or RNA sequences in applications like fluorescence in situ hybridization (FISH) or sequencing.
Quencher Molecules
Quencher molecules are another important functional attachment, often used with fluorescent dyes in diagnostic probes. A quencher absorbs the energy emitted by a nearby fluorophore, reducing or “quenching” its fluorescence. In applications such as quantitative PCR (qPCR), a probe might be designed with a dye and a quencher positioned close together. When the probe binds to its target and is cleaved, the dye and quencher are separated, leading to an increase in fluorescence that signals a positive result. “Dark quenchers,” like the Black Hole Quencher (BHQ) series, are particularly useful as they do not emit their own light, ensuring a low background signal.
Handles for Purification or Immobilization
Functional groups can also serve as “handles” for purification or immobilization. Biotin, a small molecule with a high affinity for avidin and streptavidin, is frequently attached to oligonucleotides. This biotinylation allows the oligonucleotide to be captured or isolated from complex mixtures by binding to streptavidin-coated surfaces or beads. Biotinylated oligonucleotides are widely used in affinity purification, detection assays like ELISA, and for immobilizing nucleic acids onto solid supports for microarray analysis.
Applications in Therapeutics and Diagnostics
Modified oligonucleotides have become foundational in both therapeutic and diagnostic fields.
Therapeutics
In therapeutics, antisense oligonucleotides (ASOs) are a prominent example, designed to bind to specific messenger RNA (mRNA) sequences to block the production of disease-causing proteins. For instance, nusinersen (Spinraza) is an ASO with phosphorothioate modifications used to treat spinal muscular atrophy (SMA). Other ASOs, like mipomersen, target specific mRNAs to reduce cholesterol levels.
RNA interference (RNAi) is another therapeutic approach that utilizes modified oligonucleotides, specifically small interfering RNAs (siRNAs). These double-stranded RNA molecules can trigger the degradation of target mRNA, effectively silencing gene expression. Patisiran, for example, is an FDA-approved siRNA therapeutic that targets transthyretin mRNA to treat hereditary transthyretin-mediated amyloidosis. SiRNAs are also being investigated for various other conditions.
Diagnostics
In diagnostics, modified oligonucleotides are indispensable tools for detecting and quantifying genetic material. Quantitative PCR (qPCR) assays, widely used for detecting viral or bacterial DNA/RNA (such as in COVID-19 tests), rely on fluorescently labeled oligonucleotide probes. These probes typically contain a fluorescent dye and a quencher, enabling real-time monitoring of DNA amplification by signaling when the target sequence is present.
Fluorescence In Situ Hybridization (FISH) is another diagnostic application. FISH uses fluorescently labeled DNA or RNA probes to visualize specific genetic sequences directly within cells or tissues, allowing for the detection of chromosomal abnormalities, gene amplifications, or pathogen presence. These applications demonstrate how chemical modifications transform simple nucleic acid strands into powerful agents for understanding and addressing human health challenges.