Modified Oligonucleotides in Drug Development: What You Need to Know?

The Importance of Chemical Modifications

Backbone, Sugar, and Base Modifications

Chemical modifications at the backbone, sugar, and base levels are pivotal to transforming oligonucleotides into drug-like entities. Without such enhancements, unmodified oligos suffer from poor biological stability, rapid degradation by nucleases, and limited pharmacokinetic profiles.

Illustration showing a modified oligonucleotide strand and its key roles in drug development, including targeting, therapeutic effects, and delivery optimization. (BOC Sciences Original)

• Oligo Backbone Modifications

The phosphorothioate (PS) backbone is one of the most extensively applied modifications. By replacing a non-bridging oxygen atom with sulfur in the phosphate group, PS linkages significantly enhance nuclease resistance while facilitating plasma protein binding, thereby improving half-life and systemic exposure. However, stereoisomeric complexity can influence off-target interactions and must be managed during development.

• Oligo Sugar Modifications

Modifications at the 2'-position of the ribose sugar, such as 2'-O-methyl (2'-OMe) and 2'-fluoro (2'-F), improve hybridization affinity to complementary RNA while providing enzymatic stability. These substitutions can modulate RNA-binding strength and reduce immune stimulation, supporting safer therapeutic profiles.

• Oligo Base Modifications

Altering the nucleobase can enhance sequence specificity or provide functional versatility. Locked Nucleic Acids (LNAs), for example, modify both the sugar and base conformation, increasing thermal stability (Tm) of duplex formation without compromising specificity.

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Impact on Efficacy, Stability, and Toxicity

The triad of efficacy, stability, and toxicity dictates the success of any oligonucleotide-based therapeutic. Chemical modifications serve as fine-tuning tools to balance these interdependent attributes:

• Efficacy Enhancements: Modified oligonucleotides demonstrate improved target affinity and increased resistance to off-target degradation, directly boosting functional potency. For example, LNAs raise the melting temperature (Tm) of oligo-RNA duplexes by 2–8 °C per modification, enabling shorter, more potent sequences. Strategic placement of modifications—such as flanking 2'-OMe groups around an unmodified DNA core in a gapmer—supports both effective RNase H recruitment and minimized immune activation.
• Stability Improvement: Backbone and sugar modifications significantly enhance serum and intracellular stability. Phosphorothioate linkages and 2'-modifications prevent endonuclease and exonuclease digestion, extending circulation half-life from minutes to hours or even days. Such stability is critical for systemic delivery, especially in liver- or muscle-targeted indications.
• Toxicity Modulation: While modifications improve performance, they can also introduce unwanted effects. PS linkages, for instance, may non-specifically bind proteins such as complement factors or albumin, potentially eliciting immunostimulation or hepatotoxicity. Sequence composition, stereochemistry (e.g., stereopure vs. stereorandom PS), and overall oligonucleotide architecture (e.g., wing–gap–wing design) are all essential parameters to mitigate dose-limiting toxicities. Preclinical in vivo models are indispensable for defining the safety window of each modified scaffold.

By carefully integrating these considerations, developers can engineer oligonucleotide drugs that are not only functionally potent but also clinically translatable.

Common Modifications Used in Therapeutics

Chemical modifications are no longer optional in therapeutic oligonucleotide design—they are foundational. Each class of modification confers distinct functional advantages, whether it be enhanced target affinity, metabolic stability, or delivery capability. The strategic integration of multiple modifications often defines a candidate's clinical success potential.

Phosphorothioate, 2'-OMe, 2'-F

• Phosphorothioate (PS) Backbone

The most frequently used modification in antisense oligonucleotides (ASOs), PS linkages confer nuclease resistance while allowing interaction with serum proteins. This extends plasma half-life and facilitates systemic exposure. Despite their benefits, PS linkages may increase non-specific protein binding and must be optimized to balance efficacy and tolerability. Stereopure PS oligonucleotides—where the chirality of each phosphorus center is defined—are emerging to further refine pharmacological precision.

• 2'-O-Methyl (2'-OMe)

This ribose modification enhances both nuclease resistance and RNA affinity. Often employed in the flanking wings of gapmers or in the passenger strand of siRNAs, 2'-OMe reduces immunogenicity by suppressing Toll-like receptor activation. This is particularly relevant in systemic applications where immune activation can compromise therapeutic outcomes.

• 2'-Fluoro (2'-F)

2'-F modifications are common in siRNAs and short interfering oligos. They stabilize the sugar ring, increase duplex formation, and improve resistance to hydrolytic cleavage. The 2'-F group also influences helical geometry, aiding RNA-induced silencing complex (RISC) loading and activity in RNAi applications.

Together, these three modifications represent a core modification toolkit in first- and second-generation oligonucleotide therapeutics, with multiple FDA-approved drugs incorporating them in various configurations.

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LNA, PNA, and Morpholino

• Locked Nucleic Acid (LNA)

LNAs are conformationally restricted nucleotides that lock the ribose in a C3'-endo conformation. This enhances base stacking, affinity, and thermal stability. LNAs are routinely applied to increase potency and specificity, particularly in short ASOs targeting pre-mRNA splicing or microRNAs. The inclusion of LNA nucleotides can also reduce the risk of cross-reactivity in closely homologous sequences.

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• Peptide Nucleic Acid (PNA)

PNAs substitute the sugar-phosphate backbone with a neutral peptide-like backbone, resulting in strong hybridization and complete nuclease resistance. Though their neutrality impairs passive uptake, PNAs are valuable in ex vivo and localized delivery contexts, and increasingly in multiplexed molecular diagnostics or gene editing applications.

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• Morpholino Oligomers

Widely known from the FDA-approved drug Eteplirsen, morpholinos replace the ribose-phosphate backbone with morpholine rings and phosphorodiamidate linkages. These neutral molecules are not recognized by nucleases, making them ideal for chronic indications requiring high metabolic stability. However, their lack of charge necessitates delivery via specialized carriers such as peptide conjugates or nanoparticles.

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Conjugated Peptides, Lipids, or Antibodies

Conjugation strategies aim to enhance delivery, cellular uptake, or tissue specificity—critical for therapeutic index optimization.

• Peptide Conjugation

Attaching cell-penetrating peptides (CPPs) such as TAT, penetratin, or arginine-rich domains improves membrane translocation and nuclear localization. Peptide–oligonucleotide conjugates (POCs) are being intensively studied for neuromuscular and CNS-related targets.

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• Lipid Conjugation

Lipophilic modifications like cholesterol or tocopherol enable hepatocyte uptake through receptor-mediated endocytosis. This approach is exemplified by Inclisiran, a GalNAc-conjugated siRNA targeting PCSK9, which utilizes hepatocyte-specific asialoglycoprotein receptors (ASGPR) for targeted RNAi delivery.

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• Antibody-Oligonucleotide Conjugates (AOCs)

AOCs represent a frontier class of therapeutics combining the specificity of monoclonal antibodies with the gene-silencing capability of oligonucleotides. By conjugating ASOs or siRNAs to antibodies via cleavable linkers, these constructs achieve tissue-specific delivery while preserving oligo bioactivity. BOC Sciences provides end-to-end services for AOC prototype development, including linker design, conjugation chemistry, and oligo characterization.

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As these modification strategies evolve, combinatorial approaches—such as LNA-enhanced, peptide-conjugated, PS-modified oligonucleotides—are becoming the norm. BOC Sciences leverages a comprehensive portfolio of these chemistries to support next-generation therapeutic programs across a wide range of indications.

Custom Synthesis of Modified Oligonucleotides

Broad Modification Portfolio

BOC Sciences offers a robust and versatile platform for the synthesis of chemically modified oligonucleotides tailored to specific therapeutic and research applications. Our portfolio encompasses a full spectrum of structural and functional modifications:

• Backbone: Phosphorothioate (PS), phosphorodiamidate morpholino, methylphosphonate, boranophosphate
• Sugar: 2′-O-methyl (2'-OMe), 2′-fluoro (2'-F), 2′-O-methoxyethyl (2'-MOE), Locked Nucleic Acid (LNA), Unlocked Nucleic Acid (UNA)
• Base: 5-methylcytosine, 2,6-diaminopurine, pseudouridine, inosine
• Terminal & Internal Modifications: Biotin, fluorescein, quenchers, PEGylation, cholesterol, thiol, amino, azide
• Conjugates: Cell-penetrating peptides (CPPs), GalNAc clusters, lipids, antibodies (via cleavable/non-cleavable linkers)

Our chemists provide in-depth design consultation to recommend optimal modification combinations based on sequence context, target tissue, and intended mechanism of action (e.g., RNase H-mediated cleavage vs. steric blocking).

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Scalability and Analytical Validation

From microgram to multi-gram synthesis scales, BOC Sciences maintains full process control with stringent QC systems to ensure batch consistency, purity, and performance:

• Scalable Synthesis: Automated solid-phase synthesis with capability to support milligram (discovery), gram (lead optimization), and tens of grams (IND-enabling) quantities
• Purification Technologies: Reverse-phase and ion-exchange HPLC, gel filtration, and affinity-based purification tailored to specific conjugates or impurity profiles
• Analytical Capabilities:

(1) Identity Confirmation: UPLC, MALDI-TOF MS, and ESI-MS

(2) Purity and Impurity Profiling: HPLC (A260, A280), capillary electrophoresis

(3) Structural Integrity: Duplex formation and Tm analysis

(4) Stability Testing: Serum stability assays, forced degradation studies

(5) Endotoxin and Residual Solvent Testing: In compliance with ICH Q3C and Q3D guidelines

Each oligonucleotide is delivered with a complete Certificate of Analysis (CoA) and is subjected to customizable documentation packages, including raw data files, formulation protocols, and extended stability reports upon request.

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Support for Exploratory to IND Phases

BOC Sciences functions as a critical partner to biotech and pharma clients across all stages of the preclinical pipeline—from early discovery to IND submission. Our support services are engineered to reduce development time and de-risk regulatory submissions:

Exploratory Research

• Rapid synthesis of variant libraries for SAR studies
• Support for antisense, siRNA, aptamer, miRNA mimic/inhibitor screening

Lead Optimization

• Sequence-specific modification design
• Comparative studies on toxicity, binding kinetics, and uptake

Preclinical Development (IND-Enabling)

• Scale-up synthesis under GMP-like documentation
• Release testing aligned with regulatory expectations
• Customizable reference standards and stability batches
• Technology transfer and method validation support for CDMO integration

All processes are conducted in ISO-certified facilities with rigorous documentation practices to support smooth tech transfer and pre-IND communications. With our deep expertise in nucleic acid chemistry and platform flexibility, BOC Sciences is uniquely positioned to advance your oligonucleotide drug from bench to regulated studies with precision and speed.

Related Resources

New Research on RNA Methylation Modifications (m6A)

Modified Nucleotides in mRNA Therapeutics: Types and Applications

Modified Oligonucleotides in Drug Development: What You Need to Know?

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