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Oligonucleotides (ONs) such as antisense oligonucleotides (ASOs), small interfering RNA (siRNA) and aptamer can achieve the purpose of treating diseases by targeting pathogenic RNA or protein and inhibiting its activity, but many types of oligonucleotides have such disadvantages as poor active targeting, instability in vivo and poor transmembrane ability, so on the basis of traditional oligonucleotides, the functions and properties of nucleic acid molecules are improved by covalently coupling oligonucleotides with other functional molecules, e.g., improving cell uptake, tissue delivery, bioavailability, catalytic effect, etc., and thereby improving the effect of therapeutic nucleic acid. The poor cell or tissue-specific delivery of ONs is being addressed by conjugating (covalent attachment by chemical and biological methods) natural or modified ONs to molecules like antibodies or their fragments, liposomal components, saccharides, hormones, proteins and peptides, toxins, fluorophores or photoprobes, inhibitors, enzymes, growth factors and vitamins. The conjugation technology has become a core technology in the biomedical field by precisely combining short-stranded DNA or RNA molecules with other functional molecules, such as targeting ligands, fluorescent moieties, or nanocarriers, to endow them with enhanced stability and multifunctionality. In therapy, this technology can achieve targeted delivery of siRNA, mRNA and other drugs through GalNAc ligands or antibody coupling, which significantly improves therapeutic efficacy and reduces off-target effects, and has been successfully applied in the treatment of genetic diseases and cancers; in diagnostics, fluorescently or radioactively labeled coupled probes can detect pathogenic nucleic acids or tumor markers with high sensitivity, which promotes molecular imaging and liquid biopsy; and in basic research, it serves as a tool to enable CNA and other functional molecules (e.g., fluorescent moieties or nanocarriers) to be precisely coupled with other functional molecules. In basic research, it is used as a tool to optimize the CRISPR system, track single-molecule dynamics and construct artificial biological circuits, deepening the understanding of gene regulation and cell behavior. Through interdisciplinary integration, this technology continues to break through the boundaries of nucleic acid applications, providing a core driving force for precision medicine and life science innovation.
ONs are conjugated mostly at 5'- or 3'-termini because of their easy accessibility, however the 2'-positions of ribose sugar, nucleobases and internucleotidic phosphodiester bonds are also used for conjugation. It has been suggested that conjugation through the 3'-terminus enhances the exonuclease resistance. Modifications for nucleobases have to be carefully chosen so that the hybridization properties (binding with the target sequence) are not compromised. The 2'-OH modification has been found particularly useful in antisense strategy; the ribose conformation is essentially RNA-like (sugar conformation is C3'-endo) and therefore enhances the affinity towards the target RNA. ON conjugation is challenging because subtle changes in structure may influence its biological properties, and therefore, the specific sites for conjugation should be carefully determined. The chemical characteristics of the ON and the reporter molecule to be anchored may not be compatible, and the two entities may show different stabilities during the conjugation reaction. The conjugation sites may be difficult to access while conjugating complex molecules to the ON. Several synthetic approaches have been developed for facile preparation of ON-conjugates, which can be grouped into two major categories: (1) on-support conjugation, where conjugation is achieved using support-bound ONs; and (2) solution-phase conjugation, which involves coupling of target molecule to ONs in solution.
During ON-conjugates, the first step is to rationally select the modifying groups (e.g., amino, thiol, or azide groups) according to the target application, and the chemical properties of these groups directly determine the feasibility of the conjugation strategy: the amino group can be stabilized by carboxylate coupling mediated by carbodiimide (e.g., EDC/NHS), while thiol groups are suitable for sulfhydryl-specific reactions such as maleimide or pyridine disulfide bonding, and the azide group can be efficiently conjugated to alkynes via click chemistry (e.g., CuAAC or SPAAC) to achieve bioorthogonal labeling. Second, the type of linker chemistry needs to match the functional requirements - non-cleavable linkers (e.g., stable amide or thioether bonds) are suitable for scenarios where the integrity of the coupler needs to be maintained over a long period of time (e.g., fluorescent probes or nanoparticle labeling), whereas cleavable linkers (e.g., acid-sensitive hydrazone or reduction-sensitive disulfide bonds) are commonly used in drug delivery systems to ensure controlled release of oligonucleotides within target cells. In addition, optimization of coupling efficiency requires strict control of the reaction conditions: the purity of the oligonucleotide and the coupling molecule directly affects the probability of side reactions; the pH of the reaction system needs to be adapted to the formation of specific chemical bonds (e.g., EDC-mediated coupling is usually carried out in weakly acidic conditions); and the temperature is needed to balance the reaction rate with the stability of the molecule (e.g., click chemistry often requires low temperatures to avoid catalyst inactivation). For example, in the construction of targeted siRNA couplers, the binding of azide-modified oligonucleotides to alkyne-functionalized GalNAc ligands via copper-free click chemistry (SPAAC) needs to be carried out at a physiological pH and at a medium temperature (25-37°C) in order to ensure a high coupling efficiency while maintaining the stability of the RNA secondary structure.
Some popular methods of oligonucleotide conjugation are described below:
The on-support conjugation can be achieved by the following methods: (i) incorporation of modified phosphoramidite during the automated ON synthesis; (ii) step-wise solid-phase conjugate synthesis; and (iii) on-support fragment conjugation.
Incorporation of modified phosphoramidites: This strategy involves the synthesis of nucleosidic or non-nucleosidic molecules followed by appropriate protection and phosphitylation. The modified phosphoramidite or H-phosphonate derivative is then incorporated into the ON chain by automated solid-phase synthesis. The main advantage of using this approach is that vast repertoire of modified "ready-to-use" phosphoramidite building blocks are commercially available. The disadvantage associated with this approach is that the conjugated molecule needs to be stable during the automated DNA synthesis and deprotection step. Conjugate instability during the deprotection procedure can be avoided by using protecting groups that are de-blocked under milder conditions. For instance, phenoxyacetyl group is used instead of benzoyl group for base protection because this is deprotected by ammonium hydroxide/potassium carbonate in methanol at room temperature. The other drawback is that reasonably large-scale and multistep synthesis of modified derivative is needed unless it is commercially available. Conjugation by this procedure can be useful only with target molecules that are chemically robust and could be obtained by simple synthetic procedure.
Step-wise solid-phase conjugate synthesis: The step-wise solid-phase or in line synthesis involves the complete assembly of ON fragment and the target molecule on same solid support. The ON fragment can be synthesised first followed by the target molecule or vice versa. It is also possible to incorporate a branched linker to achieve independent assembly of the two fragments. The step-wise synthesis based approaches are used for the preparation of conjugates with complex target molecules mainly peptides and carbohydrates. The advantage of using this approach is undoubtedly the ease of conjugate purification. Such approaches would be indispensable for synthesising combinatorial libraries of conjugates. Theses approaches have however very stringent requirements. The protecting groups used for the reactive functions present on the target molecule must be stable to conditions employed for ON synthesis. Removal of these protecting groups should not affect other protecting and/or anchoring group present on the ON. In addition, conjugates prepared should remain stable under the deprotection conditions. The method could be particularly cumbersome when molecules with incompatible chemistries are being conjugated such as ON and peptide. Current research efforts are directed towards the development of procedures for in-line preparation of conjugates but no single robust procedure has been developed so far in spite of several promising developments.
On-support fragment conjugation: This approach is slightly different from the in-line approach of ON conjugation. Herein, the ON's are assembled on solid-support and reactive functional moiety is introduced during the last coupling step. The protecting group blocking the reactive moiety is removed but the ON is kept protected and support bound. The target molecule modified with complementary reactive functional moiety in solution is then coupled to the support bound ON. The ON conjugates are obtained after subsequent cleavage from the support, nucleobases deprotection and purification.
Solution-phase oligonucleotide conjugation proceeds via the following reactions: (i) Conjugations via Amide or Thiourea Linkage; (ii) Conjugation via reductive amination, hydrazone or oxime linkage; (iii) Conjugation via disulfide, thioether, thiazolidine, native or metal-thiol linkage; (iv) Conjugation via cycloaddition reactions; (v) Conjugation via staudinger ligation.
ON-conjugates exhibit multidimensional applications in the biomedical field due to their flexible and functionalized design. In the therapeutic field, the delivery of antisense oligonucleotides (ASOs) can be significantly enhanced by coupling targeted ligands (e.g., GalNAc, antibodies, or cholesterol), e.g., GalNAc-ASO conjugates can achieve efficient hepatic targeting by binding to the hepatic cell surface of the desialic acid glycoprotein receptor (ASGPR), reducing systemic exposure and enhancing gene silencing efficiency; similarly, the targeting of siRNAs Similarly, the targeting of siRNA/miRNA can be further optimized by coupling aptamers or antibodies, e.g., antibody-siRNA conjugates can accurately recognize cancer cell surface antigens, promote intracellular uptake, and enhance the effect of RNA interference (e.g., Patisiran for hereditary transthyretin amyloidosis). In the diagnostic field, fluorescently labeled oligonucleotide probes (e.g., FAM or Cy5 conjugates) are widely used in real-time quantitative PCR or fluorescence in situ hybridization (FISH), which can be used to aid in pathogen detection or molecular typing of cancers by emitting detectable signals through specific binding to the target sequence. In addition, oligonucleotide conjugation technology provides new ideas for constructing smart targeted drug delivery systems: for example, coupling DNA aptamers with chemotherapeutic drugs (e.g., adriamycin), utilizing the aptamer's high affinity for tumor markers to achieve precise drug delivery, and at the same time controlling the drug release through acid-responsive or enzyme-sensitive linkers, thus reducing off-target toxicity. These applications not only highlight the versatility of oligonucleotide conjugates, but also provide the technological cornerstone for personalized medicine and precision diagnosis.
The future of oligonucleotide conjugation technology will focus on the innovative direction of precision, efficiency and multifunctionality. At the technical level, breakthroughs in novel bio-orthogonal conjugation technologies will push the coupling reaction in the direction of gentler and more efficient, such as the use of light-activated bio-orthogonal reactions to achieve spatio-temporally controllable in vivo labeling and reduce interference with biological systems. Meanwhile, advances in site-specific conjugation technologies will overcome the limitations of traditional random modifications and ensure that the key functional domains of oligonucleotides are protected from coupling interference, thus maximizing their biological activities. In the therapeutic field, the emergence of new application scenarios will reshape the mode of disease intervention: for example, efficient crossing of solid tumors or the blood-brain barrier by siRNA through coupling cell-penetrating peptide (CPP) or exosome-targeted ligands; development of dual-functional couplers to achieve gene silencing and protein degradation at the same time; or the use of precise coupling of CRISPR-Cas nuclease and delivery vectors to enhance the targeting and safety of gene-editing targeting and safety of gene editing tools. The convergence of these trends will accelerate the translation of oligonucleotide conjugation technology from the laboratory to the clinic, and open up new pathways for the precision treatment of cancer, neurodegenerative diseases and rare genetic diseases.
This paper provides a brief introduction to the preparation, application and future trends of ON-conjugates. ON-conjugates technology is an important innovative drug design strategy to expand therapeutic areas, improve drug efficacy and reduce adverse effects. After coupling oligonucleotides with antibodies, small molecule drugs, lipophilic molecules, and peptides with different functions, the functions and properties of nucleic acid molecules will be improved, such as cellular uptake, tissue delivery effect, bioavailability, and catalytic effect. However, the research and application of oligonucleotide couplers still face many challenges and there are many unknown areas to be explored. Through continuous and in-depth research and exploration, it is believed that ON-conjugates will bring new hopes and possibilities for personalized therapy and precision medicine in the future, and make greater contributions to the cause of human health.
How to overcome the problems of biostability and immunogenicity of oligonucleotides, how to realize the precise targeting and intracellular release of oligonucleotides, as well as how to reduce the cost of oligonucleotide production and improve the efficiency of its preparation are all problems to be solved. Through continuous research and exploration, we believe that ON-conjugates will bring new hopes and possibilities for personalized therapy and precision medicine in the future, and make greater contributions to the cause of human health.
Purification of oligonucleotide couplers is usually based on their physicochemical properties (e.g., molecular weight, charge, hydrophobicity) or functional group differences, and a combination of methods is used to achieve separation of high-purity products. High-performance liquid chromatography (HPLC) is the most commonly used means, reversed-phase chromatography (RP-HPLC) separates small-molecule couplers by hydrophobic interaction, while ion-exchange chromatography is suitable for separating negatively charged oligonucleotides from neutral impurities; gel electrophoresis is particularly suitable for preliminary purification of macromolecules by distinguishing couplers from unreacted chains through molecular weight differences. For nanoparticles or liposome conjugates, size exclusion chromatography (SEC) gently removes small-molecule byproducts, while affinity chromatography utilizes specific binding for targeted purification. In addition, ethanol precipitation or ultrafiltration are often used for rapid removal of salts or small molecule reagents, followed by mass spectrometry (to confirm molecular weight) and UV spectroscopy (to verify coupling efficiency) to ensure product purity.
At the forefront of oligonucleotide innovation, we offer a comprehensive range of high-purity raw materials-including modified oligonucleotides, linkers, and conjugation-ready reagents-tailored for therapeutic and diagnostic development. Our custom conjugation services are designed to meet diverse research needs, ensuring precision, scalability, and regulatory support.
Explore our oligonucleotide conjugation materials and services today, or contact us for a custom solution.
BOC Sciences provides a full spectrum of high-purity raw materials essential for precision oligonucleotide synthesis. These reagents are quality-assured, stringently tested, and optimized for both DNA and RNA chemistries.
Cap analogs are synthetic structures that mimic the 5' cap of eukaryotic mRNAs, critical for enhancing translation efficiency and stability in synthetic RNAs. We supply m⁷G(5')ppp(5')G and advanced ARCA analogs for capped RNA synthesis in in vitro transcription systems.
Controlled Pore Glass (CPG) is the solid-phase support used for oligonucleotide chain assembly. We offer standard and modified CPGs, including universal linkers and 3' modifications, with pore sizes tailored for oligos of varying lengths.
Nucleosides are the foundational units comprising a nitrogenous base and sugar, required for the synthesis of nucleotides and phosphoramidites. We provide a diverse set of protected and unprotected nucleosides for DNA, RNA, and modified oligo synthesis.
Nucleotides serve as the activated building blocks for enzymatic reactions like PCR, transcription, and ligation. Our inventory includes natural dNTPs/NTPs, as well as modified nucleotides (e.g., 5-methyl-dCTP, pseudouridine triphosphate) for specialized applications.
Phosphoramidites are the core reagents in automated solid-phase synthesis of oligonucleotides. We supply a wide range of DNA, RNA, and modified phosphoramidites, ensuring high coupling efficiency and compatibility with custom chemical modifications.
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