Custom Oligonucleotide-Cholesterol Conjugation Service

Cholesterol-Oligonucleotide Conjugation Introduction

Recently, oligonucleotide-based therapies have been developed and used to treat various rare diseases. However, the main reasons limiting the development of oligonucleotide drugs are their inadequate pharmacokinetic properties and poor cellular uptake due to difficulty in crossing the cell membrane. Based on the development and modification of chemical structures, a large number of clinical success cases have been established, which allow for sustained in vivo efficacy for several months after administration. Conjugate mediated delivery has become the most promising platform for safe and precise targeting of oligonucleotide delivery. Covalent connection of various ligands is one of the effective methods for optimizing oligonucleotide-based therapies. Various ligands widely used to form conjugates with oligonucleotides include peptides, proteins, and various small molecules such as cholesterol, tocopherol, and folate. Among them, cholesterol conjugated oligonucleotides are a well-known lipid conjugate that has been widely developed and used due to its significant alteration of the biological distribution of conjugated oligonucleotides.

Figure 1 (A) siRNA contains 4 terminal phosphate groups for modification, but only 3 sites are tolerable for bioconjugation. (B) Acid-sensitive linker cleaved by acid. (C) Disulfide linker can be cleaved in cytosol to release siRNA. (D) DsiRNA conjugate is processed by Dicer to release mature siRNA.¹

What Are the Difficulties to Develop a Therapeutic siRNA Drug?

Oligonucleotide therapeutics have a great potential to treat genetic diseases for which there are no cures with small molecule and antibody therapies. A significant challenge to the clinical development of small interfering RNA (siRNA) is the delivery of siRNA into cells in vivo. As large polyanionic macromolecules, siRNAs have poor pharmacological properties. Unmodified siRNAs are degraded rapidly in circulation (half-lives of less than 5 minutes) and cannot cross intact cell membranes. Nanoscale and molecular-scale strategies for delivery have been developed.

Cholesterol Conjugation Promotes siRNA Delivery and Distribution in Vivo

Cholesterol is one of the most extensively studied lipid components, which can achieve efficient cell and tissue delivery after direct coupling with oligonucleotides. Cholesterol accounts for 15% -30% of the cell membrane and spontaneously inserts into the lipid membrane after co incubation with cells, exerting its biological role in supporting membrane structure and fluidity. There are two main mechanisms by which cholesterol conjugation promotes siRNA uptake.

• The first method involves inserting cholesterol conjugates into the plasma membrane and internalizing oligonucleotides through endocytosis.
• The second type is that cholesterol conjugates bind to circulating plasma lipoproteins, and the uptake of siRNA is driven by their interaction with lipoprotein receptors. In vitro, cholesterol conjugated siRNA can be rapidly internalized by any cell type through EEA1 related endocytosis (within seconds after exposure).

Due to the fast internalization rate and only partial inhibition by serum, it is likely to be mainly absorbed directly through the membrane. These characteristics make cholesterol conjugated siRNA an ideal candidate for local in vivo delivery, especially to the skin, eyes, and brain parenchyma

Advantages of Cholesterol-Oligonucleotide Conjugates

Coupling cholesterol with antisense or siRNA oligonucleotides is a possible method to alter its biophysical properties:

• Improve membrane permeability or absorption mediated by specific receptors.
• The unique molecular structure is clear and can be analyzed using analytical techniques and standard quality assurance methods.
• Eliminate issues related to particle size, component dispersion, and batch differences.

Cholesterol-conjugated Oligonucleotide Design

Cholesterol Conjugation Using Different Linkers

The biophysical properties of the used linker pairs are crucial and have an impact on the pharmacokinetic behavior of the pairs. Oligonucleotide conjugation has employed various connection methods, including disulfide bonds, amide bonds, and click chemistry. Prepare various types of linkers for coupling 3 'or 5' - cholesterol with antisense oligonucleotides (ASO), including:

Selection of Ligand Attachment Sites in Oligonucleotides

Alternative ligand connections include nucleobases, particularly the 5-C atoms of pyrimidine bases such as cytosine, thymine, and uracil, the 8-C atom of adenine, and the extracellular amino group (2-N) of guanines, as well as linkers attached to the phosphate backbone. Most adapters are suitable for 3 'and 5' ends, which are technically the easiest to access, followed by 2 '- hydroxyl and nucleobases.

Applications of Cholesterol-Oligonucleotide Conjugates

Overview of What Creative Biolabs Can Provide

At Creative Biolabs, we specialize in providing a comprehensive Cholesterol-Oligonucleotide Conjugation Service designed to meet the rigorous demands of academic research and commercial drug development. If you are interested in our bioconjugation services, please feel free to contact us for more details.

Customized Conjugation Design

• Oligonucleotide selection: Supports all types of oligonucleotides (siRNA, ASO, miRNA, aptamer, guide RNA) and modifies their backbone (such as thiophosphate, 2 '- O-methyl) to enhance their stability.
• Cholesterol derivative selection: Options include cholesterol amine, cholesterol chloroformate ester, cholesterol PEG amine (for reducing aggregation), and fluorescent labeled cholesterol.
• Linker engineering: Customize connectors that can be cleaved (disulfide bonds, ester bonds, peptide bonds) or non-cleaved (amide bonds, ether bonds) according to the application.

Synthesis and Conjugation

• Using solid-phase synthesis (SPPS) technology, high-purity scalable oligonucleotide synthesis is achieved.
• Site specific binding (5 'end, 3' end, or internal position) to avoid interfering with ON activity.
• Optimize the reaction to achieve a binding efficiency of ≥ 90%.

Purification and Characterization

We provide cholesterol ON with a purity of ≥ 95% through advanced purification technology:

• HPLC (reverse phase, ion exchange) or FPLC is used to remove unreacted cholesterol, ON, and by-products.
• Comprehensive characterization to verify identity, purity, and activity:
  • Mass spectrometry (LC-MS/MS, MALDI-TOF) is used to confirm the molecular weight of the binding compound.
  • Nuclear magnetic resonance spectroscopy is used to verify the structure of cholesterol ON bonds.
  • HPLC-UV is used for purity quantification.
  • Cell based activity assays (such as siRNA knockdown efficiency, ASO binding affinity) are used to ensure biological functionality.

Choosing Creative Biolabs: Here's Why It's Smart!

• Experienced experts,strict QA and QC
• Advanced analytical equipments
• Professional technical support
• 1 on 1 customer service
• Competitive price
• Fast delivery

Frequently Asked Questions

Related Sections