Oligonucleotides (oligos) are short strands of DNA or RNA that look set to revolutionize the treatment of a range of conditions from cardiovascular diseases to cancers and central nervous system disorders (CNS).

Oligos are able to modulate gene expression in a number of ways including through gene activation, expression inhibition, splicing modulation and programmed gene editing. This means they have the potential to provide treatments that are targeted, so they produce fewer side effects.

Growth in therapeutic oligonucleotides

More than 20 synthetic oligonucleotides have already been approved and growth in this new drug category is expected to increase rapidly as confidence in it grows. A staggering 700 oligonucleotide drugs are currently in the pipeline with several already at the clinical trial stage.

There are now several classes of therapeutic oligonucleotides grouped according to their action mechanism. The main types include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs) and RNA aptamers. 

Using conjugation chemistry to enhance oligonucleotide delivery

However, there are a number of challenges that need to be overcome when delivering oligonucleotide therapies, especially to extrahepatic tissues. Issues such as tissue targeting, cellular uptake, tolerability and stability are a primary barrier to broader therapeutic application.

One powerful solution to overcoming these challenges is conjugation to a moiety such as a peptide or carbohydrate. The conjugation method you choose is likely to be influenced by a number of factors, such as the drug’s cellular target. This is because different molecules used in conjugation lend themselves to different cellular targets.

Oligonucleotide conjugation

Below, five key conjugation chemistries used for oligonucleotide drug delivery are presented. 

Thiol-Maleimide Conjugation

Covalent linkage via thiol-maleimide coupling is a widely used method in peptide and oligonucleotide chemistry, offering an efficient way to create stable conjugates for drug delivery and therapeutic applications. Maleimides are commonly employed for coupling with the reactive thiol groups of peptides or oligonucleotides via Michael addition reactions. This method is highly selective, has fast reaction kinetics, and requires mild reaction conditions, making it an ideal choice for scalable applications.

This conjugation method is frequently used to create peptide-oligonucleotide conjugates (POCs), such as cell-penetrating peptides (CPPs) attached to small interfering RNAs (siRNAs). These conjugates significantly enhance the delivery and efficacy of siRNAs by facilitating their escape from endosomes, improving cellular uptake, and enhancing subcellular distribution. The result is an increased pharmacological effect of the cargo oligonucleotide, making thiol-maleimide conjugation a powerful tool for targeted gene silencing therapies.

A notable example of thiol-maleimide conjugation in peptide-oligonucleotide delivery is the coupling of (arginine-glycine-aspartic) (cRGD)-siRNA conjugates which help improve the delivery of siRNAs via endosomal escape. They have been reported to enhance cellular uptake, subcellular distribution, and the pharmacological effects of the cargo oligonucleotide.

cRGD is a common type of cell penetrating peptide (CPP) used to target αvβ3 integrin receptors which are often upregulated in tumor cells in several cancer types. 

Disulfide Bond Formation

Another strategy of oligonucleotide conjugation  is using a disulfide bond, a popular way to form POCs. 

Disulfide bonds are formed through the oxidation of two cysteine residues, where the sulfur atoms from each cysteine’s thiol group (-SH) undergo oxidative coupling to create a covalent S-S bridge.

Covalently conjugating GLP1R to an ASO using a disulfide bridge has been shown to enhance the selective cellular uptake of ASOs in target cells. It’s also been found to improve the conjugate’s potency and induce gene expression silencing leading to a reduction in protein levels. 

This has paved the way for the treatment of conditions like diabetes caused by faulty gene expression in pancreatic β-cells.

Click Chemistry

Click chemistry, another common conjugation method, provides a powerful way of reliably conjugating moieties to oligonucleotides.That’s because click reactions can be modular, biocompatible and easily scalable. 

Click chemistry reactions use easily available reagents, are high yielding and stereospecific and they minimize the formation of by-products. What’s more, they can be carried out in an aqueous environment and they also use simple product separation techniques. 

They can be used in a wide range of applications such as antibody-oligo conjugates, as well as peptide-oligo conjugates.

Conjugation through Oxime, Thiazolidine, or Hydrazone Bonds

Another strategy for linking peptides and oligonucleotides is conjugation through oxime, thiazolidine, or hydrazone bonds. These methods offer the advantage of operating under mild conditions, making them ideal for maintaining the stability of both the peptide and oligonucleotide components.

The process involves the reaction of carbonyl compounds – such as aldehydes or glyoxylic acid derivatives – with functional groups on the peptide, such as aminooxy, 1,2-aminothiol, or hydrazine groups. The result is the formation of highly stable oxime, thiazolidine, or hydrazone bonds between the two molecules.

One of the key benefits of this method is its ability to form covalent bonds under mild conditions, making it highly efficient and suitable for sensitive biological applications. For instance, an oligonucleotide modified with a carbonyl group can be conjugated with a peptide containing an aminooxy group, producing a stable peptide-oligonucleotide conjugate. This type of conjugation is particularly useful for oligonucleotide drug delivery, where maintaining the integrity of the cargo is crucial for therapeutic success.

Conjugation through Amide Bonds

Amide bond formation is another widely used method for creating peptide-oligonucleotide conjugates. This approach involves the simple yet robust reaction of a carboxyl group on one molecule with an amine group on another, resulting in a stable and covalent linkage.

Amide bonds are preferred in many applications because of their simplicity and the strength of the bond they form. In the context of therapeutic oligonucleotides, this conjugation strategy ensures a stable, reliable connection between the oligonucleotide and peptide, which is essential for effective cellular delivery.

The mild reaction conditions for amide bond formation are another benefit, making it easy to adapt this method to a wide variety of peptide and oligonucleotide structures. As a result, amide bond conjugation has become a cornerstone of oligonucleotide drug delivery systems, offering an effective way to enhance the bioavailability and stability of these promising therapeutics.

Bachem and conjugation chemistry

The therapeutic use of oligonucleotides signifies a step-change in providing effective medicines for rare and non-rare diseases. 

However, to make this a reality, drug manufacturers have to contend with oligonucleotides’ poor pharmacokinetics and pharmacodynamics. Conjugation is a powerful strategy to help overcome these challenges. When designing effective therapeutic oligonucleotides, matching your conjugation strategy to your delivery objective is critical.

With decades of expertise, Bachem is ideally placed to be a reliable partner for those seeking to bring the next generation of oligonucleotide therapeutics to market faster.

For more information on using conjugation chemistry to improve oligonucleotide therapeutics, download our white paper or contact Bachem today and take advantage of our expertise.