Oligomerization: the What, the Why and the How 

What is oligomerization?

It is easy to think about molecules as a single distinct units, or monomers; however, in reality, monomers that are either identical or diverse can come together to form larger and more complex structures known as oligomers [1]. The number of monomers that make up a given oligomer can range between 2 and ~20 and is denoted in the terminology used to describe the complex. For instance, oligomers made up of 2 monomers are dimers, 3 monomers are trimers, 4 monomers are tetramers, and so on (Figure 1). Whilst these terms are typically used to describe proteins, other bio-molecules – particularly nucleic acids (DNA and RNA) – can undergo oligomerization [2].

Figure 1. A diagrammatic representation of a monomer and various oligomers.

In biological contexts, oligomerization is primarily driven by the self-assembly of monomers via non-covalent interactions – including hydrogen bonds, ionic interactions and van der Waals forces [3]. Such interactions may be influenced by co-factors such as metal ions/small molecules, or post-translation modifications, such as phosphorylation; both of which can promote oligomer formation. The propensity of monomers to oligomerize can also be dependent on environmental conditions; for instance, the monomeric concentration, as well as the surrounding pH and temperature.

Why is oligomerization important?

Oligomers have a number of biological implications, the importance of which is underlined by their ability to exert specific biochemical functions that cannot be elicited by monomeric molecules. These wide-ranging roles can include:

• Activity regulation. The functional activity of both proteins and enzymes can be modified by the formation of oligomers. For example, several proteins/enzymes are either activated or inhibited by dimerization, trimerization, or higher order oligomerization; their formation can induce conformational changes in active sites – altering binding affinities or co-operativity [4].
• Structural Stability. The thermodynamic and kinetic stability of biomolecules can be enhanced by oligomerization [5]. This can be driven by alterations in the exposure of hydrophobic regions and shielding motifs from degrading enzymes. This maintenance of molecular structure ensures longer-term appropriate functioning in a dynamic physiological environment.
• Signaling. Many signaling pathways rely on the oligomerization of receptors and intermediary molecules for downstream activation [6]. Oligomerization can enhance both signal amplification and specificity, ensuring that only the correct pathways (with sufficient magnitude) are activated in response to a given stimulus.
• Genetic regulation. In addition to proteins, oligomerization is a key feature of nucleic acid biology. For instance, short interfering RNAs and antisense oligonucleotides can bind complementary mRNA sequences to silence gene expression via degradation [7]. Similarly, transcription factors often function as oligomers when binding DNA, thereby controlling the transcriptional activation or repression of specific genes.

Clearly, oligomerization serves several essential biological purposes; however, it is equally important to consider the abnormal oligomerization and its role in the acceleration of disease. A good example of this are amyloid-β oligomers in Alzheimer’s disease (Figure 2). These soluble oligomers have been shown to be highly neurotoxic [8], unlike fibrils which are typically found in relatively more inert plaques. Oligomers may therefore represent a therapeutic target for new pharmaceuticals.

Figure 2. The oligomerization of Amyloid-β and the propensity of oligomers to drive neurotoxicity.

The study of oligomerization and the power of Microfluidic Diffusional Sizing (MDS)

There is a diverse toolkit of technologies which can be used to elucidate a samples state of oligomerization. These include structural, biophysical and fluorescence-based techniques. Structural methodologies such as cryo-electron microscopy (cryo-EM) and X-ray crystallography can reveal the structure of monomers and oligomers at near atomic level resolution – particularly cryo-EM; however, these methodologies are costly, time-consuming and require high levels of technical expertise. There are also a number of single-molecule spectroscopy methods available for the study of oligomerization; these include Fluorescence Resonance Energy Transfer (FRET), which detects the proximity between labelled monomers in an oligomer, and fluorescence correlational spectroscopy (FCS), which can examine the number of monomers in complex. Biophysical methods such as dynamic light scattering (DLS) and size exclusion chromatography (SEC) offer a more straightforward technique of measuring oligomers; however, the majority of biophysical techniques tend to require purified samples which prevent the investigation of oligomers in biologically relevant matrices.

Microfluidic Diffusion Sizing (MDS) is an emerging technology which enables the rapid characterization of biomolecular size (hydrodynamic radius [Rh]), as well as interaction metrics, without the need for sample purification. Given that there is a size difference between monomers, dimers, trimers and higher order oligomers., MDS-derived Rh can be used to investigate differences in the average state of oligomerization of a sample. Further to this, because size is broadly predictable (from molecular weight), the number of monomers that make up a complex can be estimated from a sample’s Rh (Table 1).

Table 1. Rh of monomers and oligomers approximated from molecular weight.

Approximate Rh taken from: What is Hydrodynamic Radius | Molecular Weight to Hydrodynamic Radius Converter

One of the biggest benefits of using MDS to investigate oligomerization is that there are no sample background restraints. This enables the behavior of molecules to be investigated under different buffer conditions (pH, concentrations etc), as well as biologically relevant samples like undiluted serum and cerebrospinal fluid – backgrounds which many other biophysical techniques struggle to handle.

Summary

Oligomerization is critical process in biology, eliciting biochemical mechanisms not available to monomeric molecules, but the inappropriate formation of oligomers may also be implicated in disease processes – particularly neurodegenerative disease. This highlights the need for more biophysical techniques to measure the oligomeric state of a sample.

To this end, MDS offers insights into the oligomerization of a sample, across any background – even 100% serum and other biologically relevant matrices. This in-solution technology makes the investigation of oligomerization quick, easy and accessible.

References

1. Naka, K., 2014. Monomers, oligomers, polymers, and macromolecules (overview). Encyclopedia of Polymeric Nanomaterials, pp.1-6. DOI: 10.1007/978-3-642-36199-9_237-1
2. Goodchild, J., 2011. Therapeutic oligonucleotides. Therapeutic Oligonucleotides: Methods and Protocols, pp.1-15. DOI:10.1007/978-1-61779-188-8_1
3. Gwyther, R.E., Jones, D.D. and Worthy, H.L., 2019. Better together: building protein oligomers naturally and by design. Biochemical Society Transactions, 47(6), pp.1773-1780.
4. Gotte, G. and Menegazzi, M., 2023. Protein oligomerization. International Journal of Molecular Sciences, 24(13), p.10648. DOI: 10.3390/ijms241310648
5. Gotte, G. and Libonati, M., 2014. Protein oligomerization. Oligomerization of Chemical and Biological Compounds, pp.239-278.
6. Milligan, G., Canals, M., Pediani, J.D., Ellis, J. and Lopez-Gimenez, J.F., 2007. The role of GPCR dimerisation/oligomerisation in receptor signalling. GPCRs: From Deorphanization to Lead Structure Identification, pp.145-162.
7. Chery, J., 2016. RNA therapeutics: RNAi and antisense mechanisms and clinical applications. Postdoc journal: a journal of postdoctoral research and postdoctoral affairs, 4(7), p.35. DOI: 10.14304/surya.jpr.v4n7.5
8. Madhu, P. and Mukhopadhyay, S., 2021. Distinct types of amyloid‐β oligomers displaying diverse neurotoxicity mechanisms in Alzheimer’s disease. Journal of cellular biochemistry, 122(11), pp.1594-1608. DOI: 10.1002/jcb.30141