Moving from bulk average to single-molecule methods for studying biomolecular interactions

Most methods for analyzing biomolecular interactions produce averaged data from many thousands of molecules or more. A new generation of single-molecule biophysical techniques are transforming how we study biomolecular interactions, and can offer unparalleled insights into molecular heterogeneity, dynamic binding, and rare events.

Understanding biomolecular interactions – the science of interactomics – is crucial for deciphering disease mechanisms and developing more effective therapeutics.

However, most techniques for studying biomolecular interactions are bulk or ensemble methods that analyze a large population of molecules simultaneously and report results representing an average value.

Increasingly, researchers are turning to single-molecule techniques such as force spectroscopy for analyzing biomolecular interactions. These approaches can reveal a more detailed and nuanced picture of molecular biology to advance our understanding of health and disease.

In this article, we explore some of these single-molecule biophysical analytical techniques and how they can be applied to study biomolecular interactions.

Bulk-averaged methods fall short for studying biomolecular interactions

Ensemble- or bulk-averaging techniques such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and microscale thermophoresis (MST) analyze biomolecular interactions by measuring the collective behavior of millions of molecules simultaneously.

While they provide an overall picture of the molecular interactions within a biological system, bulk average methods fail to capture the diversity and detail of what is really happening, including:

• Heterogeneity within molecular populations
• Dynamic processes and transitions between molecular states
• Rare or transient interactions

By contrast, single-molecule techniques can measure the behavior of individual molecules without averaging out their differences, revealing crucial details that are essential for understanding complex biological functions.

Biophysical methods for studying individual molecular interactions

Biophysical single-molecule force spectroscopy approaches can be categorized based on the type of force employed to interrogate single molecules and their interactions. The most common force spectroscopy techniques are optical tweezers (OT), magnetic tweezers (MT, also known as magnetic force spectroscopy or MFS), acoustic force spectroscopy, and atomic force microscopy (AFM).^1,2,3,4

These techniques share common features of tethering a molecule to a surface at one end, leaving the other end available for attachment to a probe. The precise position of the attached probe is measured relative to the surface, enabling the molecule of interest to be tracked.³ Typically, these techniques do not require labeling with fluorescent tags, although fluorescent tags may be utilized for some applications.

By enabling analysis of biomolecules at the level of individual interactions, single-molecule methods offer exciting new possibilities for studying and manipulating biomolecular interactions, providing unparalleled insights into health and disease.

Benefits of single-molecule approaches for analyzing biomolecular interactions

Single-molecule biophysical techniques allow researchers to identify heterogeneity and uncover sub-populations within a larger group – something that is not possible with bulk-averaged methods.

Beyond identifying heterogeneity, some of the latest single-molecule techniques enable real-time observation of dynamic processes and state transitions. This allows researchers to track time spent in each state for each individual molecule as well as the response to perturbations such as ligand binding, providing insights into the stochastic nature of biological systems.

Finally, examining individual molecules allows researchers to directly observe rare or transient events normally hidden by averaging, provided these events occur with sufficient frequency for statistical analysis.

For example, transient protein-protein interactions (PPIs), which form and break easily and are crucial for regulating the dynamics of biological networks, are typically difficult to study using bulk measurements due to their unstable and weak nature.⁵

Depixus MAGNA One™: Scalable single-molecule analysis of biomolecular interactions

Based on magnetic force spectroscopy (MFS), Depixus MAGNA One™ is a revolutionary technology transforming the study of molecular interactions through scalable single-molecule analysis.

Unlike traditional single-molecule techniques that can analyze tens or hundreds of molecules at any one time, our award-winning magnetic force spectroscopy platform enables real-time observation of thousands of individual biomolecular interactions in parallel.

This ability to simultaneously analyze thousands of biomolecular interactions leverages the strengths of single-molecule analysis while maintaining consistent conditions across a large population.

By preserving the data from each individual interaction, Depixus MAGNA One generates exceptionally detailed datasets, revealing heterogeneity, rare events, and transient interactions often missed by traditional bulk assays or smaller scale single-molecule techniques.

Using Depixus MAGNA One to distinguish binding mechanisms

A recent publication in the journal Nature Communications from Dr Jay Schneekloth and colleagues exploring the bacterial PreQ₁ RNA riboswitch exemplifies the advantage of Depixus’ scalable single-molecule MFS approach.⁶

While initial analysis suggested that the riboswitch had the same conformational response to both natural and synthetic ligands, Depixus’ magnetic force spectroscopy platform revealed the two ligands function through distinct kinetic mechanisms, altering the rate of folding and stabilizing the riboswitch in different conformations.

This distinction was only revealed with Depixus’ single-molecule MFS analysis and could not have been detected using bulk-averaged methods.

From protein-protein interactions to RNA-targeted drug development, Depixus MAGNA One provides unparalleled insights into the biomolecular mechanisms underpinning health and disease. With Depixus MAGNA One, you can see biology as it really happens.

• To discover how Depixus MAGNA One™ can be used to accelerate your research into biomolecular interactions, download our product brochure today.

Watch this short video to see Depixus’ scalable MFS technology in action

References:

1. Walter NG, Huang C-Y, Manzo AJ, Sobhy MA. Do-it-yourself guide: How to use the modern single molecule toolkit. Nat Methods. 2008; 5(6): 475–489. doi: 10.1038/nmeth.1215
2. Neuman KC, Nagy A. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat Methods. 2008; 5(6): 491–505. DOI: 10.1038/nmeth.1218
3. Tinoco I, Gonzalez RL. Biological mechanisms, one molecule at a time. Genes & Development. 2011; 25:1205–123. doi: 10.1101/gad.2050011
4. Sitters G, Kamsma D, Thalhammer G, Ritsch-Marte M, Peterman EJ, Wuite GJ (2015). Acoustic force spectroscopy. Nat Methods. 2015;12(1): 47-50. doi: 10.1038/nmeth.3183
5. Perkins JR, Diboun I, Dessailly BH, Lees JG, Orengo C. Transient Protein-Protein Interactions: Structural, Functional, and Network Properties. Structure. 2010; 18(10):1233-1243 doi: 10.1016/j.str.2010.08.007
6. Parmar S, Bume DD, Connelly CM, Boer RE, Prestwood PR, Wang Z, Labuhn H, Sinnadurai K, Feri A, Ouellet J, Homan P, Numata T, Schneekloth JS Jr. Mechanistic analysis of Riboswitch Ligand interactions provides insights into pharmacological control over gene expression. Nat Commun. 2024;15(1):8173 doi: 10.1038/s41467-024-52235-3