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Measuring protein-nucleic acid interactions with mass photometry 

Mass photometry characterizes nucleic acids, proteins and their interactions 

 

Interactions between nucleic acids and proteins are critical for many cellular processes, including DNA repair, replication and transcription. However, protein-nucleic acid interactions often result in highly heterogenous complexes, both in the type of binding partners and in the stoichiometry of the protein-DNA interactions. This heterogeneity presents a challenge to conventional bioanalytical methods.  

Mass photometry is a powerful technology that analyzes the mass distribution of the molecules present in a sample at the single-molecule level, without labels or immobilization.

A schematic showing how MassGlass CC coated slides facilitate the interaction of DNA molecules with the measuring surface.

Thanks to these strengths, mass photometry provides a detailed overview of all the molecules present in a sample as well as the complexes they form, even when they are present at low abundance. 

 

Facilitating the analysis of proteins, DNA and protein-DNA interactions are Refeyn’s functionalized MassGlass CC slides. Their cationic coating enables mass photometry analysis of negatively charged molecules, like DNA. MassGlass CC slides are also fully compatible with mass photometry analysis of proteins. 

Measuring protein-DNA affinity with mass photometry vs. SPR 

 

Mass photometry is a label-free analytical method that determines the mass of single molecules, in solution, by measuring light scattering as each molecule lands on a glass slide. This technique, with its one-minute analysis run-time, enables the precise and rapid assessment of the relative abundance of each protein species in a sample. It can provide information on oligomerization, interactions, complex formation and sample homogeneity.  

 Mass photometry histograms showing independent measurements of DNA and proteins as well as the interactions between them

SEC is a method in that separates molecules by their size (hydrodynamic radius), based on the time they take to pass through a column containing beads with pores distributed across a specific range of sizes. Coupled with UV absorbance detection (SEC-UV) and provided that molar extinction coefficients are known, it provides information on analyte concentration. Coupled to multi-angle light scattering (SEC-MALS), it can measure both the size and molecular weight of the analytes in a 30-minute analysis run-time. From this analysis, users can assess the purity of their protein samples.  

To find out how to quickly quantify protein and DNA binding affinities 

Characterizing protein-DNA interactions with
mass photometry  

In this application note, you will learn how mass photometry, along with MassGlass CC slides, can be used to successfully measure the mass of nucleic acid and protein molecules, and to quantify their abundance and the abundance of complexes they form. You will see a comparison of mass photometry measurements of proteins made using functionalized (MassGlass CC) and non-functionalized slides. Finally, you will see data proving that mass photometry can detect DNA and proteins in complex as well as separately, providing the necessary data to quantify their dissociation constant – with results consistent with SPR.   

Additional resources​ on using mass photometry to characterize protein samples 

WEBINAR

Elucidating the composition of CRISPR-Cas12f1 complexes using mass photometry

 

This webinar explores how the CRISPR-Cas12f1 effector complex recognizes and cleaves DNA. Mass photometry was used to measure the stoichiometry of two miniature Cas12f1 ribonucleoprotein complexes, AsCas12f1 and SpCas12f1 in vitro. The results indicate that the Cas protein forms a binary complex with guide RNA and remains bound when interacting with target DNA, forming a ternary complex. Overall, the results reveal the composition of a compact CRISPR-Cas system and its impact on target recognition and DNA cleavage mechanisms.

webinar Elucidating the composition of CRISPR Cas12f1 complexes using mass photometry.png
Blog - Studying protein-DNA interactions with mass photometry.png

BLOG

Studying protein-DNA interactions with mass photometry

 

In this blog post, we present a recent study that used mass photometry to investigate protein-DNA interactions. We then speak with the paper’s first author, Ananya Acharya, who explains how she and her colleagues used mass photometry to answer unique scientific questions about DNA repair mechanisms and compares it to other techniques. 

To learn more about how mass photometry can improve your bioanalytical processes

More Application Notes

Browse through our catalogue of application notes highlighting some recent case studies featuring mass photometry.

Investigating lentivirus titer with macro mass photometry

 

Mass photometry supports membrane protein purification protocols 
 

Characterizing protein oligomerization with automated mass photometry

Quantifying heterogeneous AAV capsid loading using mass photometry
 

Download the application note

download the app note
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