Abstract
Mass photometry is a recently developed methodology capable of measuring the mass of individual proteins under solution conditions. Here, we show that this approach is equally applicable to nucleic acids, enabling their facile, rapid and accurate detection and quantification using sub-picomoles of sample. The ability to count individual molecules directly measures relative concentrations in complex mixtures without need for separation. Using a dsDNA ladder, we find a linear relationship between the number of bases per molecule and the associated imaging contrast for up to 1200 bp, enabling us to quantify dsDNA length with up to 2 bp accuracy. These results introduce mass photometry as an accurate, rapid and label-free single molecule method complementary to existing DNA characterization techniques.
Highlights
Single molecule analysis has had a tremendous impact on our ability to study DNA structure, function and interactions [1]
Single molecule methods are extensively used in a variety of incarnations to study DNA–protein interactions [6], with both DNA and proteins visualized by fluorescence labelling to reach single molecule sensitivity [7]
Label-free detection of single proteins has been reported for the first time in 2014 [11,12] in the context of increasing sensitivity of interferometric scattering microscopy [13,14]
Summary
Single molecule analysis has had a tremendous impact on our ability to study DNA structure, function and interactions [1]. Further improvements to the detection methodology [15], recently lead to the development of mass photometry (MP), originally introduced as interferometric scattering mass spectrometry [16], which enables labelfree detection and imaging of single molecules, but critically their quantification through mass measurement with high levels of accuracy, precision and resolution at a lower detection limit on the order of 40 kDa. Given that biomolecules have broadly comparable optical properties in the visible range of the electromagnetic spectrum [17,18], we set out to investigate to which degree the capabilities of MP translate to nucleic acids, which would enable their detection, imaging and analysis, and provide a universal route to studying protein-DNA interactions at the single molecule level
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.