Single molecule mass photometry of nucleic acids

Abstract

Mass photometry is a recently developed methodology capable of detection, imaging and mass measurement 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 4 bp accuracy. These results introduce mass photometry as an accurate and rapid single molecule method complementary to existing DNA characterisation techniques. INTRODUCTIONSingle molecule analysis has had a tremendous impact on our ability to study DNA structure, function and interactions (1). Next generation sequencing heavily relies on single-molecule methods, be it using single molecule fluorescence (2, 3) or nanopore-based approaches (4, 5). Similarly, single molecule methods are now heavily used in a variety of incarnations to study DNA-protein interactions (6), with both DNA and proteins visualised by fluorescence labelling to reach single molecule sensitivity (7).Label-free detection and quantification would be highly desirable in this context due to the associated reduction in experimental complexity and minimisation of potential perturbations caused by the sample itself. While visualisation of single DNA molecules has been possible for decades using non-optical methods, such as electron microscopy (8) and atomic force microscopy (9), which can also be used to study mechanical properties (10), label-free optical detection has remained a considerable challenge.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). 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 not only label-free detection and imaging of single molecules, but critically also 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 therefore set out to investigate in this work to which degree the capabilities of MP translate to nucleic acids, which would enable not only their detection,

Emergence and Rearrangement of Dynamic Supramolecular Aggregates Visualized by Interferometric Scattering Microscopy

“…The resulting images consist of diffraction-limited spots with a ratiometric contrast value in the range of 0.001–0.1 ( Figure 1 ). The number of particles binding to the glass surface over a given time interval is proportional to the particle concentration, whereas the particle contrast is proportional to the mass for a variety of biological particles, such as proteins, 32 DNA, 33 molecular machines, 34 antibodies, 35 protein cages, 36 and virus capsids 29 (see Figure S15 for contrast to mass calibration). At this stage, quantitative conversion of ratiometric contrast to particle size is not possible, however, previous studies of lipid nanodiscs showed that changes in lipid mass can be accurately quantified.…”

Mass Photometry of Membrane Proteins

“…Existing techniques are very powerful, and have enabled and continue to yield detailed information on sample composition and heterogeneity. Key to MP is the combination of speed and simplicity, as well its broader capabilities, which include detection of small ligand binding 7 , quantitative evaluation of binding affinities and kinetics in a surface free manner 10,33 , nucleic acid interactions 34 and more general capabilities for characterising sample heterogeneity 9 . All of these measurements are performed in the same way as the ones presented herein: by adding small volumes (µl) of low concentration (<µM) unlabelled samples in a buffer of choice to a microscope cover slide.…”

Quantifying Protein–Protein Interactions by Molecular Counting with Mass Photometry

“…This range will enable measurements up to 100 mm affinities, making MP a powerful approach for characterizing biomolecular interactions without labels and single-molecule sensitivity in a minimally perturbative fashion. Furthermore, the applicability of MP to both nucleic acids [33] and large multimolecular machines [34] provides scope for MP becoming a universal tool for studying biomolecular interactions and dynamics in a rapid, label-free, yet single-molecule-sensitive fashion.…”