Silicon gasket

The MP experiments were conducted on Refyn Two-MP instrument (Refyn Ltd., Oxford, UK) on pre-cleaned coverslips (24 mm × 50 mm, Thorlabs Inc., Newton, NJ, USA) with serial washing with deionized water and isopropanol followed by drying. The silicon gaskets (Grace Bio-Labs, Bend, OR, USA) were cleaned in a similar process as coverslips and were placed onto coverslips for the experiments. The MP measurements were performed in an MP buffer containing 20 mM Tris pH 7.4, 100 mM KCl, 1 mM EDTA, and 10 mM MgCl₂. The calibration was performed using a protein standard mixture: of β-amylase (Sigma-Aldrich, 56, 112, and 224 kDa, St. Louis, MO, USA), and thyroglobulin (Sigma-Aldrich, 670 kDa). Before each experiment, 15 μL buffer was placed into a chamber formed by coverslip-Gasket and focus was searched and followed by locking it using autofocus function. G4 DNA, G4P proteins or their mixtures were added to the chamber and mixed by pipetting. The movies were recorded for 60 s (6000 frames) using AcquireMP (Version 2.3.0; Refeyn Ltd., Oxford, UK) using standard settings. All movies were processed and analyzed using DiscoverMP (Version 2.3.0; Refeyn Ltd., Oxford, UK). Individual molecular weights readings for each experiment were binned into 3 kDa intervals, plotted as histograms and fitted to multiple Gaussians using GraphPad Prism.

MP experiments were performed on a Refeyn TwoMP mass photometer (Refeyn Ltd, Oxford, UK). Microscope coverslips (24 mm × 50 mm, Thorlabs Inc.) were cleaned by serial rinsing with Milli-Q water and HPLC-grade isopropanol (Sigma Aldrich) followed by drying with a filtered air stream. Silicon gaskets (Grace Bio-Labs) to hold the sample drops were cleaned in the same procedure immediately prior to measurement. All MP measurements were performed at room temperature using buffer (20 mM HEPES, pH 7.5 and 150 mM NaCl). The instrument was calibrated using a protein standard mixture: β-amylase (Sigma-Aldrich, 56, 112 and 224 kDa), and thyroglobulin (Sigma-Aldrich, 670 kDa). Before each measurement, 15 μL of buffer was placed in the well to find focus. The focus position was searched and locked using the default droplet-dilution autofocus function after which 5 μL of protein samples (40 nM) was added and pipetted up and down to briefly mix before movie acquisition was promptly started. Movies were acquired for 60 s (3000 frames) using AcquireMP (version 2.3.0; Refeyn Ltd) using standard settings. All movies were processed and analyzed using DiscoverMP (version 2.3.0; Refeyn Ltd).