Determination of the Fraction and Stoichiometry of Femtomolar Levels of Biomolecular Complexes in an Excess of Monomer Using Single-Molecule, Two-Color Coincidence Detection

Abstract

We have extended the method of single-molecule fluorescence, two-color coincidence detection (TCCD) to detect coincident events due to a low fraction of a complex against a background of chance coincident events, due to monomers. We developed two complementary methods to determine the number of chance coincident events using the experimental data and without the need for additional experiments. We show that the subtraction of the chance coincidence level is essential for accurate quantification of the relative number of complexes and their stoichiometry. By performing experiments on model samples made from fluorophore-labeled duplex DNA and free dye, a linear dependence on the fraction of duplex DNA was found, independent of the level or ratio of free dye, with quantification down to a level of 0.5% and 500 fM duplex DNA. The method was then used to measure the equilibrium dissociation constant and offrate of a 9-mer duplex DNA, demonstrating the application of this method to systems with nanomolar dissociation constants. These improvements to the method of TCCD analysis significantly extend the application of TCCD to weakly bound complexes and large multicomponent biomolecular systems.

Direct characterization of amyloidogenic oligomers by single-molecule fluorescence

“…1. TCCD is capable of detecting oligomeric complexes even in the presence of monomers, based on a molecule-by-molecule analysis of the species as they diffuse through a confocal volume excited by overlapped red and blue lasers (26,27). The converged lasers allow for the independent detection of Alexa Fluor 488-or Alexa Fluor 647-labeled monomers as they pass through the confocal volume, as well as the detection of coincident bursts of fluorescence from mixed-label complexes.…”

“…Therefore, oligomeric species formed by equimolar mixtures of Alexa Fluor 488-and Alexa Fluor 647-labeled PI3-SH3 can be readily distinguished from monomeric protein molecules containing a single fluorophore. In the analysis of these data, we have applied methods developed to account for the chance coincidental background events that occur when two noninteracting PI3-SH3 molecules with different fluorophores pass through the confocal volume simultaneously; through this approach we can determine the fraction of oligomers in solution with greater accuracy (27).…”

“…The instrumentation for TCCD measurement has been reported in detail (27). Two overlapped Gaussian laser beams, at 488 nm, argon ion (35LAP321-230; Melles Griot), and 633 nm, He:Ne laser (25LHP151; Melles Griot), were directed to the back port of an inverted microscope (Nikon Eclipse TE2000-U).…”

“…We first set an optimized threshold value for each channel, donor and acceptor channels, as described (32) using a model sample, a dual-labeled 40-bp double-stranded DNA. Once the thresholds are applied to the SH3 dilutions, the coincidence events are counted and analyzed (after correction for coincident events due purely to chance) following the methodology previously reported (27). The association quotient shown in Fig.…”

Kinetic model of the aggregation of alpha-synuclein provides insights into prion-like spreading

“…The setup used for single-molecule measurements (Fig. 1B) is analogous to one previously described (54). For the FRET measurement, a collimated 488-nm laser beam (Spectra Physics NewPort Cyan) was directed through a back port of an inverted microscope (Nikon Eclipse Ti-U) at 2 mW power (measured at the back port of the microscope), where it was reflected by a dichroic mirror (Semrock DiO1 R405/488/594) and sent through an oil immersion objective (Plan Apo VC 60 x, NA 1.40; Nikon) to be focused 10 μm into the center of the microfluidic channel.…”