DNA-Origami-Based Fluorescence Brightness Standards for Convenient and Fast Protein Counting in Live Cells

Abstract

Fluorescence microscopy has been one of the most discovery-rich methods in biology. In the digital age, the discipline is becoming increasingly quantitative. Virtually all biological laboratories have access to fluorescence microscopes, but abilities to quantify biomolecule copy numbers are limited by the complexity and sophistication associated with current quantification methods. Here, we present DNA-origami-based fluorescence brightness standards for counting 5-300 copies of proteins in bacterial and mammalian cells, tagged with fluorescent proteins or membrane-permeable organic dyes. Compared to conventional quantification techniques, our brightness standards are robust, straightforward to use, and compatible with nearly all fluorescence imaging applications, thereby providing a practical and versatile tool to quantify biomolecules via fluorescence microscopy.

Keywords: Bioconjugation; Brightness standard; DNA origami; Fluorescent protein; Live cell imaging; Quantitative microscopy.

Figure 1.

A DNA-origami-based mEGFP brightness standard. (a) 3D models and TEM micrographs of monomeric DNA 6hb structures labeled with 5–100 copies of mEGFP (green) in the main body, 12 copies of Alexa Fluor 647 (red) at each end, and 4 biotin molecules along one side. The minimum spacing of the fluorophores is ~12 nm. Scale bars: 50 nm. (b) Generation of mEGFP-DNA conjugate. mEGFP-pAzF is expressed and purified from a GRO, in which the antisense TAG codon has been reassigned to encode pAzF, an azide-modified Phe. mEGFP(pAzF) was purified via immobilized metal affinity chromatography (IMAC), then reacted with alkyne-labeled DNA. Two subsequent purification steps removed unreacted proteins and DNA. (c) Gel electrophoresis (top) and widefield microscopy images (WFM, bottom) of mEGFP standards. Images are set to the same brightness scale (no saturated pixels in the original images). Scale bars: 2 μm. (d) Differential interference contrast (DIC, top) and WFM (bottom) images of B. subtilis (strain NW001) expressing dnaC-mEGFP. Circles indicate puncta picked for quantification. Scale bars: 2 μm. (e) Quantifying dnaC-mEGFP. Left: a calibration curve with intensities of DNA-origami standards and interpolated protein counts (mean±SEM) from dnaC-mEGFP puncta. Dotted lines denote 95% confidence interval. Right: frequency distribution and sum-of-two-Gaussians fit of dnaC-mEGFP puncta.

Figure 2.

A DNA-origami-based SiR brightness standard. (a) 3D models of dimeric DNA-origami nanotubes hosting 25–200 SiR molecules (magenta) in the main body, as well as Alexa Fluor 488 (Green) and TAMRA (red) at three distinct locations (12 fluorophores at each location) for barcoding. Confocal microscopy images revealed the expected barcoding patterns and corresponding increase in SiR intensity. Scale bars: 1 μm. (b) Agarose gel images of SiR standards show the expected combinations of barcoding dyes Alexa Fluor 488 and TAMRA, as well as increasing SiR intensity. (c) Confocal images of HeLa cells expressing Halo-fused clathrin light chain (CLC) after labeling with SiR-chloroalkane. Inset shows details of coated pits (small and round, green arrows) and plaques (larger and irregularly shaped, red arrows). Scale bars: 10 μm. (d) Quantifying SiR-labeled CLCs. Left: Calibration curve generated from DNA-origami-based SiR standards (SEM too small to see). Dotted lines denote 95% confidence interval. Right: Spots containing SiR-labeled CLCs binned by molecule number.