21 Fluorescent Protein-Based DNA Staining Dyes

Abstract

Fluorescent protein-DNA-binding peptides or proteins (FP-DBP) are a powerful means to stain and visualize large DNA molecules on a fluorescence microscope. Here, we constructed 21 kinds of FP-DBPs using various colors of fluorescent proteins and two DNA-binding motifs. From the database of fluorescent proteins (FPbase.org), we chose bright FPs, such as RRvT, tdTomato, mNeonGreen, mClover3, YPet, and mScarlet, which are four to eight times brighter than original wild-type GFP. Additionally, we chose other FPs, such as mOrange2, Emerald, mTurquoise2, mStrawberry, and mCherry, for variations in emitting wavelengths. For DNA-binding motifs, we used HMG (high mobility group) as an 11-mer peptide or a 36 kDa tTALE (truncated transcription activator-like effector). Using 21 FP-DBPs, we attempted to stain DNA molecules and then analyzed fluorescence intensities. Most FP-DBPs successfully visualized DNA molecules. Even with the same DNA-binding motif, the order of FP and DBP affected DNA staining in terms of brightness and DNA stretching. The DNA staining pattern by FP-DBPs was also affected by the FP types. The data from 21 FP-DBPs provided a guideline to develop novel DNA-binding fluorescent proteins.

Keywords: DNA; DNA-binding proteins; FP-DBP; fluorescent protein; microfluidic device; single-molecule.

Figure 1

Expressed FP-DBPs in test tubes and their stained DNA images. (a) HMG-FP/FP-HMG (b) tTALE-FP. FP-DBP stained λ phage DNA images were given below. Illustrations of FP-DBP binding to DNA were shown for comparison of the size of DBPs. HMG-Emerald and tTALE-Emerald structures were modeled through AlphaFold 2.1.0 (DeepMind, London, UK) [36]. DNA-binding protein motifs were colored black and were aligned to DNA by using PDB 2EZD (a) PDB 4OTO (b) in the software PyMOL.

Figure 2

Fluorescence comparison between reference and measured values. (a) HMG (b) tTALE linked FP brightness was obtained from the FPbase.org (X-axis), and FP-DBP fluorescence intensities were measured from microscopic images (Y-axis). If the DBP name was omitted, DBP was linked N-terminal to FP. The dotted lines are calculated for a linear relationship. R² was 0.48 for HMG constructs (a) and 0.08 for tTALE constructs (b). FP abbreviated names: mNG, mNeonGreen; mStb, mStrawberry; mTurq, mTurquoise2. (c) Fluorescence spectra of mNG-HMG and tTALE-mNG with and without DNA.

Figure 3

Positional effects of FP and DBP. (a) Integrated intensity comparison of HMG-RRvT vs. RRvT-HMG. The λ DNA molecules were stained with FP-DBP. (b) Stretching comparison of HMG-mNeonGreen (N-terminal) vs. mNeonGreen-HMG (C-terminal). Scale bars = 10 μm.

Figure 4

tTALE-FP staining of λ Phage DNA for A/T specific map. (a) λ Phage DNA was stained with 30 nM tTALE-Emerald. The fluorescence intensity profiles are shown along with the in silico profile of AT frequency. Stained images of (b) 40 nM tTALE-mTurquoise2 (c) 40 nM tTALE-Ypet (d) 30 nM tTALE-mStrawberry and an intensity profile were shown. A stained image of (e) 30 nM tTALE-mNeonGreen with in silico maps of TGTCTGT was shown. Fully stained DNA was treated with different concentrations of NaCl in 1× TE buffer.

Figure 5

Structural comparison of FPs. (a) MEGA generated a phylogenetic tree for five FPs: Emerald, mTruquoise2, Ypet, mNeonGreen, and mStrawberry. (b) AlphaFold 2.1.0 [36] model of Emerald(green) was compared to mTurquoise2 (cyan) structure (PDB 6YLO) and Alphafold model of Ypet (yellow). (c) structure of the Emerald (green) model was also compared to the mNeonGreen (dark green) structure (PDB 5LTR) and the mStrawberry (pink) structure (PDB 2H5P).