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1.
Figure 1

Figure 1. From: Fluorescent DNA Nanotags: Supramolecular Fluorescent Labels Based on Intercalating Dye Arrays Assembled on Nanostructured DNA Templates.

Fluorescent “nanotags” based on linear and branched DNA templates and intercalating dyes. Self-quenching is avoided by confining the dyes within separate intercalation sites. “2WJ” = 2-way junction, “3WJ” = 3-way junction, etc.

Andrea L. Benvin, et al. J Am Chem Soc. ;129(7):2025-2034.
2.
Figure 11

Figure 11. From: Fluorescent DNA Nanotags: Supramolecular Fluorescent Labels Based on Intercalating Dye Arrays Assembled on Nanostructured DNA Templates.

Fluorescence microscope images of CD3+ mouse T cells labeled with biotinylated anti-CD3-antibody and phycoerythrin-streptavidin conjugate (a) or streptavidin + biotinylated 3WJ/YOYO-1/Cy3 DNA labels (b).

Andrea L. Benvin, et al. J Am Chem Soc. ;129(7):2025-2034.
3.
Figure 9

Figure 9. From: Fluorescent DNA Nanotags: Supramolecular Fluorescent Labels Based on Intercalating Dye Arrays Assembled on Nanostructured DNA Templates.

Flow cytometry analysis of bead mixture labeled with 3WJs, YOYO-1 and either 0 or 2 Cy3 acceptors. Peaks for separate populations are unfilled; peaks for bead mixture are filled. Samples were monitored in the Cy3 (acceptor) channel.

Andrea L. Benvin, et al. J Am Chem Soc. ;129(7):2025-2034.
4.
Figure 6

Figure 6. From: Fluorescent DNA Nanotags: Supramolecular Fluorescent Labels Based on Intercalating Dye Arrays Assembled on Nanostructured DNA Templates.

FRET in a 3WJ DNA nanotag loaded with YOYO-1 intercalator dyes and covalently attached Cy3 acceptor dyes. This experiment corresponds to the format illustrated in Figure 4B. Vertical arrows indicate quenching of YOYO-1 donors and sensitized emission of Cy3 acceptors. Samples were excited at 440 nm.

Andrea L. Benvin, et al. J Am Chem Soc. ;129(7):2025-2034.
5.
Figure 2

Figure 2. From: Fluorescent DNA Nanotags: Supramolecular Fluorescent Labels Based on Intercalating Dye Arrays Assembled on Nanostructured DNA Templates.

UV melting curves recorded for a DNA 3WJ consisting of three ten base pair arms in the absence of intercalating dye as well as in the presence of mono- (YO-PRO-1) and bis- (YOYO-1) intercalating dyes.

Andrea L. Benvin, et al. J Am Chem Soc. ;129(7):2025-2034.
6.
Figure 7

Figure 7. From: Fluorescent DNA Nanotags: Supramolecular Fluorescent Labels Based on Intercalating Dye Arrays Assembled on Nanostructured DNA Templates.

FRET in a DNA 3WJ loaded with YOYO-1 intercalator (donor) dyes and 3 covalently attached terminal WellRed (acceptor) dyes. A: Overlap between donor emission and acceptor absorption spectra. (B) Efficient FRET is observed in spite of poor spectra overlap between donor and acceptor dyes. Samples were excited at 440 nm.

Andrea L. Benvin, et al. J Am Chem Soc. ;129(7):2025-2034.
7.
Figure 3

Figure 3. From: Fluorescent DNA Nanotags: Supramolecular Fluorescent Labels Based on Intercalating Dye Arrays Assembled on Nanostructured DNA Templates.

Dependence of maximum fluorescence intensity on ratio of intercalating YO groups per DNA base pair. [DNA] = 1 μM 3WJ = 30 μM base pairs for YO-PRO-1 and 0.1 μM 3WJ = 3.0 μM base pairs for YOYO-1. Spectra were recorded with excitation at 440 nm and data were normalized to the highest value at the emission wavelength (507 nm for YO-PRO-1, 510 nm for YOYO-1).

Andrea L. Benvin, et al. J Am Chem Soc. ;129(7):2025-2034.
8.
Figure 5

Figure 5. From: Fluorescent DNA Nanotags: Supramolecular Fluorescent Labels Based on Intercalating Dye Arrays Assembled on Nanostructured DNA Templates.

FRET experiments in which donor (YO-PRO-1) and acceptor (TO-PRO-3) were cointercalated at varying ratios in a DNA 3WJ template. Legend lists YO-PRO-1 and TO-PRO-3 concentrations in micromolar (e.g. 14:1 = 14 μM YO-PRO-1 and 1 μM TO-PRO-3. (A) Experimental data acquired with excitation at 440 nm. [DNA 3WJ] = 1.0 μM. (B) Simulated data corresponding to FRET efficiencies determined based on quenching of donor from spectra in (A), as described in Materials and Methods section.

Andrea L. Benvin, et al. J Am Chem Soc. ;129(7):2025-2034.
9.
Figure 10

Figure 10. From: Fluorescent DNA Nanotags: Supramolecular Fluorescent Labels Based on Intercalating Dye Arrays Assembled on Nanostructured DNA Templates.

Flow cytometry results illustrating increasing brightness with the number of base pairs in the nanotag. Samples included 50 nM DNA nanotag and YOYO-1 dye concentrations that equaled one dye per 4 DNA base pairs. Sample labeled 3WJ+Fluorescein did not have any YOYO-1, but instead had one covalently attached 5′-fluorescein label. Nanotag structural schematics are shown in Figure 1.

Andrea L. Benvin, et al. J Am Chem Soc. ;129(7):2025-2034.
10.
Figure 4

Figure 4. From: Fluorescent DNA Nanotags: Supramolecular Fluorescent Labels Based on Intercalating Dye Arrays Assembled on Nanostructured DNA Templates.

Two strategies for using Forster resonance energy transfer (FRET) to shift emission wavelength away from excitation wavelength. (A) Co-intercalated donor (blue) and acceptor (red) dyes allow efficient FRET to occur, but some donors must be displaced to accommodate acceptors. (B) Non-intercalated acceptor dyes (red spheres) linked to DNA termini allow FRET without displacing donor dyes. Note that efficient energy migration among intercalated donor dyes prior to energy transfer to an acceptor dye can occur in both formats.

Andrea L. Benvin, et al. J Am Chem Soc. ;129(7):2025-2034.
11.
Figure 8

Figure 8. From: Fluorescent DNA Nanotags: Supramolecular Fluorescent Labels Based on Intercalating Dye Arrays Assembled on Nanostructured DNA Templates.

Flow cytometry results recorded for 4 different bead populations monitoring the donor channel (a) and acceptor channel (b). Beads labeled with 3WJs but no intercalating dye gave only background signal (A). Beads labeled with 3WJs and YOYO-1 (B) gave strong signals in both channels due to the broad emission spectrum of the intercalator. Addition of one (C) or two (D) Cy3 acceptor dyes led to a decrease in intensity in the donor channel due to efficient FRET (left). A corresponding increase in fluorescence was observed in the acceptor channel due to sensitized emission from the Cy3 labels (right).

Andrea L. Benvin, et al. J Am Chem Soc. ;129(7):2025-2034.
12.
Chart 12

Chart 12. From: Fluorescent DNA Nanotags: Supramolecular Fluorescent Labels Based on Intercalating Dye Arrays Assembled on Nanostructured DNA Templates.

The intercalating dyes used for these experiments are based on the oxazole yellow chromophore (Chart 1)32. YO-PRO-1 and YOYO-1 are classified respectively as mono- and bisintercalators33 and YO-PRO-1 exhibits moderate affinity for double-stranded DNA34 (Kb = ca. 106 M−1). The binding constant for the bisintercalator has not been reported, but a similar dimeric version of the monointercalator ethidium exhibits ca. 1000-fold higher affinity35 than its monomeric counterpart, implying that YOYO-1 should bind with Kb of at least 109 M−1. The dyes also have large extinction coefficients (εmax), 52,000 M−1 cm−1 for YO-PRO-1 and 98,900 M−1 cm−1 for YOYO-1 in DNA, both at 491 nm. Organic dyes often form aggregates in which the extinction coefficient is lower than expected due to pi stacking, but the DNA templates will prevent this because each dye will bind to a distinct intercalation site. Moreover, the twist of the DNA helix will cause the transition moments of successive dyes to be misaligned. Therefore, minimal perturbation of the extinction coefficient is to be expected, and a DNA 3WJ with 15 YO-PRO-1 molecules should have an effective εmax ≈ 780,000 M−1 cm−1. Moreover, these dyes have little to no intrinsic fluorescence when free in solution but show strong fluorescence when bound to DNA (quantum yield φf = 0.44 for YO-PRO-1 and 0.52 for YOYO-1)36. Overall, the extinction coefficients and fluorescence quantum yields for the DNA nanotags will be comparable to widely used phycobiliproteins while having approximately one-tenth the molecular weight.

Andrea L. Benvin, et al. J Am Chem Soc. ;129(7):2025-2034.

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