Electroporation and Microinjection Successfully Deliver Single-Stranded and Duplex DNA into Live Cells as Detected by FRET Measurements

<div><p>Förster resonance energy transfer (FRET) technology relies on the close proximity of two compatible fluorophores for energy transfer. Tagged (Cy3 and Cy5) complementary DNA strands forming a stable duplex and a doubly-tagged single strand were shown to demonstrate FRET outside of a cellular environment. FRET was also observed after transfecting these DNA strands into fixed and live cells using methods such as microinjection and electroporation, but not when using lipid based transfection reagents, unless in the presence of the endosomal acidification inhibitor bafilomycin. Avoiding the endocytosis pathway is essential for efficient delivery of intact DNA probes into cells.</p></div

Electroporation confocal microscopy images.

<p>Left: Cy3 and Cy5 tagged DNA (<b>S1:S2</b>) duplex added to cells via electroporation and imaged using confocal microscopy. Images A/E represents the Cy3 channel; B/F the Cy5 channel the nuclear stain channel; C/G the bright field channel and D/H an overlay of all the channels. Images A–D are excited with a 543 nm laser only. Images E–H are excited with both the 543 and 633 nm lasers. Right: Intracellular fluorescence intensity from images A/B and E/F. Data are expressed as mean ± s.e.m from at least ten cells (p = 0.001 to 0.01).</p

Schematic of Cy3 and Cy5 tagged DNA.

<p>a) Complementary DNA strands are individually tagged with Cy3 and Cy5 fluorophores (<b>S1</b> and <b>S2</b> respectively). When in close enough proximity the Cy3 can donate energy to Cy5 through FRET. In this case, FRET can only occur when the two complementary strands form a duplex. b) Single strand DNA can be tagged at either end with Cy3 and Cy5 (<b>S3</b>). FRET can occur as long as the single strand remains intact.</p

Tagged oligonucleotides synthesized.

<p>Tagged oligonucleotides synthesized.</p

Fixed cell confocal microscopy images.

<p>Left: Cy3 and Cy5 tagged DNA duplex (<b>S1:S2</b>) added to fixed/permeabilised cells and imaged using confocal microscopy. Images A/E represents the Cy3 channel; B/F the Cy5 channel; C/G the bright field channel and D/H an overlay of all the channels. Images A–D are excited with the 543 nm laser. Images E–H are excited with both the 543 and 633 nm lasers. Right: Intracellular fluorescence intensity from images A/B and E/F. Data are expressed as mean ± s.e.m from at least ten cells (p<0.001).</p

Emission spectra of Cy3 and Cy5 DNA.

<p>Titration of Cy5 tagged DNA (<b>S2</b>) into Cy3 tagged DNA (<b>S1</b>), showing resulting Cy5-Cy3 FRET upon duplex formation (excitation wavelength = 554 nm). The emission intensities centred at 570 nm and 670 nm correspond to emission from Cy3 and Cy5 respectively (conditions: 1 µM DNA, 100 mM NaCl, and pH 7.0 sodium phosphate buffer). The spectra are subtracted for the spectrum of <b>S2</b> alone, excited at 554 nm, which gave a small signal caused by direct excitation of the Cy5 chromophore.</p

Chemical transfection confocal microscopy images.

<p>Left: Cy3 and Cy5 tagged DNA duplex (<b>S1:S2</b>) added to cells via chemical transfection using Lipofectamine and imaged using confocal microscopy. Images A/E represents the Cy3 channel; B/F the nuclear stain channel; C/G the Cy5 channel and D/H an overlay of all the channels. Images A–D are excited with a 543 nm laser only. Images E–H are excited with both the 543 and 633 nm lasers. Right: Intracellular fluorescence intensity from images A/C and E/G. Data are expressed as mean ± s.e.m from at least ten cells (p<0.001).</p

Photocontrolled Binding and Binding-Controlled Photochromism within Anthracene-Modified DNA

Modified DNA strands undergo a reversible light-induced reaction involving the intramolecular photodimerization of two appended anthracene tags. The photodimers exhibit markedly different binding behavior toward a complementary strand that depends on the number of bases between the modified positions. By preforming the duplex, photochromism can be suppressed, illustrating dual-mode gated behavior

Single Site Discrimination of Cytosine, 5‑Methylcytosine, and 5‑Hydroxymethylcytosine in Target DNA Using Anthracene-Tagged Fluorescent Probes

The ability to discriminate between epigenetic variants in DNA is a necessary tool if we are to increase our understanding of the roles that they play in various biological processes and medical conditions. Herein, it is demonstrated how a simple two-step fluorescent probe assay can be used to differentiate all three major epigenetic variants of cytosine at a single locus site in a target strand of DNA