Polyamide Fluorescent Probes for Visualization of Repeated DNA Sequences in Living Cells

Abstract

DNA imaging in living cells usually requires transgenic approaches that modify the genome. Synthetic pyrrole-imidazole polyamides that bind specifically to the minor groove of double-stranded DNA (dsDNA) represent an attractive approach for in-cell imaging that does not necessitate changes to the genome. Nine hairpin polyamides that target mouse major satellite DNA were synthesized. Their interactions with synthetic target dsDNA fragments were studied by thermal denaturation, gel-shift electrophoresis, circular dichroism, and fluorescence spectroscopy. The polyamides had different affinities for the target DNA, and fluorescent labeling of the polyamides affected their affinity for their targets. We validated the specificity of the probes in fixed cells and provide evidence that two of the probes detect target sequences in mouse living cell lines. This study demonstrates for the first time that synthetic compounds can be used for the visualization of the nuclear substructures formed by repeated DNA sequences in living cells.

Synthesis of mouse centromere-targeted polyamides and physico-chemical studies of their interaction with the target double-stranded DNA

“…This increase seems to result from the changes of fluorescent quantum yield rather than from the changes of absorptivity upon the interaction with DNA. 39 In contrast, the fluorescent properties of the probes labeled by cyanine fluorophores F1-TO, F1-D133 and F1-S24 are more complex despite their better affinity. While the probes with the intercalating fluorophores TO and D133 demonstrate a better specificity (F1-TO is the best one that demonstrates more than 2 times higher fluorescence intensity with the cognate duplex compared to the non-cognate one), the minor groove binders 2993SL and S24 do not increase their fluorescence intensity and emit even higher fluorescence upon the interaction with the non-cognate DNA ( Figure S8, Supporting Information).…”

“…As it was already noted also for noncyanine fluorophores, 66,67,7 an unexpectedly strong sequence-specific increase of the fluorescence intensity (36 times, 9 times more than with non-cognate duplex) was observed upon binding of F1-FITC to the cognate DNA. 39 All the probes labeled by cyanines Cy3 or Cy5 demonstrate various increase of fluorescence intensity depending on the nature of the fluorophore and of the conjugated polyamide. This increase is observed both with the non-cognate Duplex 3 and the cognate Duplex 1; however, with the cognate duplex this increase is much higher.…”

“…39 Potency of the probes for cellular DNA imaging depends both on structural factors regulating the affinity and specificity of the probes to target DNA [40][41][42][43] and on their cellular uptake and nuclear penetration. Here we present a systematic physico-chemical in vitro study of the structural factors in order to optimize combinations of polyamides and fluorophores for better DNA affinity and fluorescent properties.…”

Interaction of fluorescently labeled pyrrole-imidazole polyamide probes with fixed and living murine and human cells

“…For simultaneous detection of both FISH and polyamide probes, the FISH protocol was applied first and followed by treatment with the fluorescent polyamide probes. [15] Images were recorded with a Zeiss epifluorescence inverted microscope (Axio Observer ZI) equipped with a Hamamatsu ORCA R2 camera, a plan apochromatic 63x1.4 NA oil-immersion objective and the following filters set: 49 shift free for Hoechst or DAPI (G365/FT395/BP44/50), 38 HE shift free for FITC (BP470/40, FT495, BP525/50), home-made sets for Cy3 (BP 546/10, FF555, BP 583/22) and for Cy5 (BP643/20, FF660, BP684/24). Immersion oil of refractive index 1.518 at 23°C was used.…”

“…Recently, we have used fluorescently labeled synthetic Nmethylpyrrole-N-methylimidazole (Py-Im) polyamides that bind specifically to the minor groove of double-stranded DNA for imaging major satellite sequences, which form the basis of pericentromeric heterochromatin foci, in living mouse cells [15]. This approach does not necessitate genome modifications; the polyamides easily penetrate into the cells without toxicity and specifically recognize their target native dsDNAs.…”