Two‐Factor Fluorogenicity of Tetrazine‐Modified Cyanine‐Styryl Dyes for Bioorthogonal Labelling of DNA

Abstract

Two green fluorescent tetrazine-modified cyaninestyryl dyes were synthesized for bioorthogonal labelling of DNA by means of the Diels-Alder reaction with inverse electron demand. With DNA as target biopolymer the fluorescence of these dyes is released by two factors: (i) sterically by their interaction with DNA, and (ii) structurally via the conjugated tetrazine as quencher moiety. As a result, the reaction with bicyclononyne-modified DNA is significantly accelerated up to � 284,000 M À 1 s À 1 , and the fluorescence turn-on is enhanced up to 560 by the two-factor fluorogenicity. These dyes are cell permeable even in low concentrations and undergo fluorogenic reactions with BCN-modified DNA in living HeLa cells. The two-factor fluorescence release improves the signal-to-noise ratio such that washing procedures prior to cell imaging are not needed, which is a great advantage for live cell imaging of DNA and RNA in the future.

trans-Cyclooctene- and Bicyclononyne-Linked Nucleotides for Click Modification of DNA with Fluorogenic Tetrazines and Live Cell Metabolic Labeling and Imaging

“…These dNTPs need to be good substrates for intracellular DNA polymerases, and after their incorporation into DNA, the clickable handles of the nucleotides must be able to undergo a chemical post-labeling performed with suitably modified fluorophores in live cells. There have been many examples , of clickable nucleosides and nucleotides used in the chemical , or enzymatic − synthesis of clickable nucleic acid probes, including several dNTPs (mostly 2′-deoxyuridine nucleotides) bearing trans -cyclooctene (TCO), − bicyclononyne (BCN), or cyclopropene , moiety that undergo strain-promoted azide–alkyne cycloadditions with azides or IEDDA reactions with various tetrazines. However, these studies were so far limited to in vitro DNA labeling.…”

Two‐Factor Fluorogenic Cyanine‐Styryl Dyes with Yellow and Red Fluorescence for Bioorthogonal Labelling of DNA

“…), 7.18 (d, J = 16.1 Hz, 1H, RÀ CH=CHÀ R), 4.21 (s, 3H, RÀ CH 3 ), 3.91 (s, 3H, RÀ CH 3 ). 13 C-NMR (101 MHz, DMSO-d 6 ): δ = 153.0, 144.6, 138.5, 133.4, 132.5, 125.5, 124.0, 123.5, 122.0, 117.9, 113.0, 112.4, 110.9, 46.5, 33.6. HR-ESI-MS (m/z): [M] + calcd.…”

“…(m, 1H), 3.17 (d, J = 5.2 Hz, 2H), 2.99-2.93 (m, 2H), 2.20-2.13 (m, 3H), 1.83-1.78 (m, 2H), 1.55-1.52 (m, 2H). 13 14: Under argon atmosphere, 21 (25 mg, 81.3 μmol, 1.00 eq) was dissolved in MeOH. NaOH solution (15 g dissolved in 15 mL H 2 O) was added and stirred for 2 h at ambient temperature.…”

“…The product was obtained as a yellow solid (7 mg, 20 %) 1 H-NMR (400 MHz, DMSO-d 6 ): δ = 8.10 (s, 1H), 7.74 (s, 2H), 7.70 (s, 1H), 6.48-6.45 (m, 1H), 5.26 (s, 1H), 5.07 (s, 1H), 4.34-4.32 (m, 1H), 4.08-4.06 (m, 2H), 4.03-4.02(m, 2H), 3.82 (s, 1H), 3.58-3.55 (m, 1H), 3.51-3.48 (m, 1H), 2.19-2.14 (m, 3H), 1.84-1.80 (m, 2H). 13 DNA1 and DNA2: According to literature, DNA was synthesized via a DNA synthesizer. [10] Oligonucleotides were synthesized by using standard solid-phase phosphoramidite synthesis protocol on a H-6 DNA/RNA synthesizer by K&A Laborgeräte.…”

The Efficiency of Metabolic Labeling of DNA by Diels–Alder Reactions with Inverse Electron Demand: Correlation with the Size of Modified 2′-Deoxyuridines

“…For the kinetic investigations in vitro , a tetrazine-modified cyanine-styryl dye ( 6 ) developed by our group, which is structurally based on our previously published photostable dyes, was applied because it provides a two-factor fluorogenicity with DNA . The fluorescence of 6 is nearly completely quenched due to the attached tetrazine as quencher moiety. ,,, The fluorogenic reaction with the dienophiles in the 2′-deoxyridines 1 – 5 converts the quenching tetrazine into the nonquenching diazine (Figure ). Additionally, the fluorescence is enhanced upon DNA binding due to restricted rotation around the bridging carbon–carbon bonds in the center of the cyanine-styryl dye.…”