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  • Acknowledgments We acknowledge the Danish Council

    2020-10-22

    Acknowledgments We acknowledge the Danish Council for Independent Research (Grant no. 11-105250) and the Carlsberg Foundation (Grant no. 2011-01-0567) for funding this work.
    Introduction DNA is vital as a biological target to design diagnostic agents as well as therapeutic drugs. The recognition and sensing of DNA are important because many diseases are caused by genetic disorders. Therefore, considerable attentions have been dedicated to the development of new fluorescent probes for sensitive, rapid and cost-effective DNA detection [1]. Many small molecule pigments such as ethidium bromide (EB) [2] and Hoechst 33258 [3] are exploited to quantify DNA. EB can intercalate into DNA because the planar and aromatic moieties can slide between the neighbouring 1027 pairs [4]. And Hoechst 33258 is classical groove binding agent due to the special falcate fitting the spiral shape of the DNA groove [5,6]. Their common feature is that the intensity of the emission signal is greatly increased upon DNA binding [3,7]. Whereas, most DNA dyes and related DNA testing assays have some defaults, for example, the growing environmental ionic strength can impede combination of the small molecular dyes and DNA, and weaken the emission signal [8]. For instance, EB showed decreasing fluorescence at high ionic strength and cell membrane-impermeable, which limited the applications of the small molecules in biology and medicine [9]. Therefore, novel DNA fluorescent probes are worthy of developing for sensitive detection of DNA selectively. Totally three basic interactional means would happen when small molecules bind to DNA: intercalation, groove binding as well as electrostatic interactions. The structure of the small molecules is a determinative factor for the binding types. A planar and aromatic structure of a molecule prefers to have an intercalation with double strand DNA [4,10]. Whereas, with a stretched crescent shape, such as DAPI, Hoechst 33258, netropsin, a molecule would tightly insert into the groove of DNA [3,11]. Carbazole derivatives are potentially good dyes to stain DNA in living cells [[12], [13], [14]]. In our previous work, we investigated the interacting types of DNA and γ,γ′‑diazacarbazoles [15] and γ‑carbolines [8]. γ,γ′‑Diazacarbazole dication (DPDI) was testified to bind to DNA by two modes of intercalative and groove interactions. At the NH of DPDI, replacement of H by a phenyl group, DPPDI binds to DNA by only groove binding [15]. In groove interactions, dications on the two diazacarbazoles combined with DNA to tightly bind to the spiral shape of the DNA groove, with the group of NH or NPh on the opposite side of the groove [15]. Subsequently, two cationic γ‑carbolines (MPII and DPII), was confirmed to intercalate to DNA tightly [8]. And on the structure of MPII, the detachment of H from 5‑NH leaded to be more water soluble but low interaction strength with DNA than DPII [8]. The capability of γ,γ′‑diazacarbazoles and γ‑carbolines binding to DNA were sensitive to ionic strength [8,15]. Both γ,γ′‑diazacarbazoles and γ‑carbolines only can strongly interact with DNA in low ionic strength. We still need to develop carbazole derivatives to interact with DNA in high ionic strength for further applications in biology and medicine. Herein, to get better fluorescent probes suitable to be used in high ionic strength and for cell imaging, two cationic δ,δ′‑diazacarbazoles, 1‑Methyl‑5H‑pyrrolo[3,2‑b:4,5‑b′]dipyridinium iodide (MPDPI) and 1,5‑Dimethyl‑5H‑pyrrolo[3,2‑b:4,5‑b′]dipyridinium iodide (DPDPI, the structures of MPDPI and DPDPI are elaborated in Fig. 1) have been synthesized. DPDPI has a methyl group to replace NH on MPDPI. The spectral titrations of the two compounds with CT-DNA (Calf Thymus DNA) have shown that the two diazacarbazoles have a strong interaction with DNA. Further, CD and EB competition results disclosed that the two diazacarbazoles combined with DNA by intercalative interactions, and DPDPI have a stronger interaction than MPDPI. Furthermore, the two diazacarbazoles made the conformation of DNA compacted, which was demonstrated by images of DNA in the presence of diazacarbazoles with the atomic force microscopy (AFM). Moreover, we found that a strong boost of the fluorescence for either of the two compounds occurred when DNA was added, and with ionic strength increasing, the δ,δ′‑diazacarbazoles-DNA complexes displayed increasing fluorescence, indicating cationic δ,δ′‑diazacarbazole are promising DNA-targeted fluorescent probes in physiological conditions. Furthermore, cell uptake of DPDPI was successfully realized and strong fluorescent imaging was exhibited, indicating the potential application of δ,δ′‑diazacarbazoles for cell imaging.