N-aryl pyrido cyanine derivatives are nuclear and organelle DNA markers for two-photon and super-resolution imaging

Live cell imaging using fluorescent DNA markers are an indispensable molecular tool in various biological and biomedical fields. It is a challenge to develop DNA probes that avoid UV light photo-excitation, have high specificity for DNA, are cell-permeable and are compatible with cutting-edge imaging techniques such as super-resolution microscopy. Herein, we present N-aryl pyrido cyanine (N-aryl-PC) derivatives as a class of long absorption DNA markers with absorption in the wide range of visible light. The high DNA specificity and membrane permeability allow the staining of both organelle DNA as well as nuclear DNA, in various cell types, including plant tissues, without the need for washing post-staining. N-aryl-PC dyes are also highly compatible with a separation of photon by lifetime tuning method in stimulated emission depletion microscopy (SPLIT-STED) for super-resolution imaging as well as two-photon microscopy for deep tissue imaging, making it a powerful tool in the life sciences.


Feb 25 th 2021
Response to Reviewers We appreciate helpful comments from reviewers. We have carefully revised the manuscript according to the comments. Modified parts are shown with red characters in the text.
Reviewer #1: The authors present the pyrido cyanine molecules as molecular probes for live cell imaging of DNA. The idea of pyrido cyanine was in principle first followed by Gutsulyak and Romanko, when they synthesized methine dyes from 1-aryl-3-acetyllepidinium salts and by Leubner with 2,2'-, 2,4'-, and 4,4'-pyrido-and -azapyridocyanines. In this manuscript additional pyrido cyanines were synthesized and tested as DNA markers. The pyridine cyanines with the N-Aryl residues bind with high selectivity to DNA. The advantage of the developed dyes compared to the established Hoechst 33342 are that they allow to discriminate between mt-DNA and nuclear DNA. Although the manuscript provides results that are of interest, the paper would be more appropriate for a specialized journal in the field.
Thank you for your comments, but we can't help but think that the assessment of the novelty is not properly conducted. As this reviewer pointed out, there have been previous reportｓ on the synthesis of pyrido cyanine skeleton derivatives and we cite a appropriate paper in the text (Leubner, 1973). The novelty to be assessed in our manuscript is not the chemical structure itself, but rather its overwhelmingly superior properties as DNA probes (DNA selectivity, long-wavelength, cell permeability, organellar DNA stainability, and compatibility with advanced microscopes) when compared to widely known conventional DNA staining dyes, such as Hoechst and SYBR-Green. Given the importance of DNA fluorescent dyes as a research tool for many researchers in the life sciences, we believe our work makes a big leap in the field of molecular and cellular biology. Therefore, we are no doubt that the novelty of our work is suited to Nature Communication. Reviewer #2: the authors claimed the high selectivity of probe to DNA over RNA in term of the fluorogenic property, but the reason is not discussed at all. Similarly, its DNA binding mode and AT base-pair preference are not clearly explained.
Thank you for these important comments. In response to these comments, first, we examined a competitive experiment in DNA sequence using AATT DNA hairpin oligo and found that PC1 shares the same AATT DNA sequences with Hoechst (Fig. S3k). We also examined circular dichroism spectrum of PC1 complexed with dsDNA. As a result, The CD spectrum clearly showed a positive cotton effect in both dyes (Fig. S4), similar to that of minor-groove binders, such as Hoechst and DAPI, suggesting that PC1 binds to dsDNA with minor-groove. The similar binding mode to Hoechst would contribute the high selectivity of PC1 to DNA over RNA. Furthermore, to assess the reason why the DNA selectivity is higher than that of Hoechst, effects of (methyl)piperazine site, which is part of Hoechst, on fluorogenic properties was investigated. We synthesized a PC dye (PC 9) with a (methyl)piperazine site by replacing one of the two diethylamino groups of PC4 with (methyl)piperazine and examined the fluorogenic properties ( Fig. S2i). As a result, the fluorescent intensity of PC 9 increased 250-fold upon binding RNA, which was 5 times higher than that of PC4 (53-fold) (Fig. S2d, i), suggesting that the binding of the (methyl) piperazine moiety to RNA is thought to be a factor that reduces DNA selectivity. We have mentioned these new results in the text in revised manuscript. the author used the lifetime difference of PC1 between the nucleus and mitochondria for the selective imaging, however the mechanism of such difference is not described.
Thank you for this careful comment. The fluorescent lifetime depends on its energy dispersal to the surrounding environment. There are many possibilities, especially in living cells, but one major factor is the shortening of the fluorescent lifetime due to self-absorption between fluorescent dyes in the case of dye properties with small stokes-shift (Kristoffersen et al. 2014). First, to investigate whether the fluorescent lifetime of PC1 changes at high concentrations, we measured the fluorescent lifetime of PC1 at various concentrations of PC1 using a constant concentration of pBluescript II SK+ DNA vector. As a result, the fluorescent lifetime of PC1 was found to be shortened in a concentration-dependent manner possibly because of self-absorption (Fig. S9a). On the other hand, it is known that cationic fluorescent dyes tend to accumulate in mitochondria, which have membrane potential (Bunting et al. 1989 Biophys J). In fact, PC1 stains mitochondria DNA at less than 100 pM, while more than 1 nM of PC1 is required for staining nuclear DNA (Fig. 3). This suggests that the dye concentration is different between nuclear and mitochondrial compartments, even when cells were stained at a constant concentration. Therefore, it is possible that the increase in dye concentration may cause a shortening of the fluorescent lifetime in mitochondria due to self-absorption more easily than in the nucleus. To test this possibility, we stained cells with different PC1 concentrations and measured the fluorescent lifetime of PC1 in nuclear DNA and mitochondria DNA. We found that the fluorescent lifetime of PC1 in nuclear DNA was maintained up to 10 nM, while that in mitochondrial DNA was shortened above 100 pM (Fig. S9b). Taken together with these results, it is considered that the fluorescent lifetime of PC1 between nuclear DNA and mitochondrial DNA can be separated when staining at 1 nM, because the fluorescent lifetime in mitochondrial DNA is shortened due to self-absorption of PC1, while it is retained in nuclear DNA. We have mentioned these new results in the text in revised manuscript.