2′-Deoxy-5-(hydroxymethyl)cytidine: estimation in human cancer cells with a simple chemosensor

A pyrrole-based rhodamine conjugate (CS-1) has been developed and characterized for the selective detection and quantification of 2′-deoxy-5-(hydroxymethyl)cytidine (5hmC) in human cancer cells with a simple chemosensing method.

2 0 -Deoxy-5-(hydroxymethyl)cytidine (5hmC) is found in both neuronal cells and embryonic stem cells. It is a modied pyrimidine and used to quantify DNA hydroxymethylation levels in biological samples 1-3 as it is capable of producing interstrand cross-links in double-stranded DNA. It is produced through an enzymatic pathway carried out by the Ten-Eleven Translocation (TET1, TET2, TET3) enzymes, iron and 2-oxoglutarate dependent dioxygenase. [4][5][6][7] In the DNA demethylation process, methylcytosine is converted to cytosine and generates 5hmC as an intermediate in the rst step of this process which is then further oxidized to 5-formylcytosine (fC) and 5-carboxycytosine (caC) of very low levels compared to the cytosine level. 8 Though the biological function of 5hmC in the mammalian genome is still not revealed, the presence of a hydroxymethyl group can regulate gene expression (switch ON & OFF). Reports say that in articial DNA 5hmC is converted to unmodied cytosine when introduced into mammalian cells. 9,10 Levels of 5hmC substantially vary in different tissues and cells. It is found to be highest in the brain, particularly in nervous system and in moderate percentage in liver, colon, rectum and kidney tissues, whereas it is relatively low in lung and very low in breast and placenta. 11,12 The percentage of 5hmC content is much less in cancer and tumor tissues compared to the healthy ones. The reason behind this loss is the absence of TET1, TET2, TET3, IDH1, or IDH2 mutations in most of the human cancer cells which means decrease of methylcytosine oxidation. [13][14][15] This loss of 5hmC in cancer cells is being used as a diagnostic tool for the detection of early-stage of malignant disease. Few analytical methods [16][17][18][19] such as glucosyltransferase assays, tungsten-based oxidation systems, and TET-assisted bisulte sequencing (TAB-Seq) or oxidative bisulte sequencing (oxBS-Seq) protocols are now developed to differentiate 5hmC from other nucleotide which are naturally occurred. There are also few methods such as liquid chromatography/tandem mass spectroscopy (LC/MS-MS), which determine the level of 5hmC in mammalian cancer cell. [20][21][22] However, these procedures are highly toxic and expensive due to requirement of catalyzation through enzymes or heavy metal ion and these techniques require expertise, facilities, much time and costs even beyond standard DNA sequencing. As a result, these detection techniques are currently inappropriate for the high-throughput screening of genome-wide 5hmC levels (performance comparison is shown in Table S1, ESI †).
Among all reputed methods uorescence detection method using chemosensors is signicantly important due to its indispensable role in medicinal and biological applications. [23][24][25][26][27] Chemosensors have been effectively explored to monitor biochemical processes and assays through in situ analysis in living systems and abiotic samples with much less time and cost.
In this contribution we prepared and characterize (Scheme S1 and Fig. S1-S3, ESI †) a pyrrole-rhodamine based chemosensor (CS-1) which shows efficient and selective uorescence signal for 5hmC in aqueous medium (Scheme 1). A transparent single crystal of CS-1 ( Fig. 1) was obtained by slow evaporation of the solvent from a solution of CS-1 in CH 3 CN. It crystallizes as monoclinic with space group P2 1 /n ( Fig. S4 and Table S2, ESI †).
Spectrophotometric and spectrouorimetric titrations were carried out to understand the CS-1-5hmC interaction with 1 : 1 binding stoichiometry (Fig. S5, ESI †) upon adding varying concentrations of 5hmC to a xed concentration of CS-1 (1 mM) in aqueous medium at neutral pH. Upon the addition of increasing concentrations of the 5hmC, a clear absorption band (K a ¼ 4.47 Â 10 5 M À1 , Fig. S6, ESI †) appeared to be centered at 556 nm with increasing intensity (Fig. 2a). On the other hand, for the uorescence emission spectra of CS-1 (Fig. 2b), upon irradiation at 325 nm, an emission maxima at 390 nm was observed, which was attributed to the uorescence emission from the donor unit i.e. the pyrrole moiety of CS-1. When 5hmC were added, due to rhodamine moiety CS-1 showed a 95-fold increase in uorescence at 565 nm (K a ¼ 4.61 Â 10 5 M À1 , Fig. S7, ESI †) with the detection limit of 8 nM (Fig. S8, ESI †). The binding of 5hmC induces opening of the spirolactam ring in CS-1, inducing a shi of the emission spectrum. Subsequently, increased overlap between the emission of the energy-donor (pyrrole) and the absorption of the energy-acceptor (rhodamine) greatly enhances the intramolecular FRET process, 28,29 producing an emission from the energy acceptor unit in CS-1.
In order to establish the sensing selectivity of the chemosensor CS-1, parallel experimentations were carried out with other pyrimidine/purine derivatives such as 5-methylcytosine, cytosine, cytidine, thymine, uracil, 5-hydroxymethyluracil, adenine and guanine. Comparing with other pyrimidine/purine derivatives the abrupt uorescence enhancement was found upon addition of 5hmC to CS-1 while others do not make any uorescence changes under UV lamp (Fig. 3, lower panel). Furthermore, the prominent color change from colorless to deep pink allows 5hmC to be detected by naked eye (Fig. 3, upper panel). The above observation shows consistency with the uorescence titration experiments where no such binding of CS-1 with other pyrimidine/purine derivatives was found (Fig. S9, ESI †).
pH titration reveals that CS-1 becomes uorescent below pH 5 due to the spirolactam ring opening of rhodamine. However, it is non-uorescent at pH range of 5-13. Upon addition of 5hmC to CS-1 shows deep red uorescence in the pH range of 5-8 (Fig. S10, ESI †). Considering the biological application and the practical applicability of the chemosensor pH 7.4 has been preferred to accomplish all experiments successfully.
In 1 H NMR titration (Fig. S11, ESI †), the most interesting feature is the continuous downeld shi of aromatic protons on the pyrrole moiety of CS-1 upon gradual addition of 5hmC. This may be explained as the decrease in electron density of the pyrrole moiety upon binding with 5hmC through hydrogen bonding. Xanthene protons to be shied downeld upon spirolactam ring opening indicates the probe to coordinate with 5hmC and electrons are accumulated around 5hmC. In 13 C NMR titration the spiro cycle carbon peak at 65 ppm was shied to 138 ppm along with a little downeld shi of the aromatic region of CS-1 (Fig. S12, ESI †). This coordination led to the spiro cycle opening and changes to the absorption and emission spectra, further evident by mass spectrometry (Fig. S13, ESI †), which corroborates the stronger interaction of CS-1 with 5hmC.
The experimental ndings were validated by density functional theory (DFT) calculations using the 6-31G+(d,p) method basis set implemented at Gaussian 09 program. Energy optimization calculations presented the conformational changes at the spirolactam position of CS-1 while 5hmC takes part to accommodate a probe molecule. Aer CS-1-5hmC complexation the energy is minimized by 19.45 kcal from the chemosensor CS-1, indicating a stable complex structure ( Fig. 4 and Table S3, ESI †). This theoretical study strongly correlates the experimental ndings.
The desirable features of CS-1 such as high sensitivity and high selectivity at physiological pH encouraged us to further evaluate the potential of the chemosensor for imaging 5hmC in live cells (Fig. 5). A549 cells (Human cancer cell A549, ATCC no. CCL-185) treated with CS-1 (1 mM) exhibited weak uorescence, whereas a deep red uorescence signal was observed in the cells stained with CS-1 (1 mM) and 5hmC (10 mM), which is in good Scheme 1 5hmC-induced FRET OFF-ON mechanism of the chemosensor CS-1.   agreement with the FRET OFF-ON prole of the chemosensor CS-1 in presence of 5hmC, thus corroborating the in-solution observation (Fig. S14, ESI †). Cytotoxicity assay measurement shows that the chemosensor CS-1 does not have any toxicity on the tested cells and CS-1-5hmC complex does not exert any signicant adverse effect on cell viability at tested concentrations (Fig. S15, ESI †). As far as we are aware, this is the rst report where we are executing the possible use of the pyrrolerhodamine based chemosensor for selective recognition of 5hmC in living cells. These ndings open an avenue for future biomedical applications of the chemosensor to recognize 5hmC.
The concentration of 5hmC was also quantied from A549 human cancer cells. Lysate of 10 7 A549 cells was added to 1 mM of CS-1 and the uorescence signal was recorded. Presence of 5hmC in these cancer cells was detected with the help of CS-1-5hmC standard uorescence curve (Fig. 6) using the selective detection ability of the chemosensor CS-1.
From the standard curve it was found that the concentration of 5hmC in the tested sample was 0.034 mM present in 16.7 mm 3 A549 cell volume ( Table 1). The above result was authenticated by spiking a real sample with a known concentration of 5hmC and different uorescence signals were observed by adding known volumes from each sample to the CS-1. The recovery of the spiked samples was estimated to be over 99% (Table S4, ESI †). Assay of 5hmC was further validated from multiple samples of A549 human cancer cells using CS-1. Increasing fold of uorescence signals was also statistically validated aer calculating the Z 0 value (Table S5, ESI †). All tested samples shows the Z 0 score value more than 0.9, indicating an optimized and validated assay of 5hmC.

Conclusions
In conclusion, a chemosensor CS-1 has been developed for the rapid detection of 5hmC with low cost. The selective detection and quantication of 5hmC was successfully demonstrated in human cancer cells at neutral pH with very low concentration. With this potentiality of CS-1 one can successfully apply this method to estimate 5hmC in disease cancer tissues and other biological samples of patients with metabolic dysfunction or various carcinomas.

Conflicts of interest
There is no conicts to declare.