Wojciech M Professor kwiatek and his team studied the methylation difference between euchromatin and heterochromatin in a single chromosome by using nanoir based on PtIr, realized the non fluorescence labeled single chromosome methylation analysis and the identification of euchromatin heterochromatin, and further detected the positioning information of the anticancer drug pt-103 on a single chromosome, which is the anticancer drug The mechanism of the drug provides an intuitive observation method. This achievement (infrared spectroscopic mapping of a single metaphase chrome) was published in the Journal of nuclear acids research on July 25, 2019 (nuclear acids res. 2019, 47 (18): E108. Doi: 10.1093/nar/gkz630).

There are two different components in human single chromosome, euchromatin and heterochromatin. Heterochromatin has a stronger tendency of methylation. DNA methylation can regulate gene expression, change the mechanical properties of DNA and affect the interaction intensity between DNA and protein or drugs, thus affecting the physiological activities of cells. DNA methylation is also closely related to the occurrence of cancer. It is very important to study the degree and region of chromosome methylation. The traditional super-resolution optical method needs fluorescence labeling, and it can not completely reflect the original methylation region distribution of chromosome, and can not get the chemical composition information of the labeled region because of the labeling miss or fluorescence quenching. Because of its low resolution, the traditional micro infrared technology can not characterize the chemical information distribution of a single chromosome. Afm-ir technology based on photothermal induced resonance (PtIr) can realize the spatial distribution of chemical information with a resolution of 10 nm, which is an ideal way to study the chemical information of a single chromosome. Firstly, the infrared spectrum of hypermethylation region and hypomethylation region of single chromosome was studied. As shown in Fig. 1b, the afm-ir spectrum of chromosome can significantly see the characteristic signals corresponding to DNA (nucleic acid C = O bond vibration, ring vibration and phosphate O-P-O vibration absorption), corresponding to the characteristic absorption signals of protein (amide I and amide II characteristic absorption) and the vibration signals corresponding to the methylation of nucleic acids, which are in good agreement with the infrared spectra measured by the synchrotron radiation infrared spectroscopy (sr-ftir) on the nucleus. The unmethylated DNA spectrum obtained by atr-fitr does not contain the corresponding protein and nucleic acid methylation signals. Further analysis of the IR spectra of methylated DNA and unmethylated DNA showed that the vibration signal of methyl in the IR spectrum of chromosome mainly came from the methylation behavior of DNA in chromosome rather than the methyl vibration of protein in chromosome.

Then, the spatial distribution of methylation in a single chromosome was studied, and the relationship between the methylation of euchromatin and heterochromatin was analyzed. Figure 2 shows the results of spatial distribution analysis of chemical absorption of single chromosome by nanoir. The distribution of 1240 cm-1 absorption (phosphate O-P-O antisymmetric stretching vibration) in chromosome reflects the distribution of DNA in chromosome (Figure 2C). The distribution of 2952 cm-1 absorption (methyl antisymmetric stretching vibration) in chromosome reflects the degree of DNA methylation in chromosome (Figure 2D). Because the strong infrared absorption region of DNA can also enhance the methylation absorption signal, the heterogeneity of methylation distribution is not obvious. Therefore, we use 1240 cm-1 absorption distribution to normalize the absorption distribution of 2952 cm-1. After normalization, the 2922 cm-1 / 1240 cm-1 signal can see obvious banded structure (Fig. 2e). The region with strong signal corresponds to the highly methylated heterochromatin region in the chromosome. The characteristics of the bands in the heterochromatin region are in good agreement with the fluorescence imaging obtained by immunofluorescence staining (Fig. 2F, g).

The authors extracted a pair of X chromosomes from a single cell for identification. One of the active X chromosomes contained hypomethylated euchromatin and hypermethylated heterochromatin, while the other was almost all hypermethylated heterochromatin. The infrared absorption distribution based on afm-ir can well show the band structure of methylated heterochromatin of active X chromosome (Fig. 3b) and all highly methylated heterochromatin regions of inactive X chromosome (Fig. 3e). The distribution of chemical signals collected by afm-ir in nano micro area was verified by immunofluorescence imaging (Fig. 3C, f). The results show that afm-ir technology can acquire and analyze the chemical information of single chromosome.

The combination of pt-103, a new anticancer drug, on chromosome was studied by using the characteristics of afm-ir that can obtain chemical information at nanometer scale with high resolution. The chromosomes of pt-103 cells were extracted and analyzed by afm-ir. Pt-103 has strong infrared absorption near 2916 cm-1 (Fig. 4G). The morphological characterization and the absorption distribution at 1240 cm-1 showed that there was no significant difference between the chromosomes of conventional culture cells (control group) and those of pt-103 treatment. In the 2916 cm-1 infrared absorption distribution, the chromosome treated by pt-103 has an obvious bright spot (Fig. 4F), corresponding to the position of drug binding. Furthermore, the infrared spectra collected in the drug binding region (Fig. 4f, G position I) and in the vicinity of the binding region (Fig. 4f, G position II) showed significant heterogeneity. There was significant signal absorption of pt-103 in the drug binding area (2916 cm-1, 2848 cm-1 and 1610 cm-1). There was no significant signal absorption of pt-103 near the drug binding area. Further principal component analysis (PCA) showed that pt-103 was preferentially combined with highly methylated heterochromatin region, which showed that afm-ir technology had a broad application prospect in drug research and development, mechanism research and other fields.

This work was completed by using the unique probe resonance enhanced photothermal induced resonance (PtIr) technology of Bruker through the nanoir series equipment. The new nano IR 3 can be used for infrared spectrum acquisition and chemical information distribution imaging based on contact mode. Based on tapping IR technology, the chemical information spatial resolution of 10 nm can be achieved. It can also be used for infrared spectrum acquisition and chemical information distribution imaging. It provides researchers with high resolution infrared spectrum information on nanometer scale, and provides powerful tools for cell biology, biophysics, drug development and disease mechanism research. Fast spectrum acquisition technology can achieve second level infrared absorption spectrum acquisition, Hyperspectra function can achieve fast spectrum acquisition for each pixel of the sample, providing strong support for comprehensive understanding of the infrared information of the sample.

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