Infrared nanospectroscopic mapping of a single metaphase chromosome

Abstract

The integrity of the chromatin structure is essential to every process occurring within eukaryotic nuclei. However, there are no reliable tools to decipher the molecular composition of metaphase chromosomes. Here, we have applied infrared nanospectroscopy (AFM-IR) to demonstrate molecular difference between eu- and heterochromatin and generate infrared maps of single metaphase chromosomes revealing detailed information on their molecular composition, with nanometric lateral spatial resolution. AFM-IR coupled with principal component analysis has confirmed that chromosome areas containing euchromatin and heterochromatin are distinguishable based on differences in the degree of methylation. AFM-IR distribution of eu- and heterochromatin was compared to standard fluorescent staining. We demonstrate the ability of our methodology to locate spatially the presence of anticancer drug sites in metaphase chromosomes and cellular nuclei. We show that the anticancer 'rule breaker' platinum compound [Pt[N(p-HC6F4)CH2]2py2] preferentially binds to heterochromatin, forming localized discrete foci due to condensation of DNA interacting with the drug. Given the importance of DNA methylation in the development of nearly all types of cancer, there is potential for infrared nanospectroscopy to be used to detect gene expression/suppression sites in the whole genome and to become an early screening tool for malignancy.

Single molecule secondary structure determination of proteins through infrared absorption nanospectroscopy

“…On the other hand, infrared nanospectroscopy based on thermochemical detection (atomic force microscopy-infrared spectroscopy (AFM-IR)) measures directly the light absorbed by a sample by photothermal induced resonance 17,18 . Thus, the infrared absorption spectra produced are not affected by scattering effects or specific nanoscale selection rules, and as such they are in agreement with conventional bulk results [19][20][21][22][23][24][25][26] . To date, however, the sensitivity of AFM-IR has been limited to the measurements of large (>0.3 μm) and flat (~2-10 nm) selfassembled monolayers or biomolecular aggregates composed of several hundreds of molecules and has not demonstrated the ability to characterise single biomolecules 23,27 .…”

Ultrathin Polydopamine Films with Phospholipid Nanodiscs Containing a Glycophorin A Domain

“…[ 40,41 ] AFM‐IR is thus capable to acquire and correlate simultaneously the morphological and chemical properties of thin films with 3–8 nm thickness with a spatial resolution in the order of the radius of the AFM probe (≈10–20 nm). [ 42,43 ] The PDA film electropolymerized on gold appeared highly homogeneous (Figure 3A). After acquiring the three‐dimensional morphology of the sample, nanoscale resolved IR spectra were acquired by AFM‐IR between 1800 and 1250 cm −1 .…”

Stearoyl-CoA Desaturase 1 Activity Determines the Maintenance of DNMT1-Mediated DNA Methylation Patterns in Pancreatic β-Cells

“…We analyzed the spatial distribution of the integrated band characteristic of the stretching of bonds between phosphate and oxygen atoms in the DNA backbone in the spectral range of 1280–1215 cm −1 (υ asym (OPO)), which refers to the DNA distribution, and the amide I band in the spectral range of 1695–1630 cm −1 , that conforms to the distribution of proteins (mainly histones). We also investigated the spatial distribution of the integrated band in the spectral range of 2900–2850 cm −1 (υ sym (CH 3 )) that corresponded to the arrangement of -CH 3 groups [ 23 , 24 ]. The distribution of proteins and DNA within the chromosomes was similar in the SCD1 inhibitor-treated and control samples ( Figure 2 C).…”