Contribution of Ribonucleic Acid (RNA) to the Fourier Transform Infrared (FTIR) Spectrum of Eukaryotic Cells

Abstract

We report on an optimized protocol for the digestion of cellular RNA, which minimally affects the cell membrane integrity, maintaining substantially unaltered the vibrational contributions of the other cellular macromolecules. The design of this protocol allowed us to collect the first Fourier transform infrared (FTIR) spectra of intact hydrated B16 mouse melanoma cells deprived of RNA and to highlight the in-cell diagnostic spectral features of it. Complementing the cellular results with the FTIR analysis of extracted RNA, ds-DNA, ss-cDNA and isolated nuclei, we verified that the spectral component centered at ∼1220 cm is a good qualitative and semiquantitative marker of cellular DNA, since it is minimally affected by cellular RNA removal. Conversely, the band centered at ∼1240 cm, conventionally attributed to RNA, is only a qualitative marker of it, since its intensity is majorly influenced by other macromolecules containing diverse phosphate groups, such as phospholipids and phosphorylated proteins. On the other hand, we proved that the spectral contribution centered at ∼1120 cm is the most reliable indicator of variations in cellular RNA levels, that better correlates with cellular metabolic activity. The achievement of these results have been made possible also by the implementation of new methods for baseline correction and automated peak fitting, presented in this paper.

The quality is in the eye of the beholder: The perspective of FTIR and UV resonant Raman spectroscopies on extracted nucleic acids

“…Conversely, guanidinium hydrochloride in solution has only a strong peak in this region at about 1,670 cm −1 . This observation indicates that the dehydration of our sample is not complete, as suggested also by the position of the asymmetric band of phosphates, which is peaked at about 1,225 cm −1 , revealing that DNA is folded in physiological B‐form . The same information can be retrieved from the position of the in‐plane carbonyl stretching band of adenine–thymine base pairing, peaked at ~1,730 cm ‐1 .…”

“…The guanidinium spectrum is also characterized by a broad and relatively weak band in the 1,300–1,000 cm −1 spectral range. This band covers the contributions of asymmetric and symmetric stretching bands of phosphates moieties of DNA and RNA, which are the most important for obtaining information on nucleoside‐specific interaction and nucleic acid conformation . In fact, these bands are characteristic of vibrations localized to the sugar and sugar‐phosphate moieties that give rise to vibrational modes sensitive to backbone conformation and sugar puckering …”

“…Curve a in Figure shows the first ATR‐FTIR spectrum of the dehydration series of B16‐RNA obtained with the tailored protocol: no features of ethanol were visible in the region below 1,100 cm −1 from the beginning of the measurements. B16‐RNA spectra from the sample extracted with the tailored protocol are consistent with those reported in literature, especially in the region of phosphates vibrations, where all the fingerprint peaks of ribose‐phosphate backbone are clearly discerned . As in the case of DNA protocol, we verified the repeatability of the optimized extraction procedure by measuring several solutions prepared in this way.…”

Placenta Stem/Stromal Cell–Derived Extracellular Vesicles for Potential Use in Lung Repair

“…Total nucleic acid content of EVs comprises DNA and RNA. It was previously shown that the RNA spectrum has two clear spectral components centered at 1240 and 1220 cm −1 , while the ds‐DNA band in the same region is sharper and peaked at 1226 cm −1 . The symmetric stretching band of nucleic acid phosphate is typically centered at ≈1086 cm −1 .…”

Infrared spectroscopy as a new tool for studying single living cells: Is there a niche?

“…The purpose of this review is to highlight the potential of FTIR technique for studying the macromolecular composition and eventually alteration of cell samples, moving from fixed to hydrated or even living ones [65,71,74]. Two different acquisition techniques (Attenuated Total Reflection ATR, and Transmission) will be described, each mode offering conveniences and challenges.…”