Studying Epigenetic Modifications of DNA Using UV Resonance Raman Spectroscopy


Epigenetic modifications of DNA are crucial for regulating gene expression and are associated with several human diseases, such as cancer. Another layer of epigenetic information may lie in alternative DNA structures like G-quadruplexes, which can influence gene transcription (1). Recent research has revealed that cytosine modifications within G-quadruplex loops can impact their stability and structure.

Bruno Pagano, full Professor of Physical Chemistry at the University of Naples Federico II (Italy) and his team have turned to UV resonance Raman (UVRR) spectroscopy to better understand these interactions. Spectroscopy spoke to Prof. Pagano about his work and the potential of UVRR spectroscopy as a valuable tool for studying G-quadruplex structures in biologically relevant conditions.

Professor Bruno Pagano of the University of Naples Federico II (Italy). Image Credit: Bruno Pagano

Professor Bruno Pagano of the University of Naples Federico II (Italy). Image Credit: Bruno Pagano

Can you provide a brief overview about how alternative DNA secondary structures, such as G-quadruplexes, can influence gene transcription. Why is it important to study these structures?

DNA can adopt a variety of alternative conformations, including G-quadruplex structures that can arise under physiological conditions from guanine-rich DNA sequences. Initially regarded as a structural curiosity, recent evidence highlights their involvement in key genome functions, such as transcription, replication, and epigenetic regulation, with significant connections to cancer biology. These G-quadruplex motifs are not randomly distributed in the human genome, but they are notably enriched in gene promoters, especially in oncogenes. Recent experimental studies have mapped G-quadruplex structures in chromatin to regulatory regions upstream of the transcription start sites of actively transcribed genes in human cells, further reinforcing the connection between G-quadruplexes and transcription. These structures can either impede or facilitate the binding of transcription factors to DNA, thereby regulating gene expression. Understanding these structures is vital because it opens up opportunities for targeted gene regulation and the development of therapies for diseases in which gene expression plays a crucial role. In essence, studying G-quadruplex DNA structures provides valuable insights into the complex mechanisms underlying gene transcription and its potential applications in biomedicine.

Why did you select UV resonance Raman spectroscopy (UVRR) as the preferred technique for the analysis that you were doing?

The formation of G-quadruplex DNA structures can be detected by several spectroscopic techniques, including nuclear magnetic resonance (NMR), UV, and circular dichroism (CD). However, given that conformational variations often lead to changes in the vibrational frequencies of nucleic acids, vibrational spectroscopic techniques can represent additional valuable tools to investigate DNA structural polymorphism. Moreover, vibrational spectroscopies had already been employed to study epigenetic modifications of duplex DNA. In this regard, techniques based on surface-enhanced Raman spectroscopy (SERS) have been widely used to investigate cytosine methylation. We opted for UVRR because it could provide insights into cytosine epigenetic modifications along with conformational changes in G-quadruplex DNA, enabling label-free DNA analysis and giving the possibility to work in an aqueous solution, thus keeping the DNA in near-physiological conditions.

What were the main challenges that you and your team had to overcome in this study?

Initially, UVRR measurements were conducted using two different excitation wavelengths, namely 266 nm and 228 nm. However, the spectra collected with synchrotron radiation source at 228 nm, while potentially more suited to infer about cytosine chemical modifications, were affected by a poor signal-to-noise (S/N) ratio because of the low synchrotron radiation power, making impossible to observe spectral differences between various G-quadruplex forming sequences. This could be because of both the loss of the resonance effect of guanine and adenine and of the low excitation power and spectral resolution available with the synchrotron radiation source. Fortunately, we achieved high spectral S/N ratio when using the continuous wave UV laser source at 266 nm, allowing us to investigate the DNA structural conformations through a careful spectral line-shape analysis. Additionally, we encountered challenges related to band assignment because of the presence of overlapping vibrations. Indeed, a thorough UVRR spectroscopic analysis was necessary for the identification and unambiguous assignment of bands corresponding to the vibrations of each of the four nucleobases.

Can you talk about the role circular dichroism (CD) spectroscopy played in your study? What did the CD experiments reveal that were integral to the conclusions?

In the absence of high-resolution 3D structures of the DNA molecules, to ensure correct interpretation of the results, UVRR experiments should be performed in combination with other low-resolution techniques like CD spectroscopy. CD spectroscopy proves to be a valuable tool for examining the conformation of nucleic acids in solution. Although a CD spectrum does not provide detailed structural information, it offers a reliable means of determining the conformational state of DNA when compared to reference CD spectra. CD aids in establishing the orientation of DNA strands and the conformation of guanine glycosidic torsion angles (syn or anti) within a given G-quadruplex structure. In this study, CD was employed to discriminate among different folding topologies of G-quadruplexes that are characterized by distinctive marker bands. For example, parallel structures (with all strands having the same orientation) show a positive band at approximately 260 nm and a negative band at approximately 240 nm, whereas the spectra of antiparallel G-quadruplexes display a negative band at around 260 nm and positive band at about 290 nm. In this context, the role of CD spectroscopy was crucial in helping us to interpret the data obtained by UVRR experiments.

What is the significance of cytosine epigenetic modifications in DNA G4s? Why is it important to study these modifications?

Cytosine epigenetic modifications in DNA G-quadruplexes are of great significance because they could modulate the stability and formation of G-quadruplex structures. Methylation of cytosines, for example, can either enhance or inhibit G-quadruplex formation, thereby influencing gene regulation. Studying these modifications is crucial because they could provide a link between epigenetic regulation and the structural polymorphism of DNA. Understanding how cytosine modifications impact G-quadruplexes can shed light on their role in gene expression, potentially leading to insights into diseases associated with epigenetic dysregulation and offering avenues for targeted therapeutic interventions.

You and your team also studied conformational plasticity of DNA secondary structures (2) and RNA G-quadruplex complex binders (3). What information and conclusions did you bring from those studies to help your team with this current study?

In the current study, we have proposed to exploit the advantages of UVRR spectroscopy to investigate cytosine epigenetic modifications along with conformational changes in G-quadruplex DNA. This was possible thanks to the information and conclusions reached in our previous studies, which were fundamental as they allowed us to understand that it was possible to use UVRR to study the noncanonical nucleic acid structures and the dependence of their folding on the primary structure, the molecular environment, and interactions with drugs. Furthermore, in this study, UVRR bands were identified and assigned to characteristic G-quadruplex molecular vibrations by comparing literature data, including our previous works.

What are the next steps that you and your team plan on taking in your study of G-quadruplexes, both DNA and RNA?

The study of noncanonical DNA and RNA structures is incredibly fascinating because of their unique characteristics and biological significance. Our plans are to expand our study including other biologically relevant sequences capable of forming these structures and investigating their interactions with potential drugs. In this regard, G-quadruplex-interacting peptide molecules may represent the next-generation ligands for targeting G-quadruplex motifs located in biologically important regions.


(1) D’Amico, F.; Graziano, R.; D’Aria, F.; Russomanno, P.; Di Fonzo, S.; Amato, J.; Pagano, B. Cytosine Epigenetic Modifications and Conformational Changes in G‐quadruplex DNA: An Ultraviolet Resonance Raman Spectroscopy Study. Spectrochimica Acta Part A: Mol. Biomol. Spectrosc. 2023, 300, 122901. DOI: 10.1016/j.saa.2023.122901

(2) Amato, J.; Iaccarino, N.; D’Aria, F.; D’Amico, F.; Randazzo, A.; Giancola, C.; Cesaro, A.; Di Fonzo, S.; Pagano, B. Conformational plasticity of DNA Secondarry Structures: Probing the Conversion Between I-Motif and Hairpin Species by Circular Dichroism and Ultraviolet Resonance Raman Spectroscopies. Phys. Chem. Chem. Phys. 2022, 24, 7028–7044. DOI: 10.1039/D2CP00058J

(3) Moraca, F.; Marzano, S.; D’Amico, F.; Lupia, A.; Di Fonzo, S.; Vertecchi, E.; Salvati, E.; Di Porzio, A.; Catalanotti, B.; Randazzo, A.; Pagano, B.; Amato, J. Ligand-based Drug Repurposing Strategy Identified SARS-CoV-2 RNA G-quadruplex Binders. Chem. Commun. 2022, 58, 11913–11916. DOI: 10.1039/D2CC03135C

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