Studying Epigenetic Modifications of DNA Using UV Resonance Raman Spectroscopy

Welcome to our Spectroscopy and Archaeology Content Series

Spectroscopy and Archaeology

An Inside Look at Ancient Egyptian History

Proteins are vital components in the functioning of living organisms, and what they do depends on various interactions they have with other molecules. A new technique, called thermostable Raman interaction profiling (TRIP), can be used to analyze the sensitive detection of binding between proteins and ligands at low concentrations and low doses under conditions that mimic the body's natural environment (1). TRIP has significant implications for drug development, providing a valuable tool for researchers and pharmaceutical companies. It opens possibilities for high-throughput drug screening and real-time monitoring of the interactions between proteins and drugs (1).

Narangerel Altangerel (pictured), Zhenhuan Yi, and Marlan Scully of Texas A&M University recently used TRIP to analyze eight protein–ligand systems for a study published in 

 recently spoke to these three researchers about their findings and what the implications are for high-throughput drug screening. The research team collaborated on the responses, which have been lightly edited for style and clarity.

Dr. Narangerel Altangerel of Texas A&M University | Image Credit: © Narangerel Altangerel

Can you provide a brief overview about why studying protein interactions is important, especially it comes to drug development applications?

Proteins are the workhorses of biological systems, responsible for carrying out various functions within cells. Many diseases, such as cancer, neurodegenerative disorders, and infectious diseases, are often the result of dysregulated protein interactions. Understanding these interactions can provide crucial insights into the underlying mechanisms of disease. Identifying and characterizing protein interactions can lead to the discovery of potential drug targets. By targeting specific proteins or disrupting harmful interactions, researchers can develop drugs to treat diseases. For example, many cancer drugs target proteins involved in aberrant cell signaling pathways.

A simple, label-free screening technique capable of directly sensing interaction-in-solution involving large proteins, such as antibodies, holds promise for an important tool in the field of drug development.

Your study presents a thermostable Raman interaction profiling (TRIP) technique to screen protein binding (1). Why was this technique best for the analysis that you were conducting?

Thermostable Raman Interaction Profiling (TRIP) represents a promising innovation in the field of molecular interaction studies and drug discovery. Its ability to directly measure interactions in physiologically relevant conditions, its label-free nature, and its compatibility with aqueous samples make it a valuable tool for researchers seeking to better understand and develop treatments for various diseases by targeting specific protein-ligand interactions.

To see how the present TRIP technique compares with other optical approaches, we need only refer to Table I (1), which is, in part, given below:

What was unique about your study compared to previous studies done on this topic?

The TRIP technique offers several advantages that make it a cost-effective and efficient approach for high-throughput drug screening.

In Table I, we compare our technique to other label-free molecular diagnostic tools. The list in Table I is not intended to be exhaustive or complete, but only sufficient to give an idea of how Raman fits into the existing suite of tools available to biologists and chemists. Examination of the table illustrates that TRIP excels in operation at near-physiological conditions as mentioned above. The comparison with other techniques establishes TRIP as a general advance-screening tool for a wide variety of protein–drug interactions.

We summarize some of the key points of our research on TRIP below.

 The elimination of sample preparation steps reduces the time and resources required for experiments. Researchers can directly analyze samples without the need for complex and time-consuming preparation protocols.

 TRIP uses diluted protein samples, which can be especially important when working with expensive or limited protein resources. This not only reduces costs, but it also conserves valuable research materials.

 The ability to achieve rapid detection (within minutes) is a significant time-saver. Traditional drug screening methods can be time-intensive, whereas TRIP provides rapid results, allowing for more experiments to be conducted in a shorter period.

 TRIP directly measures binding interactions between proteins and ligands. This direct measurement avoids the need for secondary assays or readouts, simplifying the experimental process and reducing costs associated with additional assays.

 Capability of time-dependent measurement of TRIP successfully demonstrated for the binding interactions between the transthyretin (TTR) and 2,4 dinitrophenol DNP molecules in Figure 2 (which can be seen in the literature [1]).

 If TRIP can be implemented as an extension to existing systems, it minimizes the need for significant capital investments in new equipment. This cost-effective integration into existing laboratory setups makes it accessible to a broader range of research facilities.

These advantages collectively make TRIP an attractive option for high-throughput drug screening. It not only accelerates the screening process, but it also reduces the overall costs associated with drug discovery efforts. This is particularly valuable in pharmaceutical research, where the efficient identification of potential drug candidates can have a profound impact on the development of new therapies.

Usually, traditional drug screening techniques are only effective if the experimental conditions don’t influence them. What steps did you and your team need to ensure that your screening technique would not be influenced by experimental conditions?

We carefully addressed experimental conditions such as temperature, pH, buffer composition, laser power, and acquisition time across all experiments. This helped ensure that results are due to the interactions being studied rather than experimental artifacts. Performing experiments to replicate and assess the reproducibility of results is confirmed in clustering of the same protein in the PCA plots.We conducted control experiments with known protein-ligand interactions such as binding between streptavidin biotin and the bindings between protein A and antibodies to establish baseline measurements and assess the technique's accuracy. Also, we included the interactions of the SARS CoV-2 receptive binding domain (RBD) with non-binding and binding antibodies for checking positive and negative interactions.

Your study also used principal component analysis (PCA), stating that the method helped you and your team understand factors that affect spectral variation in the data you collected (1). Can you elaborate on this part of the study and explain how PCA assisted you in understanding your data?

Indeed, PCA is a valuable tool for understanding complex spectral data collected through techniques like Raman microscopy in our study. It simplifies data interpretation, aids in identifying influential variables, reduces noise, and facilitates the visualization and exploration of spectral variations. For example, spectral data collected using techniques like Raman microscopy are high-dimensional, with many variables (wavenumbers). PCA allows us to reduce the dimensionality of the data by identifying the most important directions (principal components) in the data space. These principal components are linear combinations of the original variables and capture the most significant sources of variation in the data. It helped us uncover meaningful insights and patterns within our data, contributing to a better understanding of the factors influencing molecular interactions and the performance of the TRIP technique.

What are the next steps in your work? What are a few questions this study raises that should be explored in a future study?

Research efforts are currently underway to employ alternative multivariate statistical techniques for the analysis of TRIP data. These approaches aim to facilitate the estimation of amino acid compositions and the determination of secondary protein structures across a wide range of protein sizes. Furthermore, we are carrying out TRIP studies involving a viral protein in the presence of four different drugs.

(1) Altangerel, N.; Neuman, B. W.; Hemmer, P. R.; Yakovlev, V. V.; Rajil, N.; Yi, Z. Sokolov, A. V.; Scully, M. O. Label-free drug interaction screening via Raman microscopy. 

Articles

Narangerel Altangerel, Zhenhuan Yi, and Marlan Scully of Texas A&M University recently used TRIP to analyze eight protein–ligand systems. Spectroscopy recently spoke to these three researchers about their findings and what the implications are for high-throughput drug screening. 

Using Thermostable Raman Interaction Profiling (TRIP) For Protein Binding Screening 

. Direct correspondence about this article to 

In a revolutionary development for exoplanet research, the near-infrared spectrograph (NIRISS) on the James Webb Space Telescope (JWST) has introduced the single object slitless spectroscopy (SOSS) mode, transforming our ability to study distant worlds. This innovative technology opens up a new chapter in our quest to understand the atmospheres and compositions of exoplanets. Loïc Albert and a large team of scientists and engineers have published a detailed description of this spectrograph (1).

For years, scientists have been intrigued by the possibility of studying exoplanet atmospheres to unlock the secrets of newly discovered distant worlds. The SOSS mode is a key instrument in this endeavor, allowing astronomers to perform time-series observations (TSOs) of exoplanets, which involves observing the same exoplanet over time to gather valuable data about its atmosphere and surface.

One of the early methods for studying exoplanet atmospheres was to use traditional transmission spectroscopy, proposed by Seager and Sasselov in 2000 (2). However, it wasn't until space telescopes like the Hubble Space Telescope (HST) came into play that this method gained traction. The first successful attempts at transmission spectroscopy with HST led to groundbreaking discoveries, including the detection of water vapor and other molecules in exoplanet atmospheres.

The SOSS mode was born out of the need to improve exoplanet transmission spectroscopy. It was developed as part of the NIRISS instrument on JWST, specifically designed to observe exoplanets discovered by the TESS mission. SOSS offers a unique set of advantages, including the ability to observe much brighter stars, making it a game-changer for studying exoplanet atmospheres.

The optical design of SOSS involves a zinc selenide (ZnSe) grism coupled with a cross-dispersing zinc sulfide (ZnS) prism, which projects three spectral diffraction orders onto the detector. A 

 is an optical component of a dispersive spectrograph that allows for an in-line optical configuration, and is comprised of a grating and one or more prisms. This configuration enables the observation of stars as bright as magnitude 6.7 with a resolving power of 650 at a wavelength of 1.2 μm (1200 nm). SOSS covers a wavelength range from 0.6 to 2.8 μm (600 to 2800 nm), encompassing critical spectral features such as water (H

), carbon monoxide (CO), and carbon dioxide (CO

) bands, as well as atomic lines of potassium (K) and helium (He). Note that a resolving power of 650 means that the optical instrument, in this case, the spectrograph, is capable of distinguishing between two spectral lines or features that are separated by a wavelength difference of 1/650th (approximately 0.00154) of the wavelength being observed (1.2 μm).

The SOSS mode offers several subarray modes to cater to different observation needs, ensuring its versatility and adaptability. Ground-based testing and commissioning observations have confirmed that SOSS is performing at its best, delivering results comparable to other JWST time-series modes.

One of the standout features of SOSS is its excellent white light flux stability. During a 5-hour TSO on an A-type star, designated as BD+60°1753, the data showed remarkable stability, with any fluctuations following the expected photon statistics. This stability extends down to approximately 20 ppm on a 40-minute timescale, making it ideal for detecting subtle changes in exoplanet atmospheres.

To produce high-quality data, several data processing steps are crucial, including 1/f noise correction, cosmic-ray flagging, bad pixel interpolation, and zodiacal background subtraction. These steps ensure that the analysis reaches near photon-noise limited levels.

Despite its remarkable performance, SOSS does face some challenges, such as positional excursions between visits that affect wavelength calibration. Efforts are underway to characterize and address these issues. Additionally, SOSS has demonstrated its potential as a wavefront sensor for detecting mirror segment tilt events on JWST.

The real excitement lies in the science programs now possible with SOSS TSOs. The NIRISS instrument development team has allocated nearly 200 hours of observation time to the NIRISS Exploration of the Atmospheric Diversity of Transiting Exoplanets (NEAT) program. NEAT aims to measure the carbon-to-oxygen (C/O) ratios for a diverse range of exoplanets, giving detailed information on their formation and evolution.

In conclusion, the introduction of the single object slitless spectroscopy (SOSS) mode on the near-infrared spectrograph (NIRISS) of the James Webb Space Telescope (JWST) is a major leap forward in our ability to study exoplanet atmospheres. This cutting-edge technology, with its excellent stability and versatility, opens up exciting possibilities for unraveling the mysteries of distant worlds and advancing our understanding of the cosmos.

(1) Albert, L. et al. The Near Infrared Imager and Slitless Spectrograph for the James Webb Space Telescope--III. Single Object Slitless Spectroscopy. 

, arXiv:2306.04572. DOI: 10.1088/1538-3873/acd7a3

(2) Seager, S. V.; Sasselov, D. D. Theoretical transmission spectra during extrasolar giant planet transits. 

The James Webb Space Telescope's near-infrared spectrograph (NIRISS) has introduced the single object slitless spectroscopy (SOSS) mode, enhancing our ability to study exoplanet atmospheres with high resolving power. This innovation holds great promise for advancing our understanding of distant worlds and their compositions.

Unveiling Exoplanet Secrets: The Near-Infrared Spectrograph on the James Webb Space Telescope

Patrick Lavery is an Editor for the MJH Life Sciences brands LCGC and Spectroscopy, and their respective websites, 

. He previously spent nearly a decade as a news anchor, reporter, and producer at New Jersey 101.5 FM.

A study out of the Key Laboratory of Bio-Inspired Smart Interfacial Science and Technology of Ministry of Education, within the School of Chemistry at Beihang University in Beijing, China, describes a novel platform for the simultaneous determination of five antioxidants with surface-enhanced Raman spectroscopy (SERS) that applies a sensor array based on the inverse etching of gold nanorods (AuNRs) and nanostars (AuNSs) (1).

Pile of shining gold pieces seen from above. Top view macro image of sparkling gold dust for backgrounds and textures. Selective focus and shallow depth of field. | Image Credit: © Ole - stock.adobe.com

The target antioxidants, ascorbic acid (AA), melatonin (Mel), glutathione (GSH), tea polyphenols (TPP), and uric acid (UA), are among those that are able to scavenge free radicals—in other words, generated from the same initiators used for free radical polymerization and copolymerization (1). According to the authors, whose study was published in the journal 

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

, too many free radicals can cause oxidative stress on the body, manifesting in potential cancers, diabetes, or cardiovascular or neurodegenerative diseases. But the correct balance of antioxidants matters just as much: too little AA can lead to scurvy, and too much UA causes gout.

For those reasons, a need for high-throughput discrimination of the five antioxidants resulted in the devised method described in this study. The compound 3,3′,5,5′-tetramethylbenzidine (TMB) is oxidized to TMB

) and horseradish peroxidase (HRP), which react with each other to release oxygen free radicals and, in turn, react with TMB (1). But the authors point out that antioxidants can prevent the further oxidation of TMB

, while also circumventing the etching of gold (Au) in the catalytic oxidation process—the aforementioned inverse etching, the end goal of which was to enhance the Raman SERS signal with regard to the antioxidants being studied. The antioxidants were then analyzed in three ways: heat map, hierarchical cluster, and linear discriminant analysis.

Surface-enhanced Raman spectroscopy (SERS) 

s a powerful analytical technique in which target analytes are adsorbed onto metallic nanostructures such as silver or gold, greatly enhancing the Raman scattering signal. This allows for the ultrasensitive detection of various biomolecules. By analyzing the unique Raman spectra that are obtained, SERS can provide valuable information about chemical compositions and the presence of specific substances. SERS is nondestructive by nature, and therefore is suitable for repeated measurements on small sample volumes, enabling rapid and accurate analysis.

The relationship of Au to the two-dimensional (2D) SERS sensor array proposed here is crucial, as both AuNRs and AuNSs demonstrate high electromagnetic field enhancement performances at their tips; therefore, they are seen as excellent substrates in Raman applications (1). With AuNRs and AuNSs as the sensing elements, the peak Raman intensity of TMB

 for the identification of the five antioxidants was set at 1605 cm

The authors found that the novel platform was effective in simultaneously identifying the five antioxidants intended in the study, reporting distinct SERS responses and patterns for each, all based on their individual abilities to scavenge free radicals (1). And because of the speed and sensitivity of the method, the study pointed to potential future applications of this SERS sensor array in food safety and overall human health and well-being.

(1) Xi, H.; Shi, Z.; Wu, P.; et al. A Novel SERS Sensor Array Based on AuNRs and AuNSs Inverse-Etching for the Discrimination of Five Antioxidants. 

Ascorbic acid (AA), melatonin (Mel), glutathione (GSH), tea polyphenols (TPP), and uric acid (UA) were distinguished in this experiment by three analyses: heat map, hierarchical cluster, and linear discriminant.

Novel SERS Sensor Array Based on Gold Nanorods and Nanostars Identifies Five Antioxidants

Aaron Acevedo is an assistant editor for LCGC and Spectroscopy at MJH Life Sciences.

A group of scientists from the University of Wrocław in Poland and the University of Innsbruck in Austria are studying the relationships between aliphatic ketones and water molecules in various environments. Their work was recently published in 

Close up water | Image Credit: © Andrey Armyagov - stock.adobe.com

Molecules with a carbonyl group attached can be studied through the elucidation of ketone-water interactions. Aliphatic ketones are the simplest compounds with carbonyl groups. Acetone is fully miscible with water, while higher ketones have limited solubility. This has led to extensive studies on acetone-water binary mixtures, though studies of mixtures of water with higher ketones are either rare or nonexistent, the researchers wrote.

In this study, the scientists selected nine aliphatic ketones of different sizes and structures, to examine the effects of different structural motifs on behavior of water in the mixtures. Using a mix of mid-infrared (MIR) and near-infrared (NIR) spectroscopy, the scientists first searched for bands from three nonequivalent OH groups: non-bonded, bonded to OH, and bonded to CO, as well as any new bands in these spectra that may arise due to changes in factors like temperature.

The scientists measure attenuated total reflectance-infrared (ATR-IR), MIR and NIR transmission spectra of dry and water-saturated ketones. Additionally, they determined variations in electric permittivity, 

 and density when going from dry to wet ketones. These values allowed the researchers “to gain additional insight into the changes in the properties of the solutions” (1).

The scientists found that conformational flexibility of aliphatic chains in the linear ketones enables effective shielding of a carbonyl group, which explains poor solubility in water.The water molecules become greatly involved in ketone-water interactions and less so with water-water interactions. Cyclic ketones also have higher solubility of water, allowing for clusters of water to be created when the molecules are in a water-like environment. When wet cyclic ketones rise in temperature, more ketone-water interactions are made; this contrasts with linear ketones and 2,6-dimethylcyclohexanone, where elevated temperatures lead to an increase in the population of singly bonded water at the expense of the doubly bonded one.

Density functional theory (DFT) calculations also reveal that substituting cyclohexanone with a single methyl group does not affect the strength of ketone-water interactions but greatly affects the impact of water solubility in the ketone.

(1) Czarnecki, M. A.; Beć, K. B.; Grabska, J.; Huck, C. W.; Mazurek, S.; Orzechowski, K. State of water in various environments: Aliphatic ketones. MIR/NIR spectroscopic, dielectric and theoretical studies.

 Spectrochim. Acta A Mol. Biomol. Spectrosc.

https://doi.org/10.1016/j.saa.2023.123057

A group of scientists from the University of Wrocław and the University of Innsbruck studied the relationships between aliphatic ketones and water molecules in various environments.

Researchers Study the Relationship Between Aliphatic Ketones and Water Molecules

 spoke to Maria Montes-Bayón of the Faculty of Chemistry at the University of Oviedo (Asturias, Spain) regarding her work with single cell inductively coupled plasma mass spectrometry (ICP-MS) to study the uptake and apoptotic status of nanoplatinum (IV) treated cells, specifically selenized yeast. 

Since then, Montes-Bayón was awarded the Lester W. Strock Award, given by the New England Section of the Society for Applied Spectroscopy (SAS) to recognize, as noted on the SAS website, “a selected publication of substantive research in/or application of analytical atomic spectrochemistry in the fields of earth science, life sciences, or stellar and cosmic sciences.” This second interview, where we revisit our previous conversation as well as discuss her career in general, is part of an ongoing series of interviews with the winners of awards that are presented at the annual SciX conference, which will be held this year from October 8 through October 13, in Sparks, Nevada.

Since our last talk regarding your work with ICP-MS normalized quantitative experiments using certified selenized yeast (1), has there been any additional feedback or next steps that you’d like to share?

Well, we never stop! We have moved some steps forward into a different topic in my group which is the use of nanoparticles to transport cisplatin into tumor cells. Part of the work is still unpublished, but we have developed some nice strategies to address their incorporation in 3D cell cultures, which is the natural evolution from 2D cultures and closer to tumor tissues. However, for this we need a strong collaboration from colleagues from biochemistry and biotechnology to create these cell models from different cell types. We are really excited about the outcome of this project as well.

What does your being named the recipient of Strock Award mean to you professionally? Personally?

In my case, the award is given to the contributions in speciation and metallomics, basically due to the application of atomic spectroscopy on the field of biomedicine and life science. This makes me particularly pleased since I have been trying to establish collaborative research in these fields for a long time with colleagues from different disciplines. I deeply believe that analytical chemists are well prepared to conduct multidisciplinary research and can provide interesting methodological alternatives in the field of life sciences. Therefore, the prize recognizes the effort required to make this feasible. From a personal point of view, it is particularly important to me since part of my work was conducted in Cincinnati, in the Group of Professor Joe Caruso, and this period completely changed my vision of science. I can be only grateful for those times, from both a personal and professional perspective.

When did you first determine that a career in science was the path for you?

I think this is something you do not really plan. When I finished my bachelor’s degree, the possibility to go to the UK to do my master’s degree thesis became feasible. Learning English was the first driving force to go there, but I discovered a new passion in research (working with Prof. Hywel Evans, once I could understand his accent from Wales). Then, back to Oviedo, Prof. Sanz-Medel offered me the possibility to earn my doctorate in his group, and, since then, I discovered my enthusiasm for research.

You first studied Chemistry at the University of Oviedo; you then joined the University of Plymouth (United Kingdom) in the framework of the Erasmus program for undergraduate thesis studies. Then, In April 2000, you joined the research group of Professor Joseph A. Caruso at the Department of Chemistry at the University of Cincinnati, Ohio (USA) as a Fulbright postdoctoral fellow until July 2002. What challenges did you encounter in these transitions across multiple departments?

In addition, I have been a visiting scientist in Germany for two different periods in Hamburg and Berlin (for a total of seven months). All in all, the different working environments are one of the aspects that enriches the scientific life the most. It always requires some time to learn how things are done in the different places but, finally, you gain an open vision about research, and life in general. Being in the United States, where I spent the longest time abroad, gave me the opportunity to learn how to be more independent in research, and how to communicate science more effectively. From all the places I have visited, I have taken something that helped me in the next step of my career.

Tell me a little bit about your first published article.

My first paper dealt with research based on plasmas but Microwave Induced Plasma, MIP, using different gases (2). Soon after, the first ICP-MS arrived in Oviedo, and since I had gained some experience during my time in Plymouth, Prof. Sanz-Medel (one of my doctorate supervisors) reoriented my doctorate to ICP-MS. Thus, thanks to the time in UK, my doctoral experiments focused on the use of ICP-MS for semiquantitative total elemental analysis and speciation, which resulted in very successful and productive work.

What do you consider to be your greatest strength, and how does that strength factor into your professional life?

It is hard to define these things about yourself. I think I am very enthusiastic about the research work we do in the laboratory, and this helps me to communicate it in a clear and somehow entertaining way. Such enthusiasm always helps when writing manuscripts or research projects to get funding. This is very important for both personal and professional aspects.

What do you consider to be your greatest professional accomplishment?

Maybe to keep an independent research group that is growing and attracts the interest of new PhD students, not only from the field of chemistry but also biotechnology or biochemistry.

What advice can you offer those starting out in their post-doctorate career?

I think it is summarized along this interview: one must develop capabilities to adapt to new working environments and to extract the best of each one of them; have enthusiasm about your work; and, while imagination is always welcome, one should be able to listen and understand what colleagues from other fields need.

(1) Chasse, J. Toward Normalization of Quantitative Single Cell ICP-MS Experiments

(2) Corte Rodriguez, M.; Álvarez-Fernández García, R.; Blanco, E.; Bettmer, J.; Montes-Bayón, M. Quantitative Evaluation of Cisplatin Uptake in Sensitive and Resistant Individual Cells by Single-Cell ICP-MS (SC-ICP-MS). 

 is a Full Professor in Analytical Chemistry in the Department of Physical and Analytical Chemistry at the University of Oviedo (Spain) since December 2017. She holds a bachelor’s degree in chemistry (1993, University of Oviedo), a bachelor-thesis in Analytical Chemistry (1994, University of Plymouth, UK and Oviedo), and a PhD (1999, University of Oviedo; awarded) degree. In the year 2000, she joined the research group of Prof. Joseph A. Caruso at the Department of Chemistry at the University of Cincinnati as a Postdoctoral Fulbright Fellow. She went back to Oviedo on a Senior Researcher contract and was promoted in 2008 to Associate Professor, and in 2017 was promoted to Full Professor of Analytical Chemistry. Her main research interests are development of quantitative strategies for determination of molecular biomarkers of clinical relevance at the single cell level using ICP-MS; evaluation of encapsulated metallodrugs for their potential use in chemotherapy using mass spectrometry: a focus on cellular resistance mechanisms; and analytical tools to study biomedical aspects of nanoparticles: biogenic nanoparticles and metal nano-debris from metallic implants. She has supervised 14 PhD students since 2007 and mentored 5 postdocs who built up a successful career afterwards. She has given >40 invited and plenary lectures in national and international congresses and conferences and co-authors 164 publications (80% in Q1; 32 with >50 citations), including original research articles, reviews, and several book chapters (with >5,300 total citations, H-index = 43). She has received funding through 29 international/national/regional research projects and is associate editor of the journal 

 since 2015. Currently, she has been elected as President of Spanish Society for Analytical Chemistry since 2022.

Earlier this year, Spectroscopy spoke to Maria Montes-Bayón, the 2023 recipient of the Lester W. Strock Award.

An Interview with 2023 Strock Award Recipient Maria Montes-Bayón

Researchers from Brno University of Technology and the Institute of Physical Chemistry and Chemical Physics are investigating a lumazine derivative that exhibits sensitivity to variations in dimethyl sulfoxide (DMSO) concentration (1).

Their findings, published in the journal 

, suggest the potential for non-invasive dimethyl sulfoxide (DMSO) detection 

, with significant implications for pharmaceutical applications, particularly in drug uptake monitoring.

The research focused on investigating the absorption and emission spectra of lumazine, alloxazine, and their cyanated or fluorinated derivatives in varying DMSO-water mixtures. The team modulated the spectroscopic properties by altering the water concentration in DMSO.

One of the most interesting findings came from samples containing 65% water and 35% DMSO, a composition that led to a decrease in absorption and emission intensities. This was more pronounced than the well-known water quenching effect observed in other cases. These experimental results align with previously published molecular dynamics (MD) simulations, corroborating the local bulk model concept.

The researchers believe that the sensitivity to DMSO concentration stems from changes in the local structure of the solvent, particularly the formation or dissolution of H2O clusters, as proposed by the local bulk theory. Furthermore, the study revealed the formation of water-DMSO trimers at the 35% DMSO concentration, highlighting the intricate interplay of solute and solvent molecules.

To interpret the spectroscopic measurements, the team employed density functional theory (DFT) coupled with an implicit DMSO solvent model and explicit water molecules. Their computational analysis found a strong correlation between water-flavin theoretical dimers and the experimental spectra of molecules in water-DMSO mixtures, revealing the role of intermolecular interactions in optical spectra.

Of particular interest was the solvatochromic behavior of the cyanated CN derivative, which exhibited a range of absorption maximum wavelengths between 315 and 340 nm, making it suitable for non-invasive monitoring of DMSO concentration for in vitro experiments. The CN molecule also boasted excellent water solubility and contained the biocompatible uracil moiety, suggesting its potential integration into biological systems.

This research presents a tool for scientists and pharmaceutical researchers seeking to delve deeper into the complexities of drug interactions within biological systems. The potential for non-invasive monitoring of DMSO concentrations offers a promising avenue for enhancing drug development and delivery for medical research and clinical applications.

Truksa, J.; Kratochvíl, M.; Richtár, J.; Ivanová, L.; Weiter, M.; Krajčovič, J.; Lukeš, V. Spectroscopic Behavior Differences between Lumazine and Alloxazine in the DMSO-Water Mixture. 

A group of researchers from Brno University of Technology and the Institute of Physical Chemistry and Chemical Physics are investigating flavin derivatives in water and DMSO. 

Lumazine Derivative Shows Promise for Non-Invasive Dimethyl Sulfoxide Detection

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. 

 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

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 (

) 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. 

(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. 

(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. 

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.

Developing fluorescent and photoluminescent materials requires the coupling of nanotechnology applications and material science. In a recent study conducted by researchers at several Saudi Arabian institutions, a research team describes how electrospun glass nanofibers (GNFs) enhanced polycarbonate (PC) to help create these materials (1). The findings of this study were published in the journal 

Innovative Science and Technology: Exploring Abstract Molecular Structures. Photo AI | Image Credit: © pixardi - stock.adobe.com

The key to developing these materials lies in the physical integration of lanthanide-activated aluminate (LA) nanoparticles (NPs) into transparent GNFs@PC smart sheets. Spectral investigations, utilizing photoluminescence and CIE Lab parameters, demonstrated the transformative potential of these materials (1). When exposed to UV light, the translucent GNFs@PC smart sheets undergo a rapid metamorphosis, changing their appearance to a vivid green, showcasing fluorescence activity. This transformation is both swift and reversible when the LANPs concentration is low, indicating fluorescence emission (1). However, higher concentrations of phosphors in GNFs@PC lead to longer-lasting afterglow photoluminescence and slower reversibility, expanding their versatility (1).

Under excitation at 368 nm, the GNFs@PC hybrids emit a distinct band at 518 nm, highlighting their unique luminescent properties (1). Using transmission electron microscopy (TEM), the GNFs and LANPs revealed particle sizes ranging from 11 to 26 nm and fiber diameters of 250 to 300 nm, respectively (1). The GNFs were produced through electrospinning technology. The GNFs were then incorporated into PC sheets. The benefit of doing this was to enhance their scratch resistance significantly compared to LANPs-free PC samples (1). The researchers found in this study that increasing the LANP concentration enhanced both hydrophobicity and UV protection.

The LANPs were loaded into the GNFs@PC hybrid host material to create UV-responsive smart materials (1). Electrospinning played a pivotal role in crafting the GNFs, which, in turn, reinforced the PC bulk (1). The resultant fluorescent and afterglow emission characteristics of the GNFs@PC substrates make them suitable for various applications, including smart windows and smart concrete. As a result, this technique allowed for the creation of hybrid nanocomposites featuring LANPs, GNFs, and PC that exhibit fluorescence under UV light and transparency under visible light (1).

The transparent appearance, UV protection, photostability, and hydrophobicity of the photoluminescent GNFs@PC substrates have all been achieved through this straightforward procedure. Morphological features were further analyzed using energy-dispersive X-ray spectroscopy (EDXA), X-ray fluorescence (XRF), and scanning electron microscopy (SEM), confirming the exceptional quality of the resulting materials (1).

The researchers also observed the contact angle of the light-emitting GNFs@PC hybrid nanocomposites. The contact angle of the light-emitting GNFs@PC hybrid nanocomposites increased from 148.6° to 158.7° as the LANPs ratio rose from 0.5% to 8% (1). The optimal ratio of LANPs at 1% yielded colorless GNFs@PC with maximum green emission under UV light. Furthermore, a LANPs-to-GNFs@PC ratio of 6% resulted in the brightest green phosphorescent emission, providing a vivid greenish-yellow glow even in the dark (1).

(1) Alrefaee, S. H.; Alnoman, R. B.; Alenazi, N. A.; Alharbi, H.; Alkhamis, K.; Alsharief, H. H.; El-Metwaly, N. M. Electrospun glass nanofibers to strengthen polycarbonate plastic glass toward photoluminescent smart materials. 

In a recent study, researchers from Saudi Arabia used electrospun glass nanofibers to enhance polycarbonate to create new high-performance materials.

Electrospun Glass Nanofibers Transform Polycarbonate into Persistent Luminescent Innovations

, a team of authors mostly led by researchers from Bohai University in Jinzhou, China, delved into the intricate world of confined water within subnano channels (1). Through the lens of infrared spectroscopy, they explored the hydrogen-bonded structures and temperature-related transitions of this confined water.

Infrared spectroscopy provides a measurement technique for intense, isolated, and reliable absorption bands of fundamental molecular vibrations from polymers and other organic compounds. This technique allows for univariate calibration with higher signal strength (absorptivity) required for solid-, liquid- or gas-phase measurements.

The researchers focused their attention on water confined within the subnano channels of AlPO

-11. By employing thermogravimetric analysis, they estimated that approximately 8.45 water molecules reside within each channel of a single unit cell, which revealed a complex interplay of hydrogen bonding and coordination at ambient temperature (1).

At such a temperature, the confined water exhibited two distinct coordination states: saturated and unsaturated. The saturated state resembled ice-like structures, with water molecules forming four-coordinated configurations. In contrast, the unsaturated state encompassed liquid-like structures, where water molecules exhibited coordination with the framework Al sites and a relatively free range of motion. This coordination between water molecules and the framework Al sites was found to be responsible for the ice-like structures observed in the channels above the ice melting point (1). In contrast, the presence of liquid-like structures was attributed to the strong confinement within the channels, preventing the formation of extensive tetrahedral hydrogen-bonded configurations.

When temperature was decreased, the confined water within the AlPO

-11 channels underwent a significant structural transformation. In this colder regime, isolated small water oligomers emerged, along with two novel components characterized by stronger hydrogen bonds: low-density amorphous ice-like structures and a type of low-density liquid-like structures (1). These findings offer critical insights into the structural organization and thermal dynamics of confined water in extremely narrow channels, shedding light on its behavior under unique conditions.

The research conducted by this team not only contributes to our fundamental understanding of confined water but also has implications for various fields, including biology and geology, where the behavior of water in extreme environments plays a pivotal role. Moreover, this study showcases the power of infrared spectroscopy in unraveling the mysteries of confined water, opening new avenues for further research. The investigation provided a comprehensive examination of the hydrogen-bonded structures and temperature-dependent transitions of confined water within subnano channels.

(1) Chen, S.; Wang, J.; Li, X.; et al. Hydrogen-Bonded Structures and Low Temperature Transitions of the Confined Water in Subnano Channels. 

A study out of Bohai University in China has uncovered some of the unique characteristics of water when in extreme confinement.

Using Infrared Spectroscopy to Analyze Confined Water in Subnano Channels

Scientists from the Amirkabir University of Technology in Tehran, Iran, used laser-induced fluorescence (LIF) spectroscopy to analyze opium and hashish. Through this research, which was published in 

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 

the scientists explored a novel method of drug analysis (1).

Opium, which comes from the opium poppy, is the source of many narcotics, including morphine and codeine. Hashish is made from the resin of the cannabis plant. Cannabis is one of the most common psychoactive drugs, due to its psychoactive components: delta-9-tetrahydrocannabinol (THC), cannabinol (CBN), and cannabidiol (CBD).

Opium and hashish are optically dense multi-fluorophore samples and contain fluorophores of unknown concentrations. This makes it difficult for conventional fluorescence measurement tools to analyze these substances. In this study, the researchers used LIF to properly characterize the properties of both opium and hashish.

LIF is often praised for its simplicity, quickness, and portability, making it a practical method of drug identification. In this study, LIF was extensively examined to overcome drawbacks of obtaining specific fluorescence parameters as a novel method of drug analysis. The spectroscopy process was carried out at a single-line excitation of 405 nm over a wide range of concentrations to obtain three distinctive fluorescence parameters (

) based on the modified Beer-Lambert (MBL) formalism.

 value was determined to be 0.30 and 0.15 mL/(cm∙mg) for opium and hashish, respectively. Similarly, typical 

 is obtained ar 0.390 and 1.25 mL/(cm∙mg), respectively. Furthermore, the concentration at max fluorescence intensity (

) was determined for opium and hashish to be 1.8 and 1.3 mg/mL, respectively. These results show that using this method, opium and hashish create their own characteristic fluorescence parameters.

(1) Shamsi, E.; Parvin, P.; Ahmadinouri, F.; Khazai, S. Laser-induced fluorescence spectroscopy of plant-based drugs: Opium and hashish provoking at 405 nm. 

Spectrochim. Acta A Mol. Biomol. Spectrosc.

https://doi.org/10.1016/j.saa.2023.123055

Scientists from the Amirkabir University of Technology in Tehran, Iran, used laser-induced fluorescence (LIF) spectroscopy to analyze opium and hashish, in order to achieve a novel method of drug analysis.

Featured Content

Studying Epigenetic Modifications of DNA Using UV Resonance Raman Spectroscopy

Welcome to our Spectroscopy and Archaeology Content Series

Spectroscopy and Archaeology

Spectroscopy and Archaeology