Conference Agenda

Deoxyribonucleic acid (DNA) is the carrier of genetic information for all living organisms. Even a small mutation in DNA sentence can cause many diseases. Therefore, an early and accurate diagnosis of a specific DNA mutations has a decisive role for effective treatment. Especially, when an immediate decision on treatment most needs to be made, the rapid and precise confirmation of clinical findings is vital.

BRCA1 and BRCA2 are multifunctional proteins that play an important role in maintaining the integrity of the genome, as they are involved in DNA damage repair processes by homologous recombination. Disruption of their function, due to genetic pathogenic variants in the BRCA1 and BRCA2 genes, results in increased sensitivity of cells to DNA-damaging agents. To date, more than 20,000 sequence variants have been described in BRCA1 and BRCA2 genes, with the majority being deletions or insertions leading to a change in the reading frame, and substitutions resulting in premature translation termination and the formation of a truncated protein product. Carriers of mutations in the BRCA1 gene are more likely to develop aggressive triple-negative breast cancer, and in the case of developing cancer hormone-dependent, HER2-negative cancers are more likely to be diagnosed with lower estrogen receptor levels, higher histologic grade, and higher Ki67 proliferation index than women without the mutation. Therefore, we decided to construct and test using clinical samples SERS (surface-enhanced Raman scattering) and SPR (surface plasmon resonance) sensors for the identification of some variants of BRCA1 gene mutations (5370C>T, 300T>G, 5382insC, 4154delA, 185delAG, 1799T>A, and 3819delGTAAA).

We found that, when one immobilizes ‘thiolated’ (with an attached alkanethiol moiety) capture single-stranded DNA (ssDNA) and 6-mercaptohexan-1-ol on a gold (or silver) surface, and the structure formed is incubated with a sample containing DNA complementary to the immobilized capture ssDNA, the presence of the target ssDNA induces hybridization, which causes a change in the conformation of the chains of chemisorbed ω-substituted alkanetiols (6-mercaptohexan-1-ol and the alkanethiol linking moiety via which the captured single-stranded DNA is attached to the gold surface). That change is indicated by a characteristic change in the measured SERS spectrum: the intensities of the ν(C–S) bands of the trans and gauche conformer of the Au–S–C–C chain (alkane chains of both: chemisorbed 6-mercaptohexan-1-ol and the alkanethiol linking moiety via which the captured single-stranded DNA is attached to the gold surface) and the intensity of the band due to the breathing vibration of adenine. For example, such DNA SERS sensor for detection of 1799T>A mutation in BRCA1 is characterized by the low detection limit at the level of pg μL^–1 and a wide analytical range from ca. 7 pg μL^–1 to 70 ng μL^–1. Selective hybridization of target DNA with the capture DNA immobilized strongly influence SPR response, and therefore, gene mutation identification was also realised using SPR signals. It is worth emphasizing that in both types of sensors (SERS over SPR) the same capture DNA was used.

We found that for different DNA sequences, a SPR or SERS sensor achieves greater detection sensitivity, which means that the selection of the optimal sensor type depends on the sequence of the target DNA. The proposed analytical approaches demonstrated completely new capabilities of SPR and SERS techniques and new insights into gene mutation detection.

Acknowledgments: This work was financed by the National Science Centre, Poland, project No. 2019/35/B/ST4/02752.

Interactions between prokaryotic and eukaryotic microorganisms have been shown to be essential for the proper functioning of ecosystems. For instance, Streptomyces species produce polyketides derived from arginine—referred to as arginoketides—that serve as key mediators in cross-kingdom interactions with Aspergillus fungi, ultimately triggering the synthesis of natural products. These arginoketides, which can be cyclic (e.g., monazomycin and desertomycin A) or linear (e.g., lydicamycin and linearmycin A), are produced by Streptomyces iranensis and have been observed to stimulate the orsellinic acid gene cluster in Aspergillus nidulans [1].

To investigate these phenomena, surface-enhanced Raman spectroscopy (SERS) is employed to elucidate the release of those products in the environment and how they provoke responses in other microorganisms [2]. For this purpose, a specialized silver substrate was fabricated on a silicon wafer through the galvanic replacement of silver and sulfate ions, resulting in a dendritic structure. On the nanoscale, the branching of this tree-like network creates sharp angles and narrow gaps that act as “hot spots”, thereby enhancing the Raman signal of the target molecules. The Raman and SERS spectra of compounds produced by both S. iranensis and A. nidulans exhibit distinct marker modes, which facilitate their detection and identification within microbial cultures.

Acknowledgment: Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) ‎under Germany´s Excellence Strategy – EXC 2051 – Project-ID 390713860.‎

[1] M.K. Krespach et al. (2023). Nature Microbiology, 8, 1348–1361.

[2] D. Cialla-May et al. (2022). Analytical Chemistry, 94, 86-119.

Vibrational spectroscopy has recently promisingly emerged as a potential tool for pathogens detection and structural investigation [1-5]. Infrared (IR) and the more innovative Terahertz (THz) spectroscopy are widely used for studying biomacromolecules [1,2,5,6], and for characterization of viruses [2,7]. IR spectroscopy offers several advantages over gold standards (such as ELISA, RT-PCR, bDNA). It does not require chemical pre-treatment of the sample, measurements are rapid, low-cost and unique for all viral samples. Employing a functionalized sensor platform coupled to spectroscopy it would be possible to perform large scale measurements in an open environment, not simply focusing on the extracted sample as common biochemical assays do.

Coupling the unique structural information provided by vibrational spectra with a sensor platform specifically optimized for airborne pathogens adsorption, would enable the development of a label-free, real-time ultrasensitive optical biosensor. For instance, silicon and/or metal oxides substrates are suitable to be nanostructured and engineered, and their surfaces can be modified with a dedicated bioconjugation based on different approaches. These procedures would allow to increase the selectivity and to obtain an optimized sensor capable of concentrating viral capsids, which can then be inspected with IR spectroscopy.

As a preliminary and essential step for achieving the design of a biosensor based on vibrational spectroscopy, in this work we report the IR spectroscopic characterization of viral proteins from different species of coronaviruses, considered as bioanalytes of interest. Specifically, Spike protein has been considered as biomarker, measured and investigated via Attenuated Total Reflection-IR (ATR-IR) spectroscopy.

Deeply focusing on the inspection of proteins amide I band (1600-1710 cm^-1) and its deconvolution [1,6], on one hand we proved the potential of IR spectroscopy to uniquely characterize a specific viral species; moreover, we provided a structural investigation of the protein under different external conditions, in terms of secondary structure, conformational order and hydrophilicity.

Here, we present an overview of our results obtained from a systematic and comparative study of coronaviruses viral proteins, SARS-CoV-2 individual protein domains, namely the Receptor Binding Domain (RBD), subunit 1 (S1) and 2 (S2) regions, and Spike (S) protein, as well as SARS-CoV-2 S1 variants at serological pH, by measuring the amide I absorption band (1600-1700 cm-1) using Attenuated Total Reflection Infrared (ATR-IR) spectroscopy [5-7]. Firstly, three cases of study are shown, starting from the IR characterization of SARS-CoV-2 S protein and its domains, from the RBD, through S1 and S2, up to the whole S protein. Secondary structure contents of different domains have been estimated, and results are compared with MultiFOLD+DSSP computational approach; in addition, the contribution of each domain to the hydrophilic/hydrophobic profile and to the conformational order of the whole S protein has been evaluated from amide I bands shapes and computational tools [6].

After this characterization, in a first comparative study, the IR spectral analysis of S1 proteins from MERS-CoV, SARS-CoV and SARS-CoV-2 viruses reveals notable differences in their amide I bands, related to notable differences among their structures and in the intermolecular b-sheet content. Moreover, pH dynamic of SARS-CoV-2 S1 protein sheds light on remarkable conformational changes and adaptation of S1 proteins occurring during the infectious process [1].

Finally, another case of study focused on the comparative study of Alpha, Gamma and Omicron variants of SARS-CoV-2 virus, differing for a very small number of mutations with respect to wild type species. Through Circular Dichroism (CD) and IR spectroscopy and Molecular Dynamics (MD) simulations, a comprehensive experimental and computational structural analysis is reported, revealing remarkable spectral differences and relating them to proteins conformational behaviour and functionality.

References:

[1] D’Arco, A. et al. Secondary Structures of MERS-CoV, SARS-CoV, and SARS-CoV-2 Spike Proteins Revealed by Infrared Vibrational Spectroscopy. Int. J. Mol. Sci., 24, 2023

[2] Piccirilli, F. et al. Infrared Nanospectroscopy reveals DNA structural modifications upon immobilization onto clay nanotubes. Nanomaterials, 11(5):1103, 2021

[3] Quintelas C. et al. Biotechnol. J., 13, 1700449, 2017

[4] Mancini T. et al. New Frontier in Terahertz Technologies for Virus Sensing. Electron-ics, 12(1), 135, 2023

[5] Barth A. and Zscherp C. What vibrations tell about proteins. Q. Rev. Biophys. 35(4), 369, 2022

[6] Mancini T. et al. Infrared Spectroscopy of SARS-CoV-2 Viral Protein: from Receptor Binding Domain to Spike Protein. Advanced Science 11.39, 2400823, 2024.

[7] Fardelli, E. et al. Spectro-chim. Acta A Mol. Biomol. Spectrosc., 288, 122148. 2023