Protein and Nucleic Acid Analysis

We employ advanced tools such as FTIR microscopy, FTIR spectroscopy, and UV Resonant Raman spectroscopy to offer insights into molecular conformations, stability, and interactions of proteins, nucleic acids, and peptides.

📍The protein and nucleic acid analysis services can also leverage the unparalleled power of synchrotron light as they are provided by SISSI Beamline and IUVS Beamline at Elettra Sincrotrone Trieste in Area Science Park.

Conformational and Stability Studies of Proteins

For this type of studies, diversified instrumental solutions may be proposed, based on both FTIR microscopy and FTIR spectroscopy.

  • For ultra-sensitive infrared absorption spectroscopy of biomolecules, plasmonic internal reflection microscopy will exploit the Hyperion 3000 Vis-IR microscope mounting a single point Mid-band MCT detector (10,000-600 cm-1) and coupled with INVENIO-R Fourier Transform Infrared Spectrometer (Bruker). More conventional studies on deuterated proteins and dehydrated thin layers exploits INVENIO-R interferometers and dedicated accessories: liquid cells and Attenuated Total Reflection accessories (both multiple and single reflection).

  • Exploration of protein folding, aggregation self-assembly, and the effects of pH on protein structure with UV Resonance Raman spectroscopy takes advantage of the sensitivity and molecular selectivity of this vibrational technique. For this type of studies, the modifications of the system can be monitored in “real time” and in situ, with a minimum handling of the sample, using the DUV portable Resonance Raman system (Photon Systems) quipped with excitation wavelength at 248 nm.
    Different solutions for the sampling (in bulk, using a micro-probe with micrometric resolution, in a flow cell to avoid photodegradation) are available in the lab, as well as the possibility to investigate the samples in a wide temperature range between -190 and 600 °C. Additionally, the tunable UV Resonance Raman micro-setup (Crisel Instrument) will allow to study the protein systems by varying the excitation wavelength in the range 210-270 nm in order to selective enhance specific chromophoric portions of the molecules (amide linkages, aromatic aminoacids side chains). Two sampling modalities, macro- and micro-sampling with lateral resolution of few µm, are available to achieve the specific user’s experimental requirements.

Structural Investigation of Nucleic Acids Architectures and Ligand Interactions

We employ UV Resonant Raman spectroscopy for the selective probing of nucleic acids and DNA. This method allows us to analyze the stability of DNA structures at different temperatures, investigate motif formations, and assess oxidative damage. We also explore the impact of novel green solvents on DNA stability. Additionally, structural analysis of new nucleic acid-based materials such as ionogels, DNA hydrogels, and self-assembled nanostructures can be performed by implementing operando and in situ UV Resonance Raman experiments.
The portable DUV Raman system (Photon Systems) with excitation at 248 nm offers a selective molecular sensitivity especially for guanine and adenine rich systems, with the possibility to implement temperature-dependent experiments. This approach benefits of minimum interference from solvent buffers and of the use of relatively low concentrations (micromolar) of the samples.
Different excitation wavelengths in the range 210-270 nm are also available with the tunable UV Resonance Raman micro-setup (Crisel Instrument) for the investigation of cytosine and thymine-based systems.
With UV Resonant Raman, we can also study the ligand-binding interactions of nucleic acids and their assembly, including RNA and drug molecules, to reveal the details of DNA structure and interactions, facilitating advances in genetic research and drug development. These studies can be implemented for in vitro experiments in physiological conditions through the collection of UV Raman vibrational images of living cells and pathogens.

Hydration Properties and Supramolecular Architecture of Small Peptides and Nanostructures

We xamine hydration properties and solvation of model peptides, along with their folding, aggregation and self-assembly under various conditions with UV Resonant Raman to offer a sensitive label-free approach to monitor specific chemical moieties inside the system in situ and in operando conditions.
Delve into the structural dynamics of peptides with UV Resonant Raman, advancing in the design of new peptides with therapeutic potential and understanding protein misfolding diseases is also possible thanks to the richness of information of the UVRR spectra.
The DUV portable Raman instrument (Photon Systems) offers quick and easy collection of high signal-to-noise Raman spectra, completely free from interfering fluorescence background. Different methods for sampling (macro and micro) and possibility to explore wide temperature ranges are also feasible.

Selected Publications

Conformational and Stability Studies of Proteins

  • “Binding of tyrosine kinase inhibitor to epidermal growth factor receptor: surface-enhanced infrared absorption microscopy reveals subtle protein secondary structure variations”
    P. Zucchiatti et al., Nanoscale, 2021, 13(16), pp. 7667–7677
    DOI: 10.1039/D0NR09200B
  • “FTIR investigation of the secondary structure of type I collagen: New insight into the amide III band”
    C. Stani et al., Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy, 2020, 229, 118006
    DOI: 10.1016/j.saa.2019.118006
  • “Amyloidogenic and non-amyloidogenic molten globule conformation of β-lactoglobulin in self-crowded regime”
    S. Venturi et al., International Journal of Biological Macromolecules 242 (2023) 124621
    DOI: 10.1016/j.ijbiomac.2023.124621
  • “UV Resonance Raman explores protein structural modification upon fibrillation and ligand interaction”
    M. Pachetti et al., Biophysical Journal, 2021, 120, 4575–4589
    DOI: 10.1016/j.bpj.2021.08.032

Structural Investigation of Nucleic Acids Architectures and Ligand Interactions
  • "Structure of human telomere G-quadruplex in the presence of a model drug along the thermal unfolding pathway"
    F. Bianchi et al., Nucleic Acids Research, 2018, 46, 22, pp. 11927–11938
    DOI: 10.1093/nar/gky1092
  • "Strategies and Perspectives for UV Resonance Raman Applicability in Clinical Analyses of Human Sperm RNA"
    M. Pachetti et al., International Journal of Molecular Sciences, 2021, 22, 13134
    DOI: 10.3390/ijms222313134
  • "Investigation of genomic DNA methylation by ultraviolet resonant Raman spectroscopy"
    F. D'Amico et al., Journal of Biophotonics, 2020, 13, 12, e202000150
    DOI: 10.1002/jbio.202000150

Hydration Properties and Supramolecular Architecture of Small Peptides and Nanostructures
  • "Aqueous solvation of glutathione probed by UV resonance Raman spectroscopy"
    S. Catalini et al., Journal of Molecular Liquids, 2019, 283, pp. 537-547
    DOI: 10.1016/j.molliq.2019.03.113
  • "Hydrogen Bonding and Solvation of a Proline-Based Peptide Model in Salt Solutions"
    S. Catalini et al., Scientific Reports, 2015, 5, 12129
    DOI: 10.3390/life11080824