Projects

Currently, we have applied to the Spanish Ministery of Science, Innovation and Universities for a funded project dealing with "Multimodality, multidimensionality and missing information in bioanalytical and biophysical process modeling".

This project involves several global objectives, dealing with the development of chemometric algorithms for the analysis of complex data sets, including image analysis, as well as data recorded along bioanalytical, biophysical and industrial processes.

In relation to bioanalytical and biophysical processes, these are another area where multimodal and multidimensional measurements would help to unravel the intrinsic complex behaviour of large biomolecules, such as nucleic acids. These studies demand the use of diverse experiments, not only steady-state measurements (i.e., studying the effect of variables like pH, temperature, ligands…), but also time-resolved experiments (in the scale of ms to ps) to detect fast transformations triggered by chemical agents or light. Therefore, the events associated to bioanalytical and biophysical processes need to be monitored by different instrumental platforms, able to probe the variety of chemical and structural transformations undergone by these systems. Our group has strong experience in the monitoring and modelling of bioanalytical processes. The research has been mostly focused on the study of non-canonical DNA structures, such as triplex, G-quadruplex or i-motif, because their importance associated with cancer diseases or nanotechnological applications. Although data fusion has been used to understand bioanalytical and biophysical processes, coupling data of different dimensionality and enabling the joint analysis of sensors with extremely different sampling frequency is yet to be explored in this field.

Within the framework of this project, we plan to work on the following objectives:

1. Modeling the interaction of fluorescent molecules with non-canonical DNA structures for biomedical and analytical purposes (exploiting data structures with missing blocks of information).
Finding molecules that selectively target specific DNA structures for biomedical or analytical purposes has been the objective of several studies made in past projects. To this end, multispectroscopic monitoring acquiring measurements at analogous conditions has been used and the complete information has been analyzed with appropriate multiset analysis methodologies. However, complementary information in bioanalytical process monitoring can come from techniques that operate at an extremely different sampling rate and, therefore, the joint analysis of all available information requires data configurations with missing blocks of information, i.e., blocks where the measurements of the slowest technique do not exist. The bioanalytical processes proposed involve the study of the interaction of diverse ligands of interest with DNA structures, monitored not only by means of UV and/or circular dichroism on-line spectroscopies, which produce a huge amount of data in a relative short period of time, but also with a very informative technique, NMR, which will produce a reduced number of measurements because of the off-line limitations of the technique. In this objective, we will work in collaboration with researchers from Masaryk University (Dr. Petr Taborsky, screening of natural alkaloids as selective binders for DNA), Università degli Studi di Milano (Dra. Stefania Mazzini, NMR studies of DNA and DNA:ligand systems) and University of Sofia (Dra. Diana Cheshmedzhieva and Dr. Aleksey Vasiliev, assessing the selectivity of new cyanine derivatives for DNA detection).

2. Multimodal modeling of the unfolding of multimeric DNA structures induced by temperature.
The stability of a DNA structure (or of a DNA:ligand complex) is usually identified in terms of the changes in enthalpy and entropy associated with the respective unfolding process induced by a rise in temperature. These thermodynamic variables are determined from the univariate monitoring of the unfolding process by means of a spectroscopic technique, such as molecular absorption, circular dichroism, or molecular fluorescence. In a previous project, we developed a hybrid multivariate approach for the modeling of unfolding processes involving monomeric structures (Gargallo, R. Anal. Biochem., 466 (2014) 4-15). The multivariate approach showed to be a good option to detect the presence of intermediates along the unfolding process of these monomeric structures. In the present project, we plan to expand this approach to model the unfolding processes of multimeric structures, like triplexes or multimeric G-quadruplex. The proposed program will be able to manage multimodal data, like those obtained from the coupling of molecular absorption and CD data.

3. A multimodal approach for the characterization of the G-quadruplex structure formed in the promoter region of the SMARCA4 gene by means of ultrafast spectroscopies.
A relevant achievement of previous projects was the identification of a guanine base-rich DNA sequence that forms a G-quadruplex structure whose stability is drastically increased by a slightly decrease of pH from 7.5 to 6.5 (Benabou et al. Sci. Reports, 9 (2019) 15807). The increase in temperature stability of this structure is explained by the formation of an additional C·C + pair between two cytosines present in lateral loops of the G-quadruplex structure. The formation of this pair not only increases stability but also produces a clear decrease in the conformational heterogeneity present at pH values greater than approximately 7.5. From circular dichroism spectra, it is inferred that the resulting G-quadruplex structure at pH 6.5 has a very marked antiparallel character, as opposed to the mixture observed at higher pH values, where parallel contribution is also appreciated. During the present project, time-resolved fluorescence spectroscopy will be used to assess the effect of pH, among other variables, on the stability of the G-quadruplex structure mentioned above. To date, there is only one work published on this topic that only focuses on structural aspects. This new system has an intrinsic biological interest and will serve to test the fusion of time-resolved fluorescence measurements, with a poor spectroscopic resolution (few emission wavelengths) with conventional steady-state excitation-emission fluorescence measurements through incomplete multiset analysis that will incorporate (in the time-resolved block) information on the decay models of the fluorescence curves acquired. Comparison of this approach with other algorithms designed to work specifically with time-resolved data will be carried out. In this objective, we will work with the team of Dr. Cyril Ruckebusch (Université de Lille).

4. Modeling the fluorescent properties of metal nanoclusters stabilized by cytosine-rich DNA sequences for analytical purposes (hybrid multilinear models).
Our group has experience in the synthesis and applications of silver nanoclusters (AgNCs) stabilized by DNA sequences rich in cytosine bases. These so-called DNA-AgNCs are groups of a few silver atoms embedded into a host 3D DNA structure that show interesting fluorescent properties. As example, the fluorescence emission can be tuned by modifying properly the host DNA sequence. DNA-AgNCs have a great potential in bioanalysis, as the hybridization of a fluorescent DNA-AgNCs probe to a target DNA or RNA analyte will produce variations in the emitted fluorescence, which eventually will allow its detection and/or quantitation. In this sense, we have recently proposed a method for the detection of pyrimidine-rich DNA sequences based on the use of fluorescent DNA-AgNCs probes and the formation of triplex structures. (García, J.F. et al. Spec. Chim. Acta Part A 297 (2023) 122752). In a previous work, we contributed to the development of a sensor for pyrimidine-rich sequences within the SARS-CoV-2 genome (Aviñó et al. Int. J. Molec. Sci. 23(2022) 1525) based on the formation of DNA triplex structures. In the present project, we will develop a methodology for the detection of these viral sequences based on the use of fluorescent DNA-AgNCs as analytical probes. To this end, complementary sequences of the target analyte will be designed and tagged with short cytosine-rich stretches able to stabilize fluorescent AgNCs. The hybridization of the probe to the target analyte will produce fluorescence changes that will be monitored producing Excitation-Emission Maps (EEMs). Moreover, it is intended the coupling of EEMs with other spectroscopic techniques, such as circular dichroism, in a multimodal approach.


	© Universitat de Barcelona	Edition: R.Gargallo Last update: 15.02.2024