A team of researchers from the Faculty of Physics at Alexandru Ioan Cuza University of Iasi (UAIC) has developed an innovative method to study, at the level of a single molecule, the dynamics of a special DNA structure involved in regulating gene activity.
The UAIC team, comprising Professor Loredana Mereuta and PhD student Adina Cimpanu, under the coordination of Professor Tudor Luchian, has developed a modern method that allows real-time observation of special DNA structures involved in the regulation of gene expression.
The discovery offers new insights into the molecular mechanisms that could underpin future gene therapies.
"Regulation of gene expression is essential for the life of any cell: excessive or insufficient activation of a gene can trigger a disastrous cascade of cellular events, leading to cell death or, in the case of cancer, to cellular immortality," Professor Tudor Luchian explained on the UAIC website.
In order to understand these dysfunctional mechanisms, it is essential to understand the factors that influence the stability and behaviour of DNA.
"Traditionally, DNA is viewed as a classical, straight double helix. However, in our cells DNA is far more dynamic and can adopt a variety of unusual forms, such as four-stranded structures (G-quadruplexes) or, as is the subject of this study, i-motif structures. These form in cytosine (C)-rich regions of the genome, such as telomeres (the ends of chromosomes) and in certain gene promoter sequences, and are strongly influenced by environmental pH. They are considered a 'second genetic code', involved in regulating gene activity," the team coordinator added.
According to him, the method developed by the UAIC researchers is based on the use of a nanoscopic protein that forms an extremely narrow channel in a membrane. When a very weak electric current passes through this channel, DNA molecules interacting with the nanopore cause specific changes in the current, which can be measured with remarkable precision.
Using this technique, the researchers studied a special DNA structure known as the i-motif and identified two modes of detection.
The most promising aspect of this research lies in its potential direct application to the development of new medical treatments.
According to the researchers, selectively blocking i-motifs in certain gene regions involved in diseases such as some forms of cancer could lead to the development of innovative therapies capable of regulating gene activity.
"As part of this research, we created a small molecule called PNA (peptide nucleic acid), made up of just six units, which fits perfectly with the DNA sequence under study. This molecule functions as a reversible 'genetic switch' that can be controlled to penetrate the i-motif structure and destabilise it, preventing its formation, especially before the environment becomes too acidic (a drop in pH). A major advantage of PNA is that it is far more resistant to enzymatic degradation in the body than ordinary DNA or RNA, which gives it significant therapeutic potential. Such PNA probes could be optimised - either by coupling them with peptides that facilitate cell entry or by adding additional nucleotide sequences - to target i-motif structures in the cell more effectively," Tudor Luchian further stated.
He stressed that the study helps to clarify the fundamental mechanisms of life and opens up new perspectives for future medical treatment strategies.
"Our paper therefore presents a powerful method that allows the study of a single DNA molecule in order to understand how i-motif structures interact. In addition, the study shows how special molecules, known as therapeutic PNAs, can be designed to target and control these structures. This research not only helps us better understand the fundamental mechanisms of life, but also opens up perspectives for future medical treatment strategies,' the UAIC research coordinator emphasised.
The research findings are presented in the study entitled "Nanopore-Based, Real-Time Single-Molecule Probing of i-Motif Structural Dynamics and Targeted PNA Disruption", published in the prestigious journal Nano Letters of the American Chemical Society, included in the select Nature Index ranking, which brings together the most relevant international publications in the life sciences.
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