NEW DIMENSIONS INTO PROTEIN-NUCLEIC ACIDS INTERACTIONS
Creators: name: ANNONI, CHIARA
Publisher: name: Universit� degli Studi di Milano
Year: 2014

description: In the late 19th century, scientists microscopically observed the association of proteins with DNA strands. Since then, researchers have used a variety of in vitro and in vivo assays to demonstrate that proteins interact with DNA and RNA to influence the structure and function of the corresponding nucleic acid. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination and RNA processing and translocation continues to revolutionize our understanding of cell biology, normal cell development and the mechanisms of disease. Furthermore, there is the potential for constructing new molecules with excellent functionalities by assembling the very simple elements and components that are the functional subunits of these natural biopolymers. In this work of thesis we attempted making a new step forward a better knowledge of protein-nucleic acid biopolymers, focusing on two complexes of strong interest. One of the important factors for determining the expression of the function is the structure of the biopolymer. Clarification of the relationship between the structure and the function of biopolymer is of crucial importance to the development of methods for constructing tailor-made functional molecules. The first goal of my thesis focalized on the obtainment of a structural model of a well-known protein-DNA complex: Maf DNA binding domain and it DNA target (T-MARE was used in this study) with the aim of outlining a strategy for the obtainment of activity modulators. Both in silico simulations and experimental studies were carried out leading to the definition of a modus-operandi based on a disorder-order transition of the complex which is commendatory for the protein activation. On the other hand, a number of artificial enzymes have been recently constructed by using the molecular design based on structural information, screening methods that utilize libraries or by a combination of these two methods. However, the above mentioned approaches use single proteins or single nucleic acid as the structural unit, and the activity of the constructed artificial enzymes is remarkably lower in many cases, than that of the native enzymes. The construction of functional complexes (rather than a single biopolymer) as scaffold can be considered as one potential solution to these drawbacks. Further in my studies, using the HIV-Rev peptide and RRE (Rev Responsive Element) RNA complex as a scaffold, for which the tridimensional structure was fully characterized, the assemble of ribonucleopeptidic fluorescent sensors was accomplished in a stepwise manner. In this method, a randomized nucleotide sequence was introduced into the RNA subunit of RNP to construct a RNP library on which the in vitro selection method was applied. In the second step, the Rev peptide was modified with the fluorophore without altering the affinity and specificity of the RNP receptor. In the absence of a ligand for RNP, fluorescence emission was effectively quenched in the RNP complex, but recovered upon ligand binding. RNP sensors were thus created, as the ligand-binding event can be monitored by measurement of the fluorescence signals.